Radiomics-Based Detection of Radionecrosis Using Harmonized Multiparametric MRI.

In this study, a radiomics analysis was conducted to provide insights into the differentiation of radionecrosis and tumor progression in multiparametric MRI in the context of a multicentric clinical trial. First, the sensitivity of radiomic features to the unwanted variability caused by different protocol settings was assessed for each modality. Then, the ability of image normalization and ComBat-based harmonization to reduce the scanner-related variability was evaluated. Finally, the performances of several radiomic models dedicated to the classification of MRI examinations were measured. Our results showed that using radiomic models trained on harmonized data achieved better predictive performance for the investigated clinical outcome (balanced accuracy of 0.61 with the model based on raw data and 0.72 with ComBat harmonization). A comparison of several models based on information extracted from different MR modalities showed that the best classification accuracy was achieved with a model based on MR perfusion features in conjunction with clinical observation (balanced accuracy of 0.76 using LASSO feature selection and a Random Forest classifier). Although multimodality did not provide additional benefit in predictive power, the model based on T1-weighted MRI before injection provided an accuracy close to the performance achieved with perfusion.

RhoB controls the 24 kDa FGF-2-induced radioresistance in HeLa cells by preventing post-mitotic cell death.

Farnesylated Ras oncoprotein induces a cellular resistance to ionizing radiation that can be reversed by farnesyltransferase inhibitors (FTI). We previously demonstrated that, expression of the 24 kDa FGF2 isoform in wild type ras bearing HeLa cells, induced radioresistance which was also reversed by FTI. We tested the hypothesis that wild type Ras or RhoB, which has been proposed as a potential FTI target, could control the FGF-2-induced radioresistance mechanisms. For this, we expressed inducible dominant negative forms of Ras (RasN17) and Rho (RhoBN19) in 24 kDa FGF2 transfected HeLa cells and analysed their survival after irradiation. While no cell survival modification was observed after RasN17 induction, the expression of RhoBN19 induced a radiosensitization of FGF2 radioresistant HeLa cells in the same range as the one observed after a 48 h treatment with the specific FTI, R115777. Moreover, we showed that activated RhoB but not RhoA induced radioresistance in NIH3T3 cells. The radiosensitizer effect of RhoBN19 expression was due to the induction of the radiation induced post-mitotic cell death. Taken together, these data demonstrate that 24 kDa FGF-2-induced radioresistance is controlled by Rho pathways and suggest that RhoB should be a major determinant in cellular resistance to ionizing radiation.

The radioprotective effect of the 24 kDa FGF-2 isoform in HeLa cells is related to an increased expression and activity of the DNA dependent protein kinase (DNA-PK) catalytic subunit.

We previously reported that overexpression of the 24 kDa basic fibroblast factor (or FGF-2) isoform provides protection from the cytotoxic effect of ionizing radiation (IR). DNA double-strand breaks (DSB), the IR-induced lethal lesions, are mainly repaired in human cells by non-homologous end joining system (NHEJ). NHEJ reaction is dependent on the DNA-PK holoenzyme (composed of a regulatory sub-unit, Ku, and a catalytic sub-unit, DNA-PKcs) that assembles at sites of DNA damage. We demonstrated here that the activity of DNA-PK was increased by twofold in two independent radioresistant cell lines, HeLa 3A and CAPAN A3, over expressing the 24 kDa FGF-2. This increase was associated with an overexpression of the DNA-PKcs without modification of Ku expression or activity. This overexpression was due to an up-regulation of the DNA-PKcs gene transcription by the 24 kDa FGF-2 isoform. Finally, HeLa 3A cells exhibited the hallmarks of phenotypic changes associated with the overexpression of an active DNA-PKcs. Indeed, a faster repair rate of DSB and sensitization to IR by wortmannin was observed in these cells. Our results represent the characterization of a new mechanism of control of DNA repair and radioresistance in human tumor cells dependent on the overproduction of the 24 kDa FGF-2 isoform.

The farnesyltransferase inhibitor R115777 reduces hypoxia and matrix metalloproteinase 2 expression in human glioma xenograft.

PURPOSE: The high-grade primary brain tumors, glioblastoma, of extremely bad prognosis contain large regions of hypoxia known to be involved in the chemo- and radioresistance. We demonstrated previously that radioresistant human wild-type Ras U87 glioblastoma can be radiosensitized in vitro by the specific farnesyltransferase inhibitor R115777. The aim of this study was to analyze the effect of this compound on the hypoxic status and the vascularization of this tumor. EXPERIMENTAL DESIGN: U87 xenografts bearing mice were treated with 100 mg/kg of R115777 b.i.d. during 4 days. Hypoxia was assessed by measuring the binding of hypoxic cell marker pentafluorinated 2-nitroimidazole. Immunohistochemistry was performed to analyze angiogenesis and metalloproteinase-2 expression. RESULTS: We demonstrated here that R115777 treatment induced a significant oxygenation of U87 xenografts (P<0.001) associated with a decrease of hypoxia-inducible factor 1alpha expression. This reduction of hypoxia was not due to a decrease of tumor size after R115777 treatment. This oxygenation was associated with a change in vessel morphology and with a significant decrease of the vessel density. Moreover, R115777 treatment reduced matrix metalloproteinase 2 expression in xenografts and inhibited matrix metalloproteinase 2 activity in vitro. These data strongly suggest that R115777 could increase this tumor oxygenation at least by interacting with angiogenesis. CONCLUSION: Our results demonstrate that R115777 treatment inhibits different pathways leading to the radioresistance of wild-type Ras expressing glioblastoma, including intrinsic radioresistance, hypoxia, and angiogenesis. These combined effects on glioblastoma underline the interest of associating R115777 with radiotherapy as a new treatment of these tumors of catastrophic prognosis.

Erythropoietin-induced reduction of hypoxia before and during fractionated irradiation contributes to improvement of radioresponse in human glioma xenografts.

PURPOSE: Our study investigated the influence of recombinant human erythropoietin (rHuEPO) treatment, inducing raised hemoglobin levels in nonanemic mice, on intratumor oxygenation before and during fractionated irradiation. Furthermore, the consequences of rHuEPO administration on tumor response to fractionated radiotherapy (RT) were evaluated. METHODS AND MATERIALS: Experiments were performed on two human malignant glioma (GBM Nan1 and U87) xenografted in nude mice. RHuEPO was daily delivered (0.3 IU/g/day, 5 days/week). Tumor hypoxia was assessed before (T1) and during (T6) fractionated irradiation using (1) pO(2)-Histograph (Eppendorf, Hamburg, Germany) and (2) the EF5-binding assay. Vascular density was determined using type IV collagen immunostaining. To assess RT efficacy, the irradiation schedule was 20 fractions of 2 Gy, once daily, 5 days/week over 4 weeks. RESULTS: At T1, hemoglobin levels in rHuEPO-treated mice were significantly increased. Percentage of pO(2) values <2.5 mm Hg was reduced in rHuEPO-treated tumors as compared with control groups (37.1 +/- 19.1% vs. 58.5 +/- 27.0%; p = 0.009 for GBM Nan1; 81.6 +/- 13.4% vs. 91.5 +/- 8.3%; p = 0.035 for U87). The decrease of viable hypoxic tumor cells fraction after rHuEPO was confirmed by the EF5-binding assay. Vascular density was not altered after rHuEPO treatment. At T6, rHuEPO reduced the hypoxic fraction by about 20% (p = 0.036 and p = 0.171) in GBM Nan1 and U87 irradiated tumors. RHuEPO did not influence tumor growth by itself. RT alone or combined with rHuEPO induced a significant tumor growth delay. Finally, rHuEPO significantly enhanced RT efficacy (p = 0.012 in GBM Nan1 and p = 0.037 in U87), resulting in radiopotentiation ratios of 1.21 and 1.54 for respective models. CONCLUSIONS: Our results indicate that rHuEPO, by enhancing blood oxygen-carrying capacity, decreases intrinsic tumor hypoxia and maintains its effect during fractionated irradiation in malignant glioma xenografts. Therefore, rHuEPO contributes to radiosensitize these tumors.

Integration method of 3D MR spectroscopy into treatment planning system for glioblastoma IMRT dose painting with integrated simultaneous boost.

BACKGROUND: To integrate 3D MR spectroscopy imaging (MRSI) in the treatment planning system (TPS) for glioblastoma dose painting to guide simultaneous integrated boost (SIB) in intensity-modulated radiation therapy (IMRT). METHODS: For sixteen glioblastoma patients, we have simulated three types of dosimetry plans, one conventional plan of 60-Gy in 3D conformational radiotherapy (3D-CRT), one 60-Gy plan in IMRT and one 72-Gy plan in SIB-IMRT. All sixteen MRSI metabolic maps were integrated into TPS, using normalization with color-space conversion and threshold-based segmentation. The fusion between the metabolic maps and the planning CT scans were assessed. Dosimetry comparisons were performed between the different plans of 60-Gy 3D-CRT, 60-Gy IMRT and 72-Gy SIB-IMRT, the last plan was targeted on MRSI abnormalities and contrast enhancement (CE). RESULTS: Fusion assessment was performed for 160 transformations. It resulted in maximum differences <1.00 mm for translation parameters and ≤1.15° for rotation. Dosimetry plans of 72-Gy SIB-IMRT and 60-Gy IMRT showed a significantly decreased maximum dose to the brainstem (44.00 and 44.30 vs. 57.01 Gy) and decreased high dose-volumes to normal brain (19 and 20 vs. 23% and 7 and 7 vs. 12%) compared to 60-Gy 3D-CRT (p < 0.05). CONCLUSIONS: Delivering standard doses to conventional target and higher doses to new target volumes characterized by MRSI and CE is now possible and does not increase dose to organs at risk. MRSI and CE abnormalities are now integrated for glioblastoma SIB-IMRT, concomitant with temozolomide, in an ongoing multi-institutional phase-III clinical trial. Our method of MR spectroscopy maps integration to TPS is robust and reliable; integration to neuronavigation systems with this method could also improve glioblastoma resection or guide biopsies.

Farnesyltransferase inhibitor effects on prostate tumor micro-environment and radiation survival.

BACKGROUND: Ras activation by mutation, overexpression, or receptor signaling can increase tumor cell survival after irradiation. METHODS: We examined whether inhibiting Ras activity with farnesyltransferase inhibitors (FTI) altered the radiosensitivity and tumor micro-environment in prostate tumors. RESULTS: Treatment with FTIs L-744,832 or FTI-277 reduced clonogenic survival of prostate tumor cells expressing oncogenic H-ras after irradiation. PI3-kinase/Akt and MAPK signaling pathways were downregulated by FTIs in these cells. FTI treatment reduced tumor hypoxia and also reduced MMP-9 expression in tumors with activated mutant H-ras. FTI treatment did not, however, increase apoptosis in irradiated intestine, demonstrating that acute radiation injury of this normal tissue was not enhanced by FTIs. CONCLUSIONS: FTIs can enhance the killing of prostate tumors with activated H-Ras. Together with the absence of increased acute toxicity to normal bowel, these results imply that FTI treatment should be further studied as a possible adjuvant to radiotherapy in the treatment of abdominal cancers with activated Ras signaling.

Increased expression of a COOH-truncated nucleophosmin resulting from alternative splicing is associated with cellular resistance to ionizing radiation in HeLa cells.

We previously demonstrated that transfecting HeLa cells with the 24 kDa basic fibroblast growth factor-2 (FGF-2) isoform dramatically increased cell survival after irradiation. To investigate genes implicated in this radioresistance acquisition, we compared mRNA expression between radioresistant 24 kDa FGF-2-expressing cells (HeLa 3A) and radiosensitive control HeLa PINA cells using the differential display technique. Of the 32 differentially expressed mRNAs, 1 presented a significant homology with a known gene. This 378 bp fragment presented 100% identity with exon 11 and 12 of human nucleophosmin (NPM) but differed by including a part of intron 9 in its 5' end. The differential expression of this fragment was confirmed using an RNase protection assay. We then cloned the entire corresponding mRNA and showed that it contained all the exons of NPM plus intron 9, suggesting that it was a splicing product of the NPM gene. This variant encoded for a 35-amino acid truncated NPM (NPM2). NPM2 expression was increased in HeLa 3A. To investigate NPM2's role in radioresistance acquisition, we transfected HeLa cells with NPM2 cDNA and analyzed survival after irradiation of the clones obtained. After transfection with NPM2, radiosensitive HeLa cells exhibited a dramatic increase in cell survival after irradiation. Taken together, our results demonstrate that expression of a COOH-truncated NPM form resulting from the alternative splicing of NPM mRNA is able to increase cell survival after irradiation and suggests that it might be involved in cellular response to ionizing radiation.

Farnesyltransferase inhibitor, R115777, reverses the resistance of human glioma cell lines to ionizing radiation.

We investigated for the first time the ability of farnesyltransferase inhibitors (FTI) to radiosensitize human glioma. For this, human glioma cell lines were treated with the specific FTI, R115777, 48 hr prior to a 2Gy irradiation. The treatment with R115777 decreased by 45% the SF2 value of the more radioresistant glioma cell lines (SF763 and U87) without any significant effect on the radioresistance of the radiosensitive ones (SF767 and U251-MG). This radiosensitizer effect was due to the induction of post-mitotic necrotic cell death. We then tested the hypothesis that wild-type Ras or RhoB, which has been proposed as potential FTI target, could control the glioma radioresistance. For this, we expressed inducible dominant negative forms of Ras (RasN17) and RhoB (RhoBN19) in radioresistant U87 glioma cell line and analyzed the survival after irradiation of the obtained clones. While blocking Ras pathways by expression of RasN17 did not affect the SF2 value of the U87 glioma cell line, the expression of RhoBN19 dramatically reduced the cell survival after irradiation of these cells. Taken together, these data demonstrated that RhoB, but not Ras, is implicated in glioma radioresistance. Furthermore, the R115777 differential radiosensitizer effect underlines the potential therapeutic interest of using this drug as a radiosensitizer of human glioma.