The influence of the oncogene N-ras on cell cycle delay in a human melanoma cell line with reduced radioresistance.

In a previous study we found that transfection of a human melanoma cell line with the oncogene N-ras led to increased radiosensitivity as measured by clonogenic assays. Since a shift in radiosensitivity is often correlated with altered G2/M delay, we investigated whether this was also the case in this oncogene containing melanoma cell line (IGRras). A human melanoma cell line, stably transfected with mutated N-ras, and its parental cell line transfected with the neomycin phosphotransferase gene only (IGRneo), were irradiated with 5 Gy and cell cycle distribution was measured at hourly time intervals by DNA staining with propidium iodide. Next, the effect of ionising radiation on the duration of the S-phase was determined by pulse labelling cells with BrdUrd before irradiation. Both cell lines showed a radiation induced G2/M delay, which was most prolonged for the ras transfected cell line. After 5 Gy, the S-phase duration was unaltered, although the shape of the relative movement (RM) curves was slightly different. No G1 delay was observed in either cell line. Ras transfection in a melanoma cell line leads to prolonged G2/M delay after radiotherapy. This prolongation is associated with increased radiosensitivity and not with radioresistance. These data throw doubt on the use of oncogene expression or G2/M delay as predictors of radiosensitivity.

Late ophthalmological complications after total body irradiation in non-human primates.

PURPOSE: To investigate the long-term effects of total body irradiation (TBI) on the incidence and time course of ocular complications. MATERIALS AND METHODS: Rhesus monkeys treated with TBI photon doses up to 8.5 Gy and proton doses up to 7.5 Gy were studied at intervals up to 25 years post-irradiation. They were compared with control groups with a similar age distribution. Cataract formation and ocular fundus lesions were scored according to a standardized protocol. Fluorescein angiography and histopathology was performed in selected animals. RESULTS: Cataract formation occurred after a latent period of 3-5 years. Significant cataract induction was observed for photon-doses of 8 and 8.5 Gy and beyond 20 years after proton irradiation. The severity of the lesions represents significant impairment of vision and would require cataract surgery if similar results occurred in human bone marrow transplant patients. Fluorescein angiography demonstrated a normal pattern of retinal vessels in 13 out of 14 animals (93%) from the irradiated group and in eight out of nine animals (89%) from the control group. No additional lesions apart from age-related degenerative changes could be demonstrated. Histological evaluation revealed no radiation-associated vasculopathy. CONCLUSIONS: Radiation alone for doses up to 8.5 Gy of photons does not carry a potential risk for fundus pathology, whereas clinically important cataract induction should be anticipated within 5 years after photon doses of 8.0 and 8.5 Gy and proton doses in excess of 2.5 Gy.

Results of ruthenium irradiation of uveal melanomas: the Dutch experience.

BACKGROUND AND PURPOSE: To update our results of the treatment of uveal melanomas using ruthenium applicators in 49 patients treated with graded doses and subsequently in 52 patients with maximal scleral doses of 800 Gy and an effective top dose of at least 160 Gy. MATERIAL AND METHODS: One hundred and one patients were treated with brachytherapy only, in 25 patients it was combined with transpupillary thermotherapy (TTT). RESULTS: A complete remission was found in 62.6% of the patients and in 31.3% a stable disease with an average follow-up of 74.6 months. Above a top dose of 120 Gy only in one of 95 patients continuous tumour growth after treatment was observed. Useful vision could be preserved in 51.5% of the patients. The initial tumour prominence and top dose strongly correlated with treatment outcome. CONCLUSIONS: Ruthenium application for uveal melanomas with the doses we have used is successful, with a substantial number of patients having their eyes preserved, their tumour controlled and their vision retained. Further improvements can be obtained with ruthenium irradiation with lower maximal scleral doses combined with TTT.

Radiotherapy for oesophagus carcinoma: the impact of p53 on treatment outcome.

BACKGROUND AND PURPOSE: Wildtype p53 protein plays an important role in the cellular response to ionizing radiation and other DNA damaging agents and is mutated in many human tumours. We evaluated the relationship of the immunohistochemically determined p53 protein status and the disease control with radiotherapy alone for carcinoma of the oesophagus. MATERIALS AND METHODS: Immunostaining for p53 protein was performed on paraffin-embedded specimens from 69 patients with adeno- and squamous cell carcinoma of the oesophagus. All patients were treated by radiotherapy exclusively, consisting of a combination of external irradiation and intraluminal brachytherapy, using two different dose levels. RESULTS: Fifty-four percent (37/69) of the tumours showed overexpression of the p53 protein. No difference in pre-treatment parameters for p53-positive and p53-negative cases was detected. In multivariate analysis p53 was significantly associated with overall survival (OS) next to weight loss, tumour stage and N-stage. For metastatic-free survival (MFS) p53 status proved to be the sole independent prognostic factor. The influence of p53 on local recurrence-free survival (LRFS), however, was not as strong as on OS and MFS. CONCLUSIONS: Immunohistochemically detected overexpression of mutated p53 protein in oesophagus carcinoma was an independent prognostic factor in a group of patients treated with radiotherapy alone.

Indications and treatment schedules for irradiation of benign diseases: a survey.

BACKGROUND: The interest in the treatment of benign diseases with radiotherapy has grown particularly in the Western part of the world. Questions about the effectiveness and risks of radiotherapy for these disorders have to be answered in the light of new information from clinical research, dose calculations and risk estimates. New indications for irradiation of non-malignant diseases, e.g. vascular disorders, such as macular degeneration and prevention of stent restenosis, are appearing. As a starting point for further discussions and publications in this area, the authors, with the help of the European Society for Therapeutic Radiology and Oncology (ESTRO), performed a survey of current practice and of opinion amongst ESTRO members. METHODS: A questionnaire was circulated in 1996 to 1348 institutes listed in the 1995 ESTRO directory asking whether the respondents considered a list of the 28 most common benign disorders as being a good indication for radiotherapy. Details of the frequency of such treatments and of treatment schedules used were requested. RESULTS AND CONCLUSIONS: A large variation in indications and treatment schedules was detected throughout the world. This variation confirms the inadequate information on which such treatments are based and stresses the importance of careful clinical studies and trials as well as clear understanding of any risks involved.

Cell density dependent plating efficiency affects outcome and interpretation of colony forming assays.

BACKGROUND AND PURPOSE: The usefulness of colony forming assays (CFA) has been established for almost 40 years (Puck and Marcus, J.Exp.Med. 103: 653-666, 1956). Although time-consuming and not successful for all cell lines, it is generally considered to be the gold standard of assays for testing the sensitivity of cell lines to ionizing radiation or other cytotoxic agents in vitro. We recently found for several cell lines that the plating efficiencies of both control and irradiated cells is dependent upon the density of cells seeded for colony formation; that is, increasing cell inoculum levels resulted in a non-linear relationship with colony formation, even at relatively low colony numbers. MATERIAL AND METHODS: All data from a human melanoma cell line, transfected with c-myc or N-ras, as well as from normal human diploid fibroblasts, were taken to see how this phenomenon influenced outcome and interpretation of clonogenic assays. Survival was recalculated using all data, or only data with a linear relationship between inoculum level and colony formation. RESULTS: It is found that when data with a non-linear relationship between inoculum level and colony formation are included, survival can be underestimated due to inhibition of colony formation in treated cultures. CONCLUSION: For validity, colony forming assays must be standardized to assure a constant relationship between the cell density and colony forming efficiency. This usually requires a much lower density of colonies than has been typically published for many cell survival studies.

[Electron beam irradiation is effective in the treatment of skin carcinomas; a comparison with superficial roentgen therapy]. / Elektronenbestraling effectief bij de behandeling van huidcarcinomen; een vergelijking met röntgencontacttherapie.

OBJECTIVE: To compare recurrence rates and cosmetic results after electron beam therapy versus superficial X-ray therapy for basal cell and squamous cell carcinomas of the skin. SETTING: Department of Radiotherapy, University Hospital Leiden, the Netherlands. DESIGN: Retrospective. METHODS: A retrospective analysis was performed of 389 histologically confirmed basal cell and squamous cell carcinomas of the skin (1980-1990), treated with either superficial X-rays or electron beam therapy, with regard to local recurrence rates and cosmetics scored with an arbitrary three point scale. RESULTS: Of 389 patients 19 developed a local recurrence (4.9%). For small tumours (irradiated surfaces < or = 10 cm2) a local recurrence rate of 2.2% was observed, both after electron beam therapy and after superficial X-ray therapy. Large tumours (irradiated surface > 50 cm2, treated with electron beam) had a higher recurrence rate (13.8%) as had tumours which had been treated before. The cosmetic results with electron beam treatment were better, because smaller fractions were used compared with superficial X-ray treatment. CONCLUSION: In our hands electron beam treatment was not inferior to superficial X-ray treatment, cosmetically even better and also applicable in case of larger tumours. The inferior results of electron beam therapy reported in literature may be due to technical factors.

Long-term consequences of high-dose total-body irradiation on hepatic and renal function in primates.

Radiation effects in non-human primates were studied in order to define the long-term risk of total-body irradiation (TBI) for bone marrow transplantation patients. The long-term effects of TBI could be investigated by keeping 84 monkeys of different ages, from an experiment on acute effects, under continuous observation for a period up to 25 years. The control group consisted of non-irradiated monkeys with a comparable age distribution and identical housing conditions. Since radiation was the common toxic agent, the different age groups provided the possibility to investigate the occurrence of deterministic effects after TBI. In the present study emphasis was placed on the assessment of hepatic and renal function and the associated histopathology. The values of the liver function parameters, such as alkaline phosphatase and gamma glutamyl transferase in the irradiated group were significantly increased after TBI (p < 0.05). Also the parameters of kidney dysfunction, e.g. haematocrit and blood urea nitrogen showed a significant change in the irradiated old-aged (post-irradiated interval > 15 years) cohort (p < 0.005). The impairment of the liver and renal functions, did not lead to clinical symptoms and were only associated with mild morphologic changes in the irradiated group of monkeys. In the population of bone marrow transplant patients treated with TBI, alterations in hepatic and renal function parameters after a post-irradiated interval of > 10 years can be anticipated. This could have consequences for the tolerance and toxicity of a broad range of drugs to be administered as additional medications.

Electron beam therapy is not inferior to superficial x-ray therapy in the treatment of skin carcinoma.

PURPOSE: To compare recurrence rates and cosmetic results after electron beam therapy vs. superficial x-ray therapy for nonmelanoma skin carcinoma. METHODS AND MATERIALS: A retrospective analysis was performed on 389 histologically confirmed basal cell and squamous cell carcinomas of the skin, treated with either superficial x-rays or electron beam therapy, with regard to local control rates and cosmetics, as scored with an arbitrary three-point scale. RESULTS: The overall local recurrence rate was 4.9% (19 out of 389). For small tumors (irradiated surfaces < or = 10 cm2) a local recurrence rate of 2.2% was observed, both after electron beam therapy and after superficial x-ray therapy. Tumor size and previous treatment proved to be important prognostic factors with regard to control rate. The well-fractionated electron beam therapy yielded better cosmetic results than the large fraction superficial x-ray therapy. CONCLUSION: Electron beam treatment is not inferior to superficial x-ray treatment and even better for larger tumors. The inferior results of electron beam therapy reported in literature might be due to technical factors, which are discussed.

Results of ruthenium irradiation of uveal melanoma.

The follow-up results are presented for the radiotherapeutic treatment of 49 patients with uveal eye melanoma using ruthenium-106 (106Ru/106Rh) applicators. Graded doses were applied and the degree of regression was found to be correlated with the dose at the top of the tumour. Complete regression is found in 66% of the patients treated with a top dose above 150 Gy, in which case the initial tumour prominence diminishes in about three years to a stable disease or to a flat scar. At this dose a marginal increase in complications was observed as compared with lower dose groups. Useful vision could be preserved in 75% of the eyes.

Vitamin B12 malabsorption after irradiation for gynaecological tumours.

The serum vitamin B12 levels were determined in 44 asymptomatic patients at least one year after completion of pelvic irradiation for a gynaecological tumour. Ten patients (23%) were found to have decreased levels of serum vitamin B12, of which six (14%) were assumed to be caused by malabsorption due to radiation damage to the terminal ileum as no other reason for a lower vitamin B12 level could be detected. Neither the radiation dose or volume nor the manifestation of acute or late radiation side effects appeared to be correlated with the finding of decreased levels of vitamin B12. It is concluded that even in asymptomatic patients who received pelvic irradiation, serum vitamin B12 levels can serve as a marker for radiation enteropathy. It is advocated to check vitamin B12 levels prior to the radiation treatment as well as during the follow-up of these patients.

Combined external beam and low dose rate intraluminal radiotherapy in oesophageal cancer.

Thirty-five patients with oesophageal cancer were treated with external beam irradiation (50-60 Gy) followed by a boost-dose of 15-20 Gy by means of low dose rate intraluminal brachytherapy. Of the 35 patients treated 17 (48%) were pretreated with laser therapy or dilation alone. Although the intraluminal application time was long (up to 36 h) the treatment was feasible with minor acute toxicity. The palliative effect of the combined treatment was excellent; a 6 weeks post-treatment 32 of the 35 patients were able to eat solid food. Late complications were seen in six patients (17%), of which only one was severe and probably treatment-related. The median survival was 11 months; the 1- and 2-year survival were 42% and 10% respectively. The survival was strongly dependent on local control. Distant metastases became evident in 23% of patients. The interval between external radiotherapy and brachytherapy seemed to be critical. The results were compared with 68 historical controls. A significantly better survival was observed at 6 months. It is concluded that low dose rate intraluminal brachytherapy is a useful and feasible technique to increase the total dose for obtaining a better local control. The shortcomings are discussed and ideas for further improvement are mentioned.

Dosimetry of ruthenium-106 eye applicators.

In view of the importance of clinical applications of ruthenium-106 beta-ray sources for the treatment of choroidal melanoma, experimental, and theoretical approaches are presented for the dosimetry of such sources. The absolute dose and percentage depth dose of ruthenium applicators have been measured with an extrapolation ionization chamber. For a special flat applicator the absolute dose could be measured with an accuracy of +/- 5%, which is determined by the collection efficiency of the extrapolation chamber. The percentage depth dose of concave applicators, employed in the clinical situation, could only be measured at a distance larger than 5 mm due to their geometry and the outer dimensions of the extrapolation chamber. A computer simulation was therefore developed for the absorption and scattering of electrons, taking into account the geometry and materials of the applicator, to predict the percentage depth dose at distances smaller than 5 mm. The calculated and experimentally determined depth doses are in good agreement. With the aid of the computer simulation a depth dose determination for concave applicators can be made for clinically relevant distances less than 10 mm from the source surface with an absolute accuracy of +/- 10%.

Education in radiation oncology in Europe.

The outcome of an inventory among 22 European countries with respect to the radiotherapy facilities and the training of new radiation oncologists in each country is described. The radiotherapeutic profession, which mostly prescribes also cytostatics or hormones, has become well-regulated in the last 20 years. Most radiation oncologists are also involved in the diagnostic work-up and follow-up of the cancer patient. The numbers of radiotherapists and other staff, treatment capacities, and patients are given. The training for radiation oncologists is mostly taken at the university centers, but the curricula are rather diverse.

Should all patients with malignant astrocytoma have postoperative radiotherapy?

A retrospective analysis was carried out on 96 patients with malignant astrocytoma treated during the period 1977--1986 to evaluate the contribution of postoperative radiation treatment to survival. In this material the initial Karnofsky performance score, age and extent of resection appeared to be prognostic factors, on the basis of which suggestions are given for selection of patients for radiotherapy.

Endometrial carcinoma, results of combined surgery and radiotherapy treatment of 93 patients (1979-1984).

A retrospective analysis was done on 93 cases with clinical stage I and II endometrial carcinoma. All patients in this series were treated by surgery and postoperative radiotherapy. Five-year survival reached 67%. No vaginal or vault recurrences were seen. Grade, stage and depth of myometrial invasion were prognostic parameters for survival, whereas 'age' did not show correlation with survival. Postoperative radiotherapy was performed by two different protocols, one of which appeared to be allied with significantly less late toxicity than the other.

Plasma spermidine and putrescine as markers of cancer management.

The hypothesis was tested that dying tumor cells release spermidine and putrescine into the bloodstream. Therefore, a rhabdomyosarcoma was implanted in rats, irradiated and its growth and subsequent reduction compared with spermidine and putrescine plasma concentrations of the venous effluent from the tumor side and contralateral side. In the experimental set-up used the hypothesis could not be verified. It appeared that irradiation only, implantation and growth of a tumor only and an irradiated tumor, all caused elevated spermidine and putrescine concentrations in the effluent of both sides in approximately the same order of magnitude, compared to untreated controls.