Enhancement of radiation response by roscovitine in human breast carcinoma in vitro and in vivo.

Frequent deregulation of cyclin-dependent kinase (CDK) activation associated with loss of cell cycle control was found in most of human cancers. A recent development of a new class of antineoplasic agents targeting the cell cycle emerged as a small molecule CDK inhibitor, roscovitine, which presents potential antiproliferative and antitumoral effects in human tumors. Additional studies reported that roscovitine combined with cytotoxic agents can cooperate with DNA damage to activate p53 protein. However, little is known about the biological effect of roscovitine combined with ionizing radiation (IR) in human carcinoma, and no studies were reported thus far in p53 mutated carcinoma. In the breast cancer cell line MDA-MB 231, which lacks a functional p53 protein, we found a strong radiosensitization effect of roscovitine in vitro by clonogenic survival assay and in vivo in MDA-MB 231 xenograft model. Using Pulse Field Gel Electrophoresis, a strong impairment in DNA-double-strand break rejoining was observed after roscovitine and IR treatment as compared with IR alone. Cell cycle analysis showed a G(2) delay and no increase in radiation-induced apoptosis in the cells treated with IR or roscovitine and IR. On the other hand, we found a significant induction in micronuclei frequency after roscovitine and IR treatment as compared with IR alone. This effect was also observed in BALB murine cells in contrast to SCID murine cells, which are deficient in DNA-PKcs, suggesting a possible DNA-double-strand break repair defect in the nonhomologous end joining pathways. In MDA-MB 231 cells, the radiosensitization effect of roscovitine was associated with an inhibition of the DNA-dependent protein kinase activity caused by a marked decrease in Ku-DNA binding by using the electrophoretic mobility shift assay. In conclusion, we found a novel effect on DNA repair of the CDK inhibitor roscovitine, which acts as a radiosensitizer in vitro and in vivo in breast cancer cells lacking a functional p53.

Antiviral agent Cidofovir restores p53 function and enhances the radiosensitivity in HPV-associated cancers.

High-risk human papillomaviruses (HPVs) have been associated to the development of cervical and some other human cancers. Most of them express E6 and E7 oncoproteins, able to bind to p53 and retinoblastoma (pRb) tumor suppressor proteins respectively and neutralize their function. Restoration of these pathways by blocking E6 and E7 expression would provide a selective therapeutic effect. Here, we show that a clinically approved antiviral agent Cidofovir reduced E6 and E7 expression in cervical carcinoma Me180 and head and neck squamous cell carcinoma HEP2 cells at the transcriptional level. Cidofovir induced the accumulation of active p53 and pRb associated to induction of cyclin dependent kinase inhibitor p21(WAF1/CIP1) in Me180 and HEP2 cells. p53 induction was also shown in Hela HPV-positive cervical carcinoma cell line. In addition, S phase cell cycle accumulation with concomitant decrease of cyclin A expression were associated to the antiproliferative activity of Cidofovir in HPV-treated cells. Combining Cidofovir to irradiation both in vivo and in nude mice xenografts resulted in a marked radiosensitization in HPV-positive cells, which was not observed in virus negative cells. This study provides the basis for a new anticancer strategy to enhance the antitumor effect of ionizing radiation in HPV-related cancers, without increase deleterious effects.

Influence of intravenous amifostine on xerostomia, tumor control, and survival after radiotherapy for head-and- neck cancer: 2-year follow-up of a prospective, randomized, phase III trial.

PURPOSE: To evaluate chronic xerostomia and tumor control 18 and 24 months after initial treatment with amifostine in a randomized controlled trial of patients with head-and-neck cancer; at 12 months after radiotherapy (RT), amifostine had been shown to reduce xerostomia without changing tumor control. METHODS AND MATERIALS: Adults with head-and-neck cancer who underwent once-daily RT for 5-7 weeks (total dose, 50-70 Gy) received either open-label amifostine (200 mg/m2 i.v.) 15-30 min before each fraction of radiation (n = 150) or RT alone (control; n = 153). RESULTS: Amifostine administration was associated with a reduced incidence of Grade > or =2 xerostomia over 2 years of follow-up (p = 0.002), an increase in the proportion of patients with meaningful (>0.1 g) unstimulated saliva production at 24 months (p = 0.011), and reduced mouth dryness scores on a patient benefit questionnaire at 24 months (p < 0.001). Locoregional control rate, progression-free survival, and overall survival were not significantly different between the amifostine group and the control group. CONCLUSIONS: Amifostine administration during head-and-neck RT reduces the severity and duration of xerostomia 2 years after treatment and does not seem to compromise locoregional control rates, progression-free survival, or overall survival.

Chromosomal aberrations induced by chemotherapy and radiotherapy in lymphocytes from patients with breast carcinoma.

PURPOSE: Stable chromosomal aberrations (SCAs) have been found in circulating lymphocytes from patients treated for breast carcinoma. Therefore, we tried to define their incidence in such patients, to determine an in vitro dose-effect relationship, and to correlate these data with clinical parameters. METHODS AND MATERIALS: This prospective study included 25 patients who, after surgery, underwent either radiotherapy (RT) alone (n = 15) or RT combined with chemotherapy (n = 10). SCAs were scored using the fluorescent in situ hybridization technique before RT and 4 and 12 months after RT. Dose-effect curves were established by in vitro irradiation of blood samples with 2 and 4 Gy, before and after treatment. RESULTS: In all patients, the rate of SCAs increased significantly after external irradiation. No significant decrease in SCAs was observed during the first year after RT. RT and chemotherapy had no effect on the lymphocyte in vitro dose-effect relationship. No relationship was found in the distribution of patients between the yield of SCAs scored after external irradiation and after in vitro irradiation. SCAs after RT or in vitro irradiation did not correlate with family history of breast carcinoma or acute toxicity of treatment. More significantly, the yield of SCA after external irradiation was strongly related to the irradiation of the internal mammary chain and the supraclavicular lymph node area, suggesting that the volume of irradiated blood vessels was an essential parameter in determining the rate of SCAs. CONCLUSION: A high and stable yield of SCAs persisted at least 1 year after external irradiation. The nature of the volume irradiated containing large blood vessels was the major determinant of the observed biologic dose.

Cancer mortality after radiotherapy for a skin hemangioma during childhood.

BACKGROUND AND PURPOSE: A cohort study was performed as part of a European Radiation Protection Program to investigate the carcinogenic effect of treatment with ionizing radiation in early childhood. This paper presents mortality after radiotherapy in this cohort. PATIENTS AND METHODS: The cohort comprised 7037 patients under 15 years of age treated for a skin hemangioma between 1940 and 1973 at the Institut Gustave-Roussy, among whom 4940 received radiotherapy. The vital status and causes of death were obtained as well as the mortality rates in the general French population. External and internal analyses were performed. Standardized mortality ratio (SMR) and relative risk (RR) variations according to exposure to radiotherapy or not and the type of treatment were studied. RESULTS: During the 1969-1997 follow-up period, 16 cohort patients died of cancer, 14 after radiotherapy. A non-significant excess of cancer-related mortality was observed for irradiated patients as compared to the general population (SMR=1.53; 95% CI=0.86-2.48). Treatment with (226)Ra seemed to play a significant role (RR=2.53; 95% CI=0.84-7.07) compared to no radiotherapy. CONCLUSION: This study suggests an excess risk of cancer-related mortality in patients treated during early childhood with radiotherapy for skin hemangioma, and especially with (226)Ra. These patients need to be followed up in the future.

[UCLA/RAND Cancer Prostate Index quality of life questionnaire after external beam radiotherapy for localized prostate cancer: repercussions of complications and quality of life in general]. / Questionnaire de qualité de vie UCLA/RAND Prostate Cancer Index après radiothérapie externe pour cancer de prostate localisé: retentissement des complications et qualité de vie générale.

INTRODUCTION: This study was designed to evaluate the quality of life after external beam radiotherapy for localized prostate cancer using the UCLA/RAND Cancer Prostate Index questionnaire. MATERIAL AND METHODS: An accurate translation of the questionnaire was retrospectively sent to 108 patients treated for localized prostate cancer by exclusive external beam radiotherapy between 1989 and 1999. The reference values adopted for comparison were those observed by Litwin in a control population without prostate cancer. Patient subgroups were constituted according to the presence or absence of neoadjuvant endocrine therapy and laboratory signs of progression (ASTRO). RESULTS: The response rate was 61.1%, the mean age of the patients was 71.9 years and the mean follow-up was 46.5 months. The percentage of patients "living in a couple" or with "a serious relationship" was 93.4% Three quarters of the population declared that they were "satisfied" or "very satisfied" with the treatment performed. General quality of life scores were comparable to those of Litwin's control population. A functional alteration and decreased tolerance of impairment were observed, in decreasing order, for the sexual, gastrointestinal and urinary factors. CONCLUSION: External beam radiotherapy essentially alters quality of life related to gastrointestinal and sexual functions, while the only aspect of urinary function studied by this questionnaire is continence.

[Nadir PSA and kinetics of PSA decline between the 3rd and 6th month after external beam radiotherapy for T1 T2 Nx M0 localized prostate cancer: value of the prediction of the risk of biological progression]. / PSA Nadir et cinétique de décroissance du PSA entre les 3ème et 6ème mois après radiothérapie externe pour cancer de prostate localisé T1 T2 Nx M0: intérêts dans la prédiction du risque de progression biologique.

INTRODUCTION: This study analyses the results of external beam radiotherapy in stage T1 T2 Nx M0 prostate cancer, with reference to the nadir PSA and the kinetics of PSA decline. MATERIAL AND METHODS: 65 patients with T1 T2 Nx M0 localized prostate cancer were treated by external beam radiotherapy (conventional or conformal) between 1990 and 1999. Two populations of 22 and 25 patients were distinguished according to the nadir PSA: population A with a nadir < or = 0.5 ng/ml or not yet reached, but with PSA < or = 0.5 ng/ml and population B with a nadir > 0.5 ng/ml. The various clinical and laboratory parameters and the kinetics of PSA decline (calculation based on the course of PSA between the first 3 and 6 months after irradiation) were compared by statistical tests (Chi-square, Student t test). According to the ASTRO criteria, the results in terms of absence of biochemical progression were evaluated by non-parametric Kaplan-Meier estimate. RESULTS: No biochemical progression was observed in population A with a mean follow-up of 29.5 months. The absence of biochemical progression in population B at 42 months was 52.77%. The baseline PSA (p = 0.009), the dose delivered (p = 0.027), and the kinetics of PSA decline (p = 0.0069) were identified as predictive factors. The patient with a zero kinetic developed biochemical progression, while 91.3% of patients with a kinetic < 0.35 ng/ml/month remained free of biochemical progression. A group of patients (median nadir: 0.8 ng/ml, baseline PSA < 10 ng/ml and kinetic < 0.35 ng/ml/month) was distinguished by its good prognosis. CONCLUSION: In stage T1-T2 prostate cancer, the value of the nadir PSA is an essential prognostic factor. The kinetics of PSA decline appear to have an early predictive role.

Thyroid adenomas and carcinomas following radiotherapy for a hemangioma during infancy.

BACKGROUND AND PURPOSE: A cohort study was performed to investigate the carcinogenic effect of treating skin hemangioma with ionizing radiation during early childhood. This paper presents the incidence of differentiated thyroid adenomas and carcinomas after radiotherapy in this cohort. METHODS AND MATERIALS: Of a total of 8307 patients treated for a skin hemangioma between 1940 and 1973 at the Institut Gustave-Roussy, 4767 were included in an incidence study, among whom 3795 had received radiotherapy. Seventy-three percent were less than 1-year-old at the time of treatment. External radiotherapy, Radium 226, Strontium 90, Yttrium 90, and Phosphorus 32 were used. The radiation dose received by the thyroid during radiotherapy, estimated in 3497 of the 3795 patients using specific software, was 41 mGy on average. Thyroid tumor cases were obtained by sending out a questionnaire, and were verified in pathological reports. Estimates of thyroid cancer specific incidence rates in the French population were obtained from the French cancer registry network. External and internal analyses were performed. RESULTS: During an average follow-up of 35 years, 11 patients developed a differentiated thyroid carcinoma and 44 a thyroid adenoma. The incidence of thyroid adenoma was found to be higher among taller and heavier individuals. The incidence of both thyroid carcinoma and adenoma was higher among non-smoker patients. A significant dose-response relationship was found between the radiation dose received by thyroid and the risk of thyroid cancer (Excess Relative Risk per GY, ERR/Gy: 14.7, 95%CI: 1.6-62.9) and of adenoma (ERR/Gy: 5.7, 95%CI: 0.7-19.4). CONCLUSION: This study confirms that radiation treatment performed in the past for hemangioma during infancy increased the risk of thyroid carcinoma and adenoma. Patients treated with external radiotherapy or with Radium 226 applicators for hemangiomas have to be more specifically followed up because this is the subgroup in whom the highest doses were received by the thyroid gland (more than 90% of the radiation doses were higher than 100 mGy). They are therefore more at risk of developing thyroid cancer.

Ionizing radiation to prevent arterial intimal hyperplasia at the edges of the stent: induces necrosis and fibrosis.

BACKGROUND: Although ionizing radiation has been proposed for the prevention of intimal hyperplasia in coronary and peripheral arteries in multicenter clinical trials, information is lacking on how irradiation affects arterial histology after stenting and especially how it affects the edges of the stent. We investigated intimal hyperplasia recasting with histological changes in arterial wall at the edges of the stent after arterial stenting followed by adequate external radiation for the prevention of intimal hyperplasia in pigs. MATERIALS AND METHODS: The aorta was experimentally stented in 30 pigs who were then assigned to two groups: irradiation with 20 Gy and a control group with no irradiation. The aorta was resected for morphometric and histological studies 6 weeks after procedure. RESULTS: Intimal thickness was reduced and the intima/media ratio was significantly lower in irradiated groups than in control pigs. In the irradiated group histological examination at the edges of the stent showed thin neointimal proliferation with an intact endothelium. In all sections analyzed in the 20-Gy irradiated group the vascular media at 45 days contained necrotic areas and fibrosis with calcifications. CONCLUSIONS: After arterial injury, adequate ionizing radiation effectively reduces neointimal thickening. Irradiation-induced histological changes include previously undetected recasting with necrosis and fibrosis at the arterial edges of the stent. The parietal recasting we observed in animal arteries irradiated at high doses is unclear and a cause of concern especially after clinical spontaneous dissection was recently reported. The use of ionizing radiation for the prevention of arterial restenosis awaits confirmation with a long-term follow-up including specific experimental histological analyses.

Chronological changes in morphometry and histology in the rabbit vascular wall after external radiation for the prevention of intimal hyperplasia.

BACKGROUND: Although ionizing radiation has been proposed for the prevention of intimal hyperplasia in coronary and peripheral arteries, information is lacking on how irradiation affects arterial histology and neointimal smooth-muscle cell proliferation-the hallmark of restenosis. We chronologically investigated early histological changes and quantitative changes in arterial wall cell proliferation after arterial injury followed by external radiation for the prevention of intimal hyperplasia in rabbits. MATERIALS AND METHODS: The aorta was experimentally injured in 26 rabbits who were then assigned to two groups: irradiation with 20 Gy and a control group with no irradiation. The aorta was resected for morphometric and histological studies at 3, 7, 15, 30, and 45 days after experimental injury. RESULTS: Intimal thickness was reduced and the intima/media ratio was significantly lower in irradiated groups than in control rabbits. In the irradiated group histological examination showed delayed neointimal proliferation with an intact endothelium. In the 20-Gy irradiated group the vascular media at 7 days contained necrotic areas and delayed fibrosis with calcifications. There was no statistical difference between the number of proliferating cells in the irradiated groups and the control group. Proliferating cells reached maximum numbers later in irradiated groups than in control rabbits (45 days versus 3 days). CONCLUSION: After arterial injury, external irradiation at 20 Gy effectively reduced aortic neointimal thickening. Irradiation-induced histological changes include recasting with rapid necrosis and delayed fibrosis. Radiation-induced parietal recasting with necrosis, fibrosis, and calcifications might worsen in time. Although irradiation after arterial injury leaves proliferative smooth-muscle cells within the arterial wall quantitatively unchanged in the early days after the procedure, it then induces a delayed reaction (observed over 45 days in our study). Whether neointimal hyperplasia is merely delayed or will ultimately develop causing restenosis awaits confirmation from experimental and clinical studies with a long-term follow-up.

Salvage radiotherapy for biochemical recurrence after radical prostatectomy: a study of 62 patients.

OBJECTIVES: To determine the predictive factors of prostate-specific antigen (PSA) recurrence after salvage radiotherapy (RT) for biochemical recurrence following radical prostatectomy (RP) to identify patients who may benefit from this treatment. METHODS: From June 1992 to January 2002, 62 patients experiencing PSA recurrence after RP were treated with RT at a dose of 65 Gy. No patient received hormonal therapy. PSA recurrence after RT was defined as three consecutive increased PSA measurements. The risk of experiencing PSA recurrence after RT was analyzed according to 10 factors: patient age, pre-RP PSA level, pathologic stage, Gleason score, surgical margin status, PSA nadir after RP, time to PSA recurrence after RP, pre-RT PSA level, PSA nadir after RT, and length of follow-up after RT. RESULTS: With a mean follow-up of 44 months (range 3 to 110), 23 patients (37.1%) experienced PSA recurrence after RT. Using univariate analysis, six factors were found to be predictive of PSA recurrence after RT: the length of follow-up after RT (P <0.0001), PSA nadir after RP (P = 0.0004), time to PSA recurrence after RP (P = 0.003), pre-RP PSA level (P = 0.008), Gleason score (P = 0.011), and pre-RT PSA level (P = 0.028). Using multivariate analysis, only the Gleason score (P = 0.015) and length of follow-up after RT (P = 0.02) were found to be predictive of PSA recurrence after RT. A Gleason score greater than 7 was a significant predictor of PSA recurrence after salvage RT (P = 0.04). CONCLUSIONS: In our experience, the Gleason score and length of follow-up were the sole independent predictors of PSA recurrence after salvage RT. Our findings suggest that patients with a Gleason score of 7 or less are more likely to benefit from salvage RT after RP and that the durability of the PSA response may be only transient.

External beam ionizing radiation for inhibition of myointimal hyperplasia after dilatation and anastomoses: experimental models and results.

In recent years there has been intensive research on the use of ionizing radiation for inhibition of intimal hyperplasia (IH). Results have clearly established that beta ionizing radiation delivered from an endoluminal source after angioplasty inhibits intimal restenosis. This effect has been confirmed by recent multicenter clinical trials in patients undergoing coronary dilatation. The purpose of this study was to determine if gamma radiation therapy delivered superficially from an external source also reduced smooth muscle cell proliferation in two animals models-the first involving experimentally induced restenosis and the second involving anastomosis between a prosthesis and artery. Ultimately we hope to develop a therapeutic application for patients undergoing peripheral anastomoses, especially in the lower extremities. Two different animal models were used in this two-stage study. The first-stage rabbit model (model 1) involved balloon injury of the aorta to validate the dose effect of external beam irradiation. The second-stage porcine model (model 2) involved aortic bypass followed by external beam irradiation of the distal anastomosis site. In model 1 a total of 56 rabbits were studied. They were divided into five groups including one control group in which external radiation was not applied after balloon injury and four test groups in which external radiation was applied in a single fraction on day 0 at four different doses: 10 grays, 15 grays, 20 grays, and 25 grays. In model 2, a total of 24 pigs underwent aortic bypass with a 6-mm PTFE graft followed by irradiation of the distal end-to-side anastomosis at a dose of 20 grays on day 0. In both models specimens were harvested after 6 weeks and studied histologically after staining with HES and orcein, histomorphometrically by measuring intimal hyperplasia, and immunohistochemically using actin and factor VIII/von Willebrand factor (F VIII/vWF). The zones of study on the anastomosis were separated into base of the artery to the tip and heel of the anastomosis and the edge of the arteriotomy. Measurements were compared using the Mann Whitney test. In the first-stage model designed to study IH in rabbits, mean intimal and medial thickness values and the intima-to-media ratio showed no difference between the control group and the groups irradiated at doses of 10 grays and 15 grays (p = 0.111, p = 0.405, and p = 0.14); (p = 0.301, p = 0.206, and p = 0.199). Conversely, there was a significant difference between the control group and the groups irradiated at 20 grays and 25 grays (p < 0.0001, p = 0.107 and p = 0.008; p = 0.008, p = 0.155, and p = 0.008). Histological examination demonstrated extensive changes in the wall with high-grade fibrosis after application of ionizing radiation. In the second-stage swine model, irradiation significantly inhibited development of IH at the level of anastomosis both at the base of the artery (p < 0.01) (tip 0.06 vs. 0.27 mm and heel 0.04 vs. 0.36) and at the level of the arteriotomy at the suture site (p < 0.001) (0.13 vs. 0.86 mm). Immunochemical analysis of the thickened zones showed a positive reaction of endothelial cells to smooth muscle actin and F VII/vWF. Like irradiation applied using an endoluminal source, superficial gamma ionizing radiation from an external source inhibits IH. Analysis of the dose effect showed that the overall dose must be between 15 and 20 grays. External radiation also reduces overall IH at the anastomosis between a prosthesis and artery. Although these experimental data are promising, further study will probably be necessary before attempting to undertake clinical trials using external beam radiation therapy for patients undergoing peripheral anastomoses.

Cyclooxygenase-2 inhibitor NS398 enhances antitumor effect of irradiation on hormone refractory human prostate carcinoma cells.

PURPOSE: We examined the potential therapeutic effect of NS398, a selective cyclooxygenase-2 (COX-2) inhibitor, combined with irradiation on human prostate adenocarcinoma DU145 cells. MATERIALS AND METHODS: The effect on tumor growth, proliferation testing and clonogenic survival was determined to evaluate its antitumor effect when exposed to NS398 or combined with irradiation. Immunoblotting analyses were done to detect the expression of COX-2, nuclear factor-kappaB p50 and Rel A p65 because evidence suggested a biological association of COX-2 with alterations in these markers. Reverse transcriptase-polymerase chain reaction was also performed to show its effect on the transcription level of COX-2. RESULTS: Exposure of DU145 cells to NS398 alone suppressed proliferation in a dose and time dependent manner. Examination of the NS398 effect on the radiation response showed marked enhancement of radiosensitivity. Western blot indicated that NS398 down-regulated the expression of COX-2, nuclear factor-kappaB, p50 and Rel A p65, whereas the effect was more pronounced when combined with irradiation. Reverse transcriptase-polymerase chain reaction showed that the NS398 antitumor effect was associated with COX-2 transcription inhibition. Importantly COX-2 expression was enhanced by irradiation but this phenomenon was abolished when cells were exposed to NS398. Inhibition of tumor growth in animal model was observed when mice were treated with NS398 alone and irradiation alone, and this effect was maximal when treated with NS398 and irradiation. CONCLUSIONS: These results suggest that NS398 could be used as a potential therapeutic agent combined with irradiation for prostate adenocarcinoma.

Morphometric and histological changes in the vascular wall after external radiation for the prevention of intimal hyperplasia.

BACKGROUND: Recent reports describe spontaneous dissections and aneurysms after coronary and peripheral artery irradiation for the prevention of intimal hyperplasia. We investigated histological changes and the vasomotor reaction in the vascular wall after external radiation for the prevention of intimal hyperplasia in rabbits. MATERIALS AND METHODS: The aorta was experimentally injured in 34 rabbits who were then assigned to one of three groups: irradiation with 20 Gy; with 25 Gy; and a control group with no irradiation. Before the arterial injury and 45 days later, vasomotor function was assessed with an intravascular ultrasound catheter. The aorta was resected for morphometric and histological studies. RESULTS: After injury and irradiation, vasomotor responses were significantly lower in the two irradiated groups (P < 0.05). Intimal thickness and the intima/media ratio were significantly lower in irradiated groups. In the irradiated group histological examination showed reduced intimal proliferation with an intact endothelium. In the 20-Gy irradiated group the vascular media contained necrotic areas, and in the 25-Gy irradiated group, severe fibrosis. CONCLUSION: After arterial injury, external irradiation at 20 and 25 Gy effectively reduces aortic intimal and medial thickening. Histological changes include recasting with necrosis and fibrosis causing a decreased vasomotor response. Further investigations are needed to confirm medial necrosis and replacement with fibrosis. Because the irradiation doses in this study match those currently used and also recommended for experimental and clinical use, if confirmed in humans parietal recasting might possibly explain the reported spontaneous dissections and aneurysm formation after irradiation.