Radiation Response In Prostate Cancer Research Articles

Targeting S6K/NFκB/SQSTM1/Polθ signaling to suppress radiation resistance in prostate cancer

In this study we have identified POLθ-S6K-p62 as a novel druggable regulator of radiation response in prostate cancer. Despite significant advances in delivery, radiotherapy continues to negatively affect treatment outcomes and quality of life due to resistance and late toxic effects to the surrounding normal tissues such as bladder and rectum. It is essential to develop new and effective strategies to achieve better control of tumor. We found that ribosomal protein S6K (RPS6KB1) is elevated in human prostate tumors, and contributes to resistance to radiation. As a downstream effector of mTOR signaling, S6K is known to be involved in growth regulation. However, the impact of S6K signaling on radiation response has not been fully explored. Here we show that loss of S6K led to formation of smaller tumors with less metastatic ability in mice. Mechanistically we found that S6K depletion reduced NFκB and SQSTM1 (p62) reporter activity and DNA polymerase θ (POLθ) that is involved in alternate end-joining repair. We further show that the natural compound berberine interacts with S6K in a in a hitherto unreported novel mode and that pharmacological inhibition of S6K with berberine reduces Polθ and downregulates p62 transcriptional activity via NFκB. Loss of S6K or pre-treatment with berberine improved response to radiation in prostate cancer cells and prevented radiation-mediated resurgence of PSA in animals implanted with prostate cancer cells. Notably, silencing POLQ in S6K overexpressing cells enhanced response to radiation suggesting S6K sensitizes prostate cancer cells to radiation via POLQ. Additionally, inhibition of autophagy with CQ potentiated growth inhibition induced by berberine plus radiation. These observations suggest that pharmacological inhibition of S6K with berberine not only downregulates NFκB/p62 signaling to disrupt autophagic flux but also decreases Polθ. Therefore, combination treatment with radiation and berberine inhibits autophagy and alternate end-joining DNA repair, two processes associated with radioresistance leading to increased radiation sensitivity.

Effect of 1α,25-Dihydroxyvitamin D3 on the Radiation Response in Prostate Cancer: Association With IL-6 Signaling.

Radiotherapy (RT) is the main treatment modality for prostate cancer (PCa). This study investigated the role of IL-6 in biological sequelae following irradiation and highlighted the effects of 1α,25-dihydroxyvitamin D3 (calcitriol) on the radiation response of PCa and its relationship with IL-6 signaling. Human and murine PCa cell lines were used to examine the response to irradiation in vitro and in vivo. The relationship of IL-6 expression with clinicopathologic characteristics in 104 PCa patients treated with definite RT was also examined. We also investigated the changes in radiation response after calcitriol supplementation and the relationship between calcitriol and IL-6 signaling by conducting cellular and animal experiments. Based on clinical samples, the positivity of IL-6 staining is a significant predictor of biochemical failure-free survival for PCa patients treated with definite RT. Data from preclinical models showed that inhibition of IL-6 increased the response of PCa to radiation, which was associated with increased oxidative DNA damage, attenuated EMT and MDSC recruitment, and decreased tumor regrowth. Moreover, increased vitamin D3 levels by calcitriol supplementation or induction by UVB-radiation was associated with inhibited IL-6 signaling and increased the response to irradiation observed in animal models. These data demonstrate that IL-6 play a critical role in the radiation response of PCa, which involved tumor cell killing and altering the tumor microenvironment. Directly targeting IL-6 signaling or vitamin D3 supplement with oral or light treatment could be a promising strategy to increase the response of PCa to radiation.

Ablation of LGR4 signaling enhances radiation sensitivity of prostate cancer cells

AimOur previous study has shown that leucine-rich repeat containing GPCR-4 (LGR4, or GPR48) LGR4 plays a role in cell migration, invasion, proliferation and apoptosis of prostate cancer (PCa). In this study, we aimed to explore whether LGR4 would affect radiation response in PCa. Materials and methodsLGR4 expression was silenced by shRNA transfection. qRT-PCR was employed to determine mRNA expression of LGR4 and DNA damage repair genes. Western blot was used to evaluate protein expression of LGR4, RSPO1-4, androgen receptor (AR), cyclic AMP response-element binding protein (CREB1), γH2A.X, and H2A.X. Cell proliferation was detected by CCK-8 assay and apoptosis was assayed by flow cytometry. Additionally, a xenograft model was also established to validate the role of LGR4 in PCa cells after radiation. Key findingsLGR4 expression was enhanced in PCa cells by radiation treatment in dose- and time-dependent means. RSPO1-4 were also upregulated post-radiation. Furthermore, LGR4 knockdown exacerbated apoptosis, reduced cell viabilities and strengthened nuclear γH2A.X staining in AR positive PCa cells but not in AR negative cells in the presence of radiation. Likewise, LGR4 ablation diminished AR and CREB1 expression induced by radiation. In contrast, RSPO1 stimulation augmented cell viabilities, promoted AR and CREB1 expression, and upregulated DNA repair gene expression, which could be reversed by enzalutamide, except for AR expression. Additionally, LGR4 knockdown further suppressed tumor growth and AR/CREB1 expression but enhanced γH2A.X expression in xenografts. SignificanceIn all, our study suggested that LGR4 might serve as an important regulator of radiation sensitivity in PCa.

A Genomic Approach to Integration of T Category and the Tumor Immune Microenvironment As Part of A Decision Algorithm for Radiation in Prostate Cancer

The nature of the tumor immune microenvironment (TIME) has been shown to have prognostic significance and implications for therapeutic response in many different cancers. Emerging data suggests that radiation may alter the TIME by promoting accumulation of immunosuppressive regulatory T-cells (Treg). Radiation is a mainstay of treatment for prostate cancer (PCa), but the importance of TIME composition with respect to radiation response in PCa has not been fully elucidated. Here, we leverage clinical and genomic data from The Cancer Genome Atlas (TCGA) to further characterize the TIME in a cohort of irradiated T2-T3 PCa patients, and assess relationships between stage, TIME composition, and recurrence free survival (RFS) in the setting of radiation therapy. Tumors treated with radiation were stratified by T2 vs. T3 disease, as well as “High” or “Low” values for measurement of T-regulatory (Treg) cell-specific markers, calculated relative to the mean values of 490 tissue samples. Normalized RNA Seq by expectation maximization (RSEM) values from the TCGA were collected along with expression data for Treg specific markers (CD25, FOXP3, and CTLA4). Clinical data was obtained from the Broad Institute’s Firebrowse tool. RFS was defined as length of time from end of treatment to biochemical recurrence and/or locoregional recurrence and was assessed using Kaplan-Meier estimates. Survival curves were compared using the log-rank test. 490 PCa specimens were retrieved from the TCGA Pan-Cancer Atlas. Of these, 106 tumors were treated with radiation. Patients with T3 disease (n = 67) on initial pathologic diagnosis and high intratumoral co-expression of CD25, FOXP3, and CTLA4 treated with radiation demonstrated significantly decreased estimated 8-year RFS compared to those patients with T3 disease lacking high co-expression of all three markers (p = 0.0084). There was no significant difference in estimated 8-year RFS after radiation in T2 patients (n = 39) with high co-expression of all Treg specific markers compared to those lacking high co-expression of these markers (p = 0.40). Various studies have attempted to decipher the interplay of radiation and Treg cells, the majority having been conducted on mouse models. Our analysis of human specimens revealed that radiation in PCa with extracapsular extension (T3 disease) and Treg specific marker elevation portends significantly worse prognosis than irradiated T3 disease without Treg specific marker elevations - this relationship was not seen in T2 disease. These results suggest a prognostic benefit from risk stratification of PCa patients by both T category and Treg specific activation markers, and warrant investigation into immune checkpoint inhibitors such as anti-CTLA4 antibody as part of the treatment paradigm in this patient subset to enhance anti-tumor effects by inhibiting Treg activity.

MicroRNA-based biomarkers of the radiation response in prostate cancer.

163 Background: Intermediate and high-risk prostate cancer can be cured with radiation (RT) to the prostate and pelvic lymph nodes with androgen deprivation therapy (ADT), but both acute and late toxicity of the GU and GI systems are common. There are no biomarkers predicting radiation outcomes, limiting the opportunity to best personalize prostate radiation therapy. Methods: A prospectively enrolled single arm trial for locally advanced prostate cancer patients (T1-T4N0-N1M0) treated with definitive RT (74Gy IMRT) plus ADT was studied. Biologic samples were available in 108 of 138 patients. Toxicity was recorded using the RTOG morbidity grading system. We applied a panel of microRNA-based germline mutations shown to predict cancer therapy endpoints. Machine learning techniques were used to simultaneously identify prognostic features and perform classification of the biomarkers. Upsampling nested LOO-CV was used to assess performance and generality. Independent Fisher’s exact tests were performed to identify statistically significant marginal associations. Three classifiers were studied: logistic regression with elastic net regularization (EN-LR), classification trees (CT), and random forests (RF), with corresponding hyper-parameters of regularization weights (EN-LR), minimum split and bucket level sample size (CT), number of trees and mtry (RF). Normalized on the simplex, feature importance was defined as absolute value of regression weights for EN-LR, and cumulative decrease in Gini impurity for primary and surrogate splits at each node/splits for CT and RF. Results: Grade 2 or higher toxicity included acute GI (11%), acute GU (34%), late GI (3%) and late GU (16%). GI and GU toxicity and acute and late toxicity had unique predictive biomarkers. The top three marginal genetic associations for late GU toxicity were microRNA site variants in CD6 and CD274 (PDL1)(p.val < 0.01) and BRCA2 (p.val = 0.014). Using RF, CT and EN_LR we could predict late GU toxicity with up to 70% sensitivity, 96% specificity, and 90% accuracy. Conclusions: We have identified microRNA-based biomarkers that can predict late GU toxicity. Work incorporating patient reported outcomes and to identify biomarkers for additional endpoints is ongoing.

Exendin-4 enhances radiation response of prostate cancer.

Exendin-4, one of the most widely used antidiabetic drugs, has recently been reported to have potential antitumor effects in cancers. Prostate cancer (PC) is one of the most common cancers in male patients with type 2 diabetes mellitus, and radiotherapy plays a vital role in the therapy of PC. Whether exendin-4 has the potential to enhance PC response to ionizing radiation (IR) remains unknown. We aimed to explore whether exendin-4 radiosensitizes PC cells. GLP-1 receptor (GLP-1R) expression in PC tissue samples and cell lines were analyzed, Human prostate cancer cells (PC3 and LNCap) were treated with IR and exendin-4, and subjected to proliferation, clone formation, cell cycle, immunoblotting, and immunohistochemical analysis. An in situ prostate tumor of animal model was established. We found that GLP-1R was expressed in human PC tissues and cell lines. 1-100 nM exendin-4 promoted the anti-proliferation effects of IR in vitro and in vivo, and enhanced radiation-induced G2/M cycle arrest in PC cells in a dose-dependent manner. Furthermore, Ex-4 increased AMPK phosphorylation, decrease the levels of p-mTOR, cyclin B, and p34cdc2 . Our study suggested exendin-4 radiosensitizes PC cells via activation of AMPK A and subsequent inhibition of p-mTOR, cyclin B, and p34cdc2 activation.

MiR-875-5p counteracts epithelial-to-mesenchymal transition and enhances radiation response in prostate cancer through repression of the EGFR-ZEB1 axis

Radiotherapy is one of the main treatment choices for non-metastatic prostate cancer (PCa), although development of radioresistance limits its effectiveness. Mounting evidence supports the ability of microRNAs to interfere with different radioresistance-associated pathways, suggesting their potential as radiosensitizers. Here, we demonstrate that reconstitution of miR-875-5p, whose expression is down-regulated in PCa clinical samples and directly correlates with that of E-cadherin, was able to enhance radiation response in PCa cell lines and xenografts through EGFR direct targeting. Consistent with the established role of EGFR in sustaining epithelial-to-mesenchymal transition (EMT) and promoting DNA repair following radiation-induced nuclear translocation, we found that miR-875-5p reconstitution in PCa cells counteracted EMT and impaired DNA lesion clearance. Down-regulation of the EMT-inducing transcription factor ZEB1, which also plays a role in homologous recombination-mediated repair of DNA lesions by regulating CHK1 expression, was found to be a major determinant of miR-875-5p-induced radiosensitization, as confirmed by phenocopy experiments showing that siRNA-mediated ZEB1 knock-down was able to reproduce the microRNA radiosensitizing effect. Overall, our data support the clinical interest in developing a novel therapeutic approach based on miR-875-5p reconstitution to increase PCa response to radiotherapy.

Long non-coding RNA urothelial carcinoma associated 1 (UCA1) mediates radiation response in prostate cancer.

Radioresistance remains a significant obstacle in the treatment of Prostate Cancer (PCa). To simulate the clinical scenario of irradiation resistance (IRR), we created DU145-IRR PCa cell lines by treatment with 2 Gy daily IR for 59 fractions. DU145-IRR cells acquired an aggressive phenotype as evidenced by increased clonogenic survival, tumorigenic potential and invasiveness. We performed transcriptome profiling to discover dysregulated genes in DU145-IRR cells and identified the long non-coding RNA (lncRNA), Urothelial carcinoma-associated 1 (UCA1). We first investigated the role of UCA1 in radiation response and found that UCA1 abundance was significantly higher in DU145-IRR cells compared to control cells. UCA1 siRNA-knockdown reversed the aggressive phenotype and significantly increased sensitivity to IR. UCA1 depletion inhibited growth, induced cell cycle arrest at the G2/M transition and decreased activation of the pro-survival Akt pathway. We then studied the clinical significance of UCA1 expression in two independent cohorts of PCa patients: MSKCC (130 patients) and CPC-GENE (209 patients). UCA1 over-expression was associated with decreased 5-year disease-free survival in MSKCC patients (HR = 2.9; p = 0.007) and a trend toward lower biochemical recurrence-free survival in CPC-GENE patients (HR = 2.7; p = 0.05). We showed for the first time that UCA1 depletion induces radiosensitivity, decreases proliferative capacity and disrupts cell cycle progression, which may occur through altered Akt signaling and induced cell cycle arrest at the G2/M transition. Our results indicate that UCA1 might have prognostic value in PCa and be a potential therapeutic target.

Mechanistic Insights into Molecular Targeting and Combined Modality Therapy for Aggressive, Localized Prostate Cancer.

Radiation therapy (RT) is one of the mainstay treatments for prostate cancer (PCa). The potentially curative approaches can provide satisfactory results for many patients with non-metastatic PCa; however, a considerable number of individuals may present disease recurrence and die from the disease. Exploiting the rich molecular biology of PCa will provide insights into how the most resistant tumor cells can be eradicated to improve treatment outcomes. Important for this biology-driven individualized treatment is a robust selection procedure. The development of predictive biomarkers for RT efficacy is therefore of utmost importance for a clinically exploitable strategy to achieve tumor-specific radiosensitization. This review highlights the current status and possible opportunities in the modulation of four key processes to enhance radiation response in PCa by targeting the: (1) androgen signaling pathway; (2) hypoxic tumor cells and regions; (3) DNA damage response (DDR) pathway; and (4) abnormal extra-/intracell signaling pathways. In addition, we discuss how and which patients should be selected for biomarker-based clinical trials exploiting and validating these targeted treatment strategies with precision RT to improve cure rates in non-indolent, localized PCa.

TGF-β1 mediates the radiation response of prostate cancer

Radiotherapy is the main treatment modality for prostate cancer. This study investigated the role of TGF-β1 in biological sequelae and tumor regrowth following irradiation, which are critical for the clinical radiation response of prostate cancer. Human and murine prostate cancer cell lines, and corresponding hormone-refractory (HR) cells, were used to examine the radiation response by clonogenic assays in vitro and tumor growth delay in vivo. Biological changes after irradiation, including cell death and tumor regrowth, were examined by experimental manipulation of TGF-β1 signaling. The correlations among tumor radiation responses, TGF-β1 levels, and regulatory T cells (Tregs) recruitment were also evaluated using animal experiments. HR prostate cancer cells appeared more radioresistant and had higher expression of TGF-β1 compared to hormone-sensitive (HS) cells. TGF-β1 expression was positively linked to irradiation and radioresistance, as demonstrated by in vitro and in vivo experiments. Inhibition of TGF-β1 increased tumor inhibition and DNA damage after irradiation. When mice were irradiated with a sub-lethal dose, the regrowth of irradiated tumors was significantly correlated with TGF-β1 levels and Tregs accumulation in vivo. Furthermore, blocking TGF-β1 clearly attenuated Tregs accumulation and tumor regrowth following treatment. These data demonstrate that TGF-β1 is important in determining the radiation response of prostate cancer, including tumor cell killing and the tumor microenvironment. Therefore, concurrent treatment with a TGF-β1 inhibitor is a potential therapeutic strategy for increasing the radiation response of prostate cancer, particularly for more aggressive or HR cancer cells. • HR prostate cancer cells appeared more radioresistant and had higher expression of TGF-β1. • TGF-β1 was positively linked to the radiation resistance of prostate cancer. • Tumor regrowth following irradiation was significantly correlated with TGF-β1 and Tregs levels. • Blocking TGF-β1 significantly attenuated RT-induced DNA repair and Tregs. • TGF-β1 inhibitor increases the radiation response of HR cancer cells.

TGF-β1 mediates the radiation response of prostate cancer.

Radiotherapy is the main treatment modality for prostate cancer. This study investigated the role of TGF-β1 in biological sequelae and tumor regrowth following irradiation, which are critical for the clinical radiation response of prostate cancer. Human and murine prostate cancer cell lines, and corresponding hormone-refractory (HR) cells, were used to examine the radiation response by clonogenic assays in vitro and tumor growth delay in vivo. Biological changes after irradiation, including cell death and tumor regrowth, were examined by experimental manipulation of TGF-β1 signaling. The correlations among tumor radiation responses, TGF-β1 levels, and regulatory T cells (Tregs) recruitment were also evaluated using animal experiments. HR prostate cancer cells appeared more radioresistant and had higher expression of TGF-β1 compared to hormone-sensitive (HS) cells. TGF-β1 expression was positively linked to irradiation and radioresistance, as demonstrated by in vitro and in vivo experiments. Inhibition of TGF-β1 increased tumor inhibition and DNA damage after irradiation. When mice were irradiated with a sub-lethal dose, the regrowth of irradiated tumors was significantly correlated with TGF-β1 levels and Tregs accumulation in vivo. Furthermore, blocking TGF-β1 clearly attenuated Tregs accumulation and tumor regrowth following treatment. These data demonstrate that TGF-β1 is important in determining the radiation response of prostate cancer, including tumor cell killing and the tumor microenvironment. Therefore, concurrent treatment with a TGF-β1 inhibitor is a potential therapeutic strategy for increasing the radiation response of prostate cancer, particularly for more aggressive or HR cancer cells. • HR prostate cancer cells appeared more radioresistant and had higher expression of TGF-β1. • TGF-β1 was positively linked to the radiation resistance of prostate cancer. • Tumor regrowth following irradiation was significantly correlated with TGF-β1 and Tregs levels. • Blocking TGF-β1 significantly attenuated RT-induced DNA repair and Tregs. • TGF-β1 inhibitor increases the radiation response of HR cancer cells.

The role of IL-6 in the radiation response of prostate cancer

BackgroundHormone-resistant (HR) prostate cancers are highly aggressive and respond poorly to treatment. IL-6/STAT3 signaling has been identified to link with the transition of HR and aggressive tumor behavior. The role of IL-6 in the radiation response of prostate cancer was investigated in the present study.Material and methodsThe murine prostate cancer cell line (TRAMP-C1) and the hormone-resistant cell sub-line, TRAMP-HR, were used to assess the radiation response using in vitro clonogenic assays and tumor growth delay in vivo. Biological changes following irradiation were investigated by means of experimental manipulation of IL-6 signaling. Correlations among IL-6 levels, tumor regrowth, angiogenesis and myeloid-derived suppressor cell (MDSC) recruitment were examined in an animal model.ResultsHR prostate cancer cells had a higher expression of IL-6 and more activated STAT3, compared to TRAMP-C1 cells. HR prostate cancer cells had a greater capacity to scavenge reactive oxygen species, suffered less apoptosis, and subsequently were more likely to survive after irradiation. Moreover, IL-6 expression was positively linked to irradiation and radiation resistance. IL-6 inhibition enhanced the radiation sensitivity of prostate cancer, which was associated with increased p53, RT-induced ROS and oxidative DNA damage. Furthermore, when mice were irradiated with a sub-lethal dose, inhibition of IL-6 protein expression attenuated angiogenesis, MDSC recruitment, and decreased tumor regrowth.ConclusionThese data demonstrate that IL-6 is important in the biological sequelae following irradiation. Therefore, treatment with concurrent IL-6 inhibition is a potential therapeutic strategy for increasing the radiation response of prostate cancer.

TU‐A‐BRC‐02: Hyperpolarized 13C MRSI for Assessing Radiation Response of Prostate Cancer in Transgenic Mice

Purpose: To assess the tumor response to radiation in transgenic mouse model of prostatic adenocarcinoma (TRAMP) by hyperpolarized MR spectroscopic imaging (MRSI) of [1–13C]pyruvate.Methods: The TRAMP mouse model used in this study develops an autochthonous tumor in the prostate at the onset of puberty. The progression of tumors was volumetrically monitored by T2‐weighted MRI at 7 T. Four mice with tumor volumes of 400–1500 mm3 underwent hyperpolarized 13C MRSI at 3 T. Two of them received fractionated X‐ray radiation (200 kV, 20 mA, 1.21 cGy/s, 1 mm Cu HVL) doses of 10 and 20 Gy to the tumors, followed by the 13C MRSI at the fourth day of postirradiation. Dynamic 13C MRSI was performed following an intravenous bolus injection of 300 μL of 80 mM hyperpolarized [1–13C]pyruvate to obtain the transient fate of 13C‐labeled metabolites in a 3D volume encompassing the tumor with a nominal spatial resolution of 2.7×2.7×3 mm3. The average time‐resolved signal intensities of pyruvate and its downstream products lactate and alanine were measured in the tumors from reconstructed metabolic images. Metabolic response was characterized in terms of lactate/pyruvate ratio and apparent rate constant of pyruvate‐to‐lactate conversion, kpl, by fitting data to a two‐site exchange model. Results: Based on MRI volumetry, the TRAMP tumors exponentially grew with doubling time of 7±3 d and shrank in response to radiation by 50% in volume within 4±1 d. The untreated tumors that grew to larger volumes over a similar postirradiation period exhibited increased lactate/pyruvate ratio and kpl compared to the treated tumors. Alanine was in noise levels and hence not considered for analysis. Conclusions: Preliminary data indicate the feasibility of utilizing hyperpolarized 13C MRSI to assess therapeutic response of prostate cancer to radiation in TRAMP mice. Further investigations are underway to obtain additional data.This work is partly supported by DOD PCRP (W81XWH‐10‐10498) research award.

Antagonists of growth hormone-releasing hormone potentiate radiation response in prostate cancer.

10597 Background: Knockdown of GHRH gene expression suppresses the proliferation of cancer cell lines and antagonists of GHRH (GHRH-A) inhibit growth of experimental human cancer. The effect of GHRH antagonists on radiation response is not completely elucidated. Therefore, experiments of GHRH antagonists (JMR-132 and MIA-606) given after tumor radiation were conducted. Methods: Two experiments involving athymic nu/nu nude mice implanted with PC-3 human tumor xenografts were done. In the first experiment 40 mice (10 animals in each group) were treated as follows: Group 1 control group (no treatment), Group 2 JMR-132 alone, Group 3 radiation (XRT) alone, and Group 4 XRT followed by JMR-132 for 3 weeks (XRT-JMR-132). JMR-132 was injected s.c. at 10 μg/day s.c. daily for 3 weeks. The radiation dose was 8 Gy in a single fraction. In the second experiment 36 nude mice were divided into 4 groups as follows: Group 1 control group (no treatment), Group 2 MIA-606 alone, Group 3 radiation alone (XRT) and Group 4 XRT followed by MIA-606 (XRT-MIA-606). MIA-606 was injected s.c. at 2 μg/day daily for 4 weeks. The radiation dose was a single fraction of 2 Gy. In both experiments, the implanted tumor was exposed to the x-ray beam and the rest of the animal's body was shielded by a lead block. Results: No significant differences were seen when GHRH-A JMR-132 was added after radiation. However, the use of new, more potent GHRH antagonist MIA-606 resulted in a significant potentiation of the inhibitory effect of radiation on the PC3 human prostate cancer. At 4 weeks, an 83% tumor growth inhibition was seen when MIA-606 was given after radiation as compared to a 70% inhibition when radiation was given alone (p < 0.05). MIA-606 alone led to a 39% tumor growth inhibition at 4 weeks compared to control. Conclusions: These results indicate, for the first time, that GHRH antagonists given after radiation may potentiate the inhibitory effect of radiation on tumors. This finding has significant clinical implications for determining the optimal sequence of these drugs with radiation. No significant financial relationships to disclose.