Jonathan Ramsay

Cellular localisation of the ataxia-telangiectasia (ATM) gene product and discrimination between mutated and normal forms.

The recently cloned gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) is involved in DNA damage response at different cell cycle checkpoints and also appears to have a wider role in signal transduction. Antibodies prepared against peptides from the predicted protein sequence detected a ∼ 350 kDa protein corresponding to the open reading frame, which was absent in 13/23 A-T homozygotes. Subcellular fractionation, immunoelectronmicroscopy and immunofluorescence showed that the ATM protein is present in the nucleus and cytoplasmic vesicles. This distribution did not change after irradiation. We also provide evidence that ATM protein binds to p53 and this association is defective in A-T cells compatible with the defective p53 response in these cells. These results provide further support for a role for the ATM protein as a sensor of DNA damage and in a more general role in cell signalling, compatible with the broader phenotype of the syndrome.

Intrinsic radiation sensitivity may not be the major determinant of the poor clinical outcome of glioblastoma multiforme

Abstract Purpose: Many radiobiologic mechanisms may contribute to the clinical radiation resistance of Glioblastoma Multiforme. One of them is considered to be an unusually low intrinsic radiation sensitivity. This is a collaborative study between three laboratories to evaluate the intrinsic radiation sensitivity of 85 cell lines derived from human malignant gliomas as the major cause of the poor clinical results of radiation treatment to these tumors. Methods and Materials: Fifty-one cell lines were early passage. The distribution by histologic type was: 58 glioblastoma, 17 anaplastic astrocytoma, six oligodendroglioma and four astrocytoma grade 2. The intrinsic radiation sensitivity will be expressed by the surviving fraction at 2 Gy (SF 2 ). The SF 2 has been determined for single dose irradiation for cell lines on exponential phase, under aerobic conditions, growing on plastic. The patient age, Karnofski Status, histological grade, survival, dose of irradiation for 50 patients are investigated for correlation with SF 2 of the corresponding newly established cell lines. Results: The mean SF 2 of the 85 cell lines was 0.46 (0.12 – 0.87). The mean SF 2 by histologic type was 0.50, 0.34, 0.54 and 0.38 for glioblastoma, anaplastic astrocytoma, oligodendroglioma and astrocytoma grade 2 cell lines, respectively. No correlation was found between SF 2 and the patient age or Karnofski status. The difference in SF 2 between the 58 glioblastoma and 17 anaplastic astrocytoma cell lines was significant p = 0.002. The difference in actuarial survival between glioblastoma and anaplastic astrocytoma patients was borderline of significance ( p = 0.08). The difference in SF 2 of cell lines derived from these two groups of patients was of borderline significance ( p = 0.08). The difference in radiation sensitivity for anaplastic astrocytoma and glioblastoma cell lines was clearly reflected in the difference in survival for the two groups of patients from where the cell lines were derived. However, no correlation was found between SF 2 and survival within each grade. In a multivariate analysis the age, grade and Karnofski status were found to be significant prognostic values for survival with a p values of 0.032, 0.03 and 0.038, respectively, however, the In SF 2 was not significant ( p = 0.40). The mean SF 2 of the 6 oligodendroglioma cell lines (0.54) was comparable to that of glioblastoma multiforme (0.50). The high SF 2 for oligodendroglioma does not accord with the much better clinical outcome of these tumors. Conclusions: These data on 85 malignant glioma cell lines show a very broad distribution of SFZ values for irradiation in vitro . SF 2 reflected the difference in sensitivity between AA (Grade 3) and GBM (Grade 4). This may suggest that the parameter SF 2 is useful to discriminate between the sensitivity of different grades or types of histology in vitro . However, SF 2 was not a predictor of the clinical outcome on individual basis for malignant gliomas. The in vitro studies will need to be supplemented by physiologic characterization of the tumors in vivo . Such conclusions would limit the predictive value of current radiation sensitivity assays based on in vitro dose-survival measurement for at least high grade malignant gliomas.

The sap from Euphorbia peplus is effective against human nonmelanoma skin cancers.

Background  The sap from Euphorbia peplus, commonly known as petty spurge in the U.K. or radium weed in Australia, has been used as a traditional treatment for a number of cancers.

TESTING FOR MUTATIONS OF THE ATAXIA TELANGIECTASIA GENE IN RADIOSENSITIVE BREAST CANCER PATIENTS

BACKGROUND AND PURPOSE To determine if clinical radiosensitivity in breast cancer patients is related to mutations of the ataxia telangiectasia gene (ATM). MATERIALS AND METHODS Fifteen patients who had developed a severe late reaction to a standard radiotherapy schedule were examined for evidence of increased in vitro radiosensitivity using the MTT assay. Mutation analysis was performed using a protein truncation assay. RESULTS No mutations were detected in the 15 patients despite evidence of increased in vitro radiosensitivity. CONCLUSIONS Testing for the ATM gene is unlikely to be useful for predicting clinical response to radiotherapy in breast cancer patients.

Normal tissue radiosensitivity in breast cancer patients

PURPOSE To investigate whether in vitro radiosensitivity of lymphocytes derived from a blood sample will predict late effects from radiotherapy in breast cancer patients. METHODS AND MATERIALS Blood samples were collected from consenting patients who had received radiotherapy for breast cancer. Lymphocytes were extracted and transformed by the Epstein-Barr virus. The resulting lymphoblastoid cell lines (LCLs) were assessed for in vitro radiosensitivity using a tetrazolium-based colorimetric assay (MTT). Survival curves were generated using doses of 0.5 to 2 Gy. For each analysis an LCL derived from an individual with the radiosensitive condition ataxia-telangiectasia (AT) was run as control. Some patients also consented to give skin biopsies from which fibroblast cultures were derived. Clonogenic assays were performed to generate survival curves using doses in the range of 0.5 to 4 Gy. Comparison was made with the data obtained from LCLs. Late effects of radiotherapy were assessed using the Radiation Therapy Oncology Group (RTOG) scoring scheme and compared with the in vitro radiosensitivity data. RESULTS LCLs from 56 breast cancer patients were assessed for in vitro radiosensitivity. Surviving fraction to 2 Gy (SF2) generated from survival curves ranged from 0.04-0.35 with coefficient of variation for the whole group of 41%. None of the LCLs equalled the sensitivity of the AT line, but 16% showed equal or greater sensitivity to a line derived from an AT heterozygote. Comparison of LCL and fibroblast radiosensitivity showed reasonable correlation for 12 paired samples (r = 0.64). The majority of patients showed no or minimal effects after radiotherapy (Grade 0, 1 effects) but seven developed a Grade 2 reaction and four a Grade 3 or 4 reaction. Patients with a Grade 2-4 reaction were found to be more sensitive in vitro than those with a Grade 0-1 reaction (p < 0.02). CONCLUSIONS The use of the MTT assay to assess LCL radiosensitivity has demonstrated considerable heterogeneity amongst the breast cancer population. The presence of a proportion of patients showing in vitro sensitivity but normal clinical response to radiotherapy would limit the usefulness of the assay for predictive purposes.

Radiation sensitivity of merkel cell carcinoma cell lines

PURPOSE Merkel cell carcinoma (MCC), being a small cell carcinoma, would be expected to be sensitive to radiation. Clinical analysis of patients at our center, especially those with macroscopic disease, would suggest the response is quite variable. We have recently established a number of MCC cell lines from patients prior to radiotherapy, and for the first time are in a position to determine their sensitivity under controlled conditions. METHODS AND MATERIALS Some of the MCC lines grew as suspension cultures and could not be single cell cloned; therefore, it was not possible to use clonogenic survival for all cell lines. A tetrazolium based (MTT) assay was used for these lines, to estimate cell growth after gamma irradiation. Control experiments were conducted on lymphoblastoid cell lines (LCL) and the adherent MCC line, MCC13, to demonstrate that the two assays were comparable under the conditions used. RESULTS We have examined cell lines from MCC, small cell lung cancer (SCLC), malignant melanomas, Epstein Barr virus (EBV) transformed lymphocytes (LCL), and skin fibroblasts for their sensitivity to gamma irradiation using both clonogenic cell survival and MTT assays. The results show that the tumor cell lines have a range of sensitivities, with melanoma being more resistant (surviving fraction at 2 Gy (SF2) 0.57 and 0.56) than the small cell carcinoma lines, MCC (SF2 range 0.21-0.45, mean SF2 0.30, n = 8) and SCLC (SF2 0.31). Fibroblasts were the most sensitive (SF2 0.13-0.20, mean 0.16, n = 5). The MTT assay, when compared to clonogenic assay for the MCC13 adherent line and the LCL, gave comparable results under the conditions used. CONCLUSION Both assays gave a range of SF2 values for the MCC cell lines, suggesting that these cancers would give a heterogeneous response in vivo. The results with the two derivative clones of MCC14 (SF2 for MCC14/1 0.38, MCC14/2 0.45) would further suggest that some of them may develop resistance during clonogenic evolution.

Radiation response of xenografts of a human squamous cell carcinoma and a glioblastoma multiforme : a progress report

Human cell lines derived from squamous cell carcinomas of the pharynx (FaDu and HSCC6) and glioblastoma multiforme (U87, A2, A7, MMC-1, MMC-2) have been studied in vitro as monolayers in exponential (all 7 cell lines) or plateau phase (FaDu and U87), and as 1 mm diameter spheroids in vitro (FaDu and U87) and as 6 mm diameter xenografts growing in the legs of athymic NCr(nu/nu) nude mice (FaDu, HSCC6, U87, A7 cells). For SF2s and D values, there was broad overlap of values between SCC and glioma cell lines. In contrast, the D0 values were higher for U87, A2, A7, and MCC-1 than the two SCC cell lines, while the extrapolation numbers were greater for the two SCC lines than any of the glial tumor lines (these differences were not regularly significant). Complete dose response assays for local control of FaDu, HSCC6, U87, and A7 xenografts have been performed under conditions of normal blood flow and clamp hypoxia for tumors growing in mice which had received 6 Gy WBI at 24 hr before transplantation. Under the latter circumstances, irradiations have been performed on FaDu and U87 as single doses or as 2, 4, or 8 equal doses; for the fractionated irradiation, treatments were given on a BID basis with 4 hr between the treatments on any 1 day. For irradiation of 1 mm diameter spheroids, radiation was administered as single doses under conditions of equilibration with AIR. The TCD50 for the FaDu was significantly higher and the dose response curve steeper for tumors growing in immune suppressed (6 Gy WBI 24 hr prior to transplantation) than in control nude mice. Tumors, exponential or plateau phase cells, and spheroids derived from U87 were significantly and substantially more resistant under all conditions and fractionation schedules than for FaDu. Thus, the in vitro results do not indicate a clearly greater resistance by the glioma cell lines, while the more limited TCD50 data (single dose and 8 fractions irradiation) show more resistance in vivo by the glial tumors. We noted that the TCD50 values for U87 and A7 glial tumors overlap those for spontaneous tumors of the C3H mouse but are higher than the human squamous cell carcinoma xenografts in the nude mice. Substantial additional data from xenografts are needed to determine if the higher TCD50 values for GBMs, especially for fractionated irradiation, is a regular finding and is of sufficient magnitude to be pursued by studies to explain the observed differences.

MUTATION ANALYSIS OF BRCA1 AND BRCA2 CANCER PREDISPOSITION GENES IN RADIATION HYPERSENSITIVE CANCER PATIENTS

PURPOSE The dose intensity of radiotherapy (RT) used in cancer treatment is limited in rare individuals who display severe normal tissue reactions after standard RT treatments. Novel predictive assays are required to identify these individuals prior to treatment. The mechanisms responsible for such reactions are unknown, but may involve dysfunction of genes involved in the sensing and response of cells to DNA damage. The breast cancer susceptibility genes BRCA1 and BRCA2 are implicated in DNA damage repair and the control of genome stability. The purpose of this study was to determine if clinical radiation hypersensitivity is related to mutations of the BRCA1 and BRCA2 genes. Such information is of potential use in the clinical management of BRCA mutation carriers and their families. METHODS AND MATERIALS Twenty-two cancer patients who developed severe normal tissue reactions after RT were screened for mutations of BRCA1 and BRCA2, using various methods including protein truncation testing, direct DNA sequencing, and a PCR-based BRCA1 exon 13 duplication test. RESULTS No mutations were detected in the 22 patients tested, despite screening for the majority of commonly described types of mutations of BRCA1 and BRCA2. CONCLUSION These early results suggest that genes other than BRCA1 and BRCA2 probably account for most cases of clinical radiation hypersensitivity, and that screening for mutations of BRCA1 and BRCA2 is unlikely to be useful in predicting response to radiotherapy. However, it has not been excluded that some BRCA1 or BRCA2 heterozygotes might experience unexpected RT toxicity; further BRCA mutation screening on radiation sensitive individuals is warranted.

Experimental studies on the incidence of metastases after failure of radiation treatment and the effect of salvage surgery

FSaII, a spontaneous fibrosarcoma, and SCCVII, a spontaneous squamous carcinoma, were studied as early generation isotransplants in the right leg of C3Hf/Sed mice. Animals successfully treated, in respect to local control by surgery alone for 6 mm diameter tumors, had an incidence of distant metastases of 2.6% for the FSaII, and 8% for the SCCVII. For 12 mm tumors the incidence of metastases was 14.3% and 41%, respectively. Animals successfully treated with radiation alone for 6 mm tumors had an incidence of distant metastases of 3.1% for the FSaII, and 6.9% for the SCCVII. Animals that developed local recurrence after radiation therapy were treated with salvage surgery when the recurrent tumor was either 6 mm or 12 mm. For those successfully treated the incidence of metastases was 12.5% for the FSaII, and 43% for the SCCVII when salvage surgery was performed on 6 mm tumors. When surgery was delayed and performed on 12 mm tumors, the incidence was 46.6% and 70.3%, respectively. The results indicate that after failure of radiation treatment there is a high incidence of metastases from the recurrent tumors. The incidence, however, can be reduced considerably by carrying out early salvage surgery.

RADIOSENSITIVITY TESTING OF HUMAN MALIGNANT GLIOMAS

Radiotherapy remains the main treatment modality for patients with malignant gliomas and is the only treatment which significantly prolongs survival. Clonogenic and tetrazolium based colorimetric assays (MTT) of early passage cultures have been performed following 2 Gy doses of x-rays in order to determine if in vitro radiosensitivity is a factor in response to treatment. Of 47 biopsies received, 39 were established in primary culture. A value of surviving fraction to 2 Gy (SF2) was obtained in 85% of growth assays and 64% of clonogenic assays. The mean SF2 value for the MTT was 0.56 which was significantly higher than the 0.42 obtained for the clonogenic assay. There was, however, reasonable qualitative agreement in assessing relative radiosensitivity/radioresistance (r = 0.7). Mean SF2 values for grade 3 tumors were 0.52 (MTT) and 0.35 (clonogenic) as against mean SF2 values of 0.63 (MTT) and 0.47 (clonogenic assay) for grade 4 tumors. In 24 patients with adequate follow-up, no direct correlation was found between SF2 and survival, although mean SF2 values for patients surviving greater than 18 months was significantly less (p = 0.01) than patients surviving less than 18 months as determined by the MTT assay.