Analysis of Gene Expression Using Gene Sets Discriminates Cancer Patients with and without Late Radiation Toxicity

BACKGROUND: Radiation is an effective anti-cancer therapy but leads to severe late radiation toxicity in 5%–10% of patients. Assuming that genetic susceptibility impacts this risk, we hypothesized that the cellular response of normal tissue to X-rays could discriminate patients with and without late radiation toxicity. METHODS AND FINDINGS: Prostate carcinoma patients without evidence of cancer 2 y after curative radiotherapy were recruited in the study. Blood samples of 21 patients with severe late complications from radiation and 17 patients without symptoms were collected. Stimulated peripheral lymphocytes were mock-irradiated or irradiated with 2-Gy X-rays. The 24-h radiation response was analyzed by gene expression profiling and used for classification. Classification was performed either on the expression of separate genes or, to augment the classification power, on gene sets consisting of genes grouped together based on function or cellular colocalization. X-ray irradiation altered the expression of radio-responsive genes in both groups. This response was variable across individuals, and the expression of the most significant radio-responsive genes was unlinked to radiation toxicity. The classifier based on the radiation response of separate genes correctly classified 63% of the patients. The classifier based on affected gene sets improved correct classification to 86%, although on the individual level only 21/38 (55%) patients were classified with high certainty. The majority of the discriminative genes and gene sets belonged to the ubiquitin, apoptosis, and stress signaling networks. The apoptotic response appeared more pronounced in patients that did not develop toxicity. In an independent set of 12 patients, the toxicity status of eight was predicted correctly by the gene set classifier. CONCLUSIONS: Gene expression profiling succeeded to some extent in discriminating groups of patients with and without severe late radiotherapy toxicity. Moreover, the discriminative power was enhanced by assessment of functionally or structurally related gene sets. While prediction of individual response requires improvement, this study is a step forward in predicting susceptibility to late radiation toxicity

Expression of the progenitor marker NG2/CSPG4 predicts poor survival and resistance to ionising radiation in glioblastoma

Glioblastoma (GBM) is a highly aggressive brain tumour, where patients respond poorly to radiotherapy and exhibit dismal survival outcomes. The mechanisms of radioresistance are not completely understood. However, cancer cells with an immature stem-like phenotype are hypothesised to play a role in radioresistance. Since the progenitor marker neuron-glial-2 (NG2) has been shown to regulate several aspects of GBM progression in experimental systems, we hypothesised that its expression would influence the survival of GBM patients. Quantification of NG2 expression in 74 GBM biopsies from newly diagnosed and untreated patients revealed that 50% express high NG2 levels on tumour cells and associated vessels, being associated with significantly shorter survival. This effect was independent of age at diagnosis, treatment received and hypermethylation of the O6-methylguanine methyltransferase (MGMT) DNA repair gene promoter. NG2 was frequently co-expressed with nestin and vimentin but rarely with CD133 and the NG2 positive tumour cells harboured genetic aberrations typical for GBM. 2D proteomics of 11 randomly selected biopsies revealed upregulation of an antioxidant, peroxiredoxin-1 (PRDX-1), in the shortest surviving patients. Expression of PRDX-1 was associated with significantly reduced products of oxidative stress. Furthermore, NG2 expressing GBM cells showed resistance to ionising radiation (IR), rapidly recognised DNA damage and effectuated cell cycle checkpoint signalling. PRDX-1 knockdown transiently slowed tumour growth rates and sensitised them to IR in vivo. Our data establish NG2 as an important prognostic factor for GBM patient survival, by mediating resistance to radiotherapy through induction of ROS scavenging enzymes and preferential DNA damage signalling

Enhancement of radiation effectiveness by hyperthermia and incorporation of halogenated pyrimidines at low radiation doses as compared with high doses: implications for mechanisms

Application of the linear-quadratic (LQ) model for description of mammalian cell survival curves to evaluate radiosensitization in dependence on dose. Data on clonogenic assays concerning the combined effects of radiation with hyperthermia and halogenated-pyrimidines, were analyzed according to the LQ formula for cell survival: S(D)/S(0) = exp-(αD+βD(2)). Effects of these agents on the linear parameter α and the quadratic parameter β are compared to evaluate differences depending on the applied dose, and the possible relations to mechanisms of radiosensitization. The values of the linear parameter α, which determines the effectiveness at low doses, are for most cell lines and in most conditions more increased than the values of the parameter β which has a higher contribution at larger radiation doses. These results can be explained by the assumption that the probability of interaction of two DNA double-strand breaks (DSB) produced in close proximity by individual ionizing particles is more enhanced than interaction of DSB produced at larger distances by two separate particles. The observed differences between values of α and β imply that the radiation enhancement factors are larger at low doses mostly applied in clinical radiotherapy as compared with larger doses as mostly evaluated in experimental studie

Analysis of enhancement at small and large radiation doses for effectiveness of inactivation in cultured cells by combining two agents with radiation

To evaluate the enhancement effect of two combined radiation-sensitizing agents in mammalian cells at small doses as compared to large doses using the linear-quadratic (LQ) mathematical model. Data on clonogenic assays concerning the radio-enhancement effects of combined halogenated pyrimidines and hyperthermia or combined cisplatin and hyperthermia, as published in earlier reports, were analyzed according to the LQ-formula: S(D)/S(0) = exp-(αD + βD(2)). Effects of sensitizing agents on the linear parameter α and the quadratic parameter β are compared in order to evaluate differences depending on the applied dose, the possible relations to mechanisms of radiation sensitization and to derive suggestions for applications. The values of the linear parameter α, which determines the effectiveness at low doses, are for all cell lines and all conditions more increased than the values of the parameter β which has a higher contribution at larger radiation doses. The combination of hyperthermia with halogenated pyrimidines to radiation as well as the combination of hyperthermia and cisplatin to radiation significantly increases the value of the linear parameter α, as compared to radiation alone or radiation combined with a single agent. The radiation enhancement factors of the values of linear and quadratic parameters demonstrate that the sensitizing agents have a larger effect on the linear parameter which is dominant at low radiation doses as is used in fractionated-radiation treatment in the clinic. Moreover, the effect is even further increased when two radiation sensitizers are use

Colour junctions as predictors of radiosensitivity: X-irradiation combined with gemcitabine in a lung carcinoma cell line

Purpose. To determine whether measurement of colour junctions by fluorescent in situ hybridisation (FISH) can predict radiosensitisation of gemcitabine (2'-2'-difluorodeoxycytidine). Methods. Human lung carcinoma cells (SW-1573) were irradiated with X-rays with or without incubation of 10 nM gemcitabine for 24 h. Cell survival was determined with clonogenic assay. Colour junctions were measured by whole chromosome FISH of chromosomes 2 and 18 and were scored according to the PAINT method. Results. A clear radiosensitisation by gemcitabine was observed on cell survival. A significant decrease in the number of colour junctions was observed after gemcitabine treatment compared with radiation treatment alone. The correlation between colour junction induction and cell survival was high for both with and without gemcitabine, but the gemcitabine-sensitised curve did not coincide with the non-sensitised curve. Conclusions. Gemcitabine-induced radiosensitisation is not predicted by induction of colour junctions in cultured SW-1573 cells. This reduces the clinical applicability of this predictive assay for radiotherapy in combination with gemcitabin

The impact of the time interval between radiation and hyperthermia on clinical outcome in patients with locally advanced cervical cancer

The benefit of hyperthermia combined with radiotherapy is well-acknowledged for patients with locally advanced cervical cancer (LACC) (1-3). However, recently a discussion evolved on the optimal time interval between radiotherapy and hyperthermia. Kroesen et al. (4) recently reported a retrospective analysis of factors influencing clinical results of treatment with radiotherapy and hyperthermia in a large cohort of locally advanced cervical cancer patients (LACC) at ErasmusMC in Rotterdam. They concluded that there is no detrimental effect of prolonged intervals on clinical outcome within a time frame of 4 h between radiotherapy and hyperthermia. Kroesen et al. thereby explicitly dismissed the findings of Van Leeuwen et al. (5) in a smaller cohort of LACC patients treated at the Academic Medical Center (AMC) of the University of Amsterdam. In that study longer time intervals and lower tumor temperatures were both found to have a highly negative effect on in-field tumor control (time interval: P = 0.021, in multivariable analysis p = 0.007) and overall survival (idem: P = 0.015, in multivariable analysis p = 0.012), where it is important to note that the median time intervals between radiotherapy and hyperthermia were ∼60 and ∼90 min for the short and long time interval subgroups of patients, respectively