N. Chavaudra

Distribution of radiation sensitivities for human tumor cells of specific histological types: Comparison of in vitro to in vivo data☆☆☆

The radiosensitivities of human tumor cell lines, grouped into 6 histological categories, have been studied using data from the published literature. The parameters alpha, beta, n, D0, D, and the surviving fraction to 2 Gy (S2) and 8 Gy (S8) were calculated. Only the two parameters mainly derived from the initial part of the survival curve, alpha and D, together with S2, provided data which were correlated with the clinical radioresponsiveness of each histological group. Thus, there are intracellular factors which influence clinical radioresponsiveness whose relative importance varies from one histological cell type to another. The value of D gave the most precise characterization of the average group radiosensitivity. It was possible to compare the in vivo radiosensitivities of non-severely hypoxic cells with those of tumor cells irradiated in vitro for 7 tumor lines grown as xenografts in mice. The average radiosensitivity was 1.9 times less in vivo than in vitro. This difference indicates that, in addition to the intrinsic factors of radioresistance demonstrated in vitro, and independently of severe hypoxia, there are other factors which specifically reduce radiosensitivity in vivo.

Relationship between the growth rate of human metastases, survival and pathological type

Abstract Data have been collected for 203 patients on the growth rate of pulmonary metastasis and survival, defined as delay between the diagnosis of the primary tumour and death. These cancers when found to be disseminated were not treated. The distribution of the survival times was found to be log-normal. The survival of the patient increased as the doubling time (D.T.) of the tumour increased. This group of patients was subdivided according to five histological types. A similar correlation between survival and doubling time was found in the five sub groups. As previously shown, the histologic subgroups have different mean D.T., but it was found that the increase of mean survival from one subgroup to another is shorter that what would be expected from the increase of D.T. Furthermore, for the same D.T., survival is longer for patients with a cancer of the histologic type with the shortest mean D.T. Assuming the growth rate of the tumours remain constant, it is possible to calculate the number of times the volume of the tumour doubles during the survival time. When comparing the various histological subgroups, this mean number increases as the mean growth rate becomes more rapid.

In vivo measurement of the potential doubling time by flowcytometry in oropharyngeal cancer treated by conventional radiotherapy

PURPOSE Experimental and clinical studies suggest that the pre-treatment potential doubling time could be predictive of tumor control in patients treated by conventional radiotherapy and could help to identify the rapidly growing tumors for which accelerated radiotherapy is required. METHODS AND MATERIALS To test this hypothesis, we studied prospectively 48 patients with a squamous cell carcinoma of the oropharynx and treated by conventional radiotherapy (70 Gy/7 weeks). The duration of S phase, the labeling index and the potential doubling time were obtained by flowcytometry measurements of a tumor biopsy obtained after injection of 200 mg bromodeoxyuridine to the patient. RESULTS Three parameters were significantly associated with an increased risk of relapse namely the tumors size (T4; p < 0.01), the nodal status (> or = N2; p < 0.05) and the site of the primary within the oropharynx (p = 0.08). The S phase, labeling index, DNA index and potential doubling time were not significantly associated with an increased risk of relapse. However when considering only the T2 subgroup of patients, high labeling indexes and short potential doubling time were associated with an increased risk of relapse: the mean pre-treatment potential doubling time of the tumors which relapsed was 3.21 versus 5.5 days when there was no evidence of local relapse (p < 0.05). The mean labeling index for the group of tumors associated with a tumor recurrence was 11.7% compared to 7.3% when there was no evidence of relapse (p = 0.02). CONCLUSION Factors other than proliferation play a role in determining the outcome of oropharyngeal cancers treated by conventional radiotherapy. However there was a significant correlation between short potential doubling time, high labeling index and tumor recurrence in the T2 subgroup of patients. The finding of significance for potential doubling time and labeling index in the T2 subset of tumors may be a reflexion of the more homogeneous nature of these tumors with regard to prognostic variables.

Influence of Nucleoshuttling of the ATM Protein in the Healthy Tissues Response to Radiation Therapy: Toward a Molecular Classification of Human Radiosensitivity

PURPOSE Whereas post-radiation therapy overreactions (OR) represent a clinical and societal issue, there is still no consensual radiobiological endpoint to predict clinical radiosensitivity. Since 2003, skin biopsy specimens have been collected from patients treated by radiation therapy against different tumor localizations and showing a wide range of OR. Here, we aimed to establish quantitative links between radiobiological factors and OR severity grades that would be relevant to radioresistant and genetic hyperradiosensitive cases. METHODS AND MATERIALS Immunofluorescence experiments were performed on a collection of skin fibroblasts from 12 radioresistant, 5 hyperradiosensitive, and 100 OR patients irradiated at 2 Gy. The numbers of micronuclei, γH2AX, and pATM foci that reflect different steps of DNA double-strand breaks (DSB) recognition and repair were assessed from 10 minutes to 24 hours after irradiation and plotted against the severity grades established by the Common Terminology Criteria for Adverse Events and the Radiation Therapy Oncology Group. RESULTS OR patients did not necessarily show a gross DSB repair defect but a systematic delay in the nucleoshuttling of the ATM protein required for complete DSB recognition. Among the radiobiological factors, the maximal number of pATM foci provided the best discrimination among OR patients and a significant correlation with each OR severity grade, independently of tumor localization and of the early or late nature of reactions. CONCLUSIONS Our results are consistent with a general classification of human radiosensitivity based on 3 groups: radioresistance (group I); moderate radiosensitivity caused by delay of nucleoshuttling of ATM, which includes OR patients (group II); and hyperradiosensitivity caused by a gross DSB repair defect, which includes fatal cases (group III).

Dose-rate effect on radiation-induced DNA double-strand breaks in the human fibroblast HF19 cell line

We measured DNA double-strand breaks (dsbs) immediately after exposure of a non-transformed human fibroblast cell line (HF19) to gamma-rays (0-40 Gy) at four dose-rates (10, 1, 0.1, and 0.01 Gy/min) at 37 degree C using clamped homogeneous electric field (CHEF) gel electrophoresis. The shape of the dose-response curves, which could be approximated by a straight line over the range 0-20 Gy for irradiation at 4 degree C, became curvilinear when irradiation was carried out at 37 degree C at 10, 1, 0.1, and 0.01 Gy/min and reached a plateau at 10 Gy after irradiation at 0.01 Gy/min. We present a mathematical analysis that predicts the results of irradiation at 37 degree C from dsb induction and repair data obtained at 4 degree C, followed by incubation for repair at 37 degree C. The model assumes that the rate of dsb rejoining changes continuously with repair time and that it is independent of dose and dose-rate in the range 10-40 Gy. The model also assumes a linear induction of dsb with dose at 4 degree C and dsb induction is independent of dose-rate and of temperature during irradiation. Independent measurements of dsb induction at 4 degree C and of repair rate accurately predict the dsb levels after irradiation at 37 degree C, during which both phenomena occur simultaneously.

Changes in labeling indices of human tumors after irradiation

Abstract The time courses of the in vitro labeling index (LI) and of the tumor cell density (CD) were studied in 53 patients after one or several sessions of irradiation. Prior to irradiation, a significant positive correlation was observed between LI and CD. In these tumors the depression in LI was greater and of longer duration than in experimental tumors, it increased progressively during the course of fractionated irradiation. The histologic type and the site of the tumor appeared to influence the course of LI and CD. A temporary increase of LI was observed in only 7 of 31 patients irradiated in one or two sessions and in only 2 of 22 patients who were followed during a fractionated course of radiotherapy. In the 9 patients whose LI increased after irradiation, the pretreatment LI and CD were significantly lower than in the other patients. No relationship was observed between pretreatment LI or CD values and either tumor regression with radiotherapy or 5-year survival. However, a significant fall in LI after irradiation was generally observed in patients who had no noticeable tumor regression and in those who did not survive 5 years; no significant or a less significant fall in LI was observed in tumors which regressed after radiotherapy and in patients who survived at 5 years.

Radiosensitivity, blood perfusion and tumour oxygenation after perflubron emulsion injection

The effect of 90% and/or 100% w/v perflubron (perfluorooctyl bromide (PFOB); Alliance Pharmaceutical Corp.) emulsions on radiosensitivity, tumour relative perfusion and oxygenation was studied using EMT6 tumours in nude mice. Perflubron (2-15 ml/kg) emulsion was injected. The mice inhaled carbogen for 30 min and 60 min prior to irradiation. The radiosensitizing effect of the 90% w/v emulsion was maximal at 4 ml/kg. The tumour relative perfusion diminished after injection of both 100% and 90% w/v emulsions in carbogen-breathing mice at a dose of 15 ml/kg. This drop could explain the lack of efficiency of these treatments at this high concentration. Lastly, tumour oxygenation was increased after administration of perflubron emulsion plus carbogen.

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.

Estimation of clonogenic cell fraction in primary cultures derived from human squamous cell carcinomas

Tumor clonogenic cell content is believed to play an important role in the outcome of radiotherapy. However, there is no proven method to assess the number of clonogens in human tumors accurately. All currently available assays employ in vitro plating efficiency or in vivo TD50 (the average number of cells needed to induce tumors in 50% of injected mice) to estimate the tumor clonogenic ability. In this study, a monolayer mass primary culture system was used to estimate the clonogenic cell fraction in human tumors. For this purpose, 25 growth curves were performed for 25 tumor specimens derived from 21 head and neck and 4 cervical squamous cell carcinomas. The exponential portion of each growth curve was extrapolated through the ordinate (day 0) to estimate the clonogenic cell fraction; this method is only an estimate because it assumes no lag phase before exponential growth of clonogenic cells. The mean clonogenic cell fraction, expressed as clonogens/tumor cells inoculated, was relatively low (mean: 0.71%, range: 0.11-9.28), and the variation was wide (coefficient of variation = 148%). On the other hand, the doubling time of the growing population was 1.46 days and exhibited a very narrow range (0.98-2.24, coefficient of variation = 24%). The mean and range of clonogenic cell fraction were found to be in agreement with published values of soft agar colony forming efficiencies in both murine and human tumors. However, further investigation is necessary to determine how accurately this method measures the relative clonogenic cell content in human tumors. Clinical correlations between clonogenic cell fraction values and the response to radiotherapy are still too early to determine.

The initial slope of human tumor cell survival curves: Its modification by the oxic cell sensitizer β-Arabinofuranosyladenine

The initial slope of the survival curve, which is a characteristic of each tumor cell line, varies with the histological group of the tumor. It is one of the factors on which clinical radioresponsiveness depends. The DNA dependant DNA polymerase inhibitor beta-ara A acts as an oxic cell sensitizer. This study was carried out on human tumor cell lines to look for a correlation between the degree of radiosensitization induced by beta-ara A and the radiosensitivity of a given cell line. Six human tumor cell lines with different radiosensitivities were used (the survival rate at 2 Gy and D ranged from 20 to 73% and from 1.2 to 3.2 Gy, respectively). beta-ara A had a major toxic effect on all cell lines but this varied greatly from one cell line to another and was concentration dependant; this toxic effect was taken into account when calculating the surviving fractions. For all cell lines, beta-ara A acted as an oxic radiosensitizer and the radiosensitization was concentration dependant. Analysis of the survival curves of the 6 cell lines using the linear quadratic model showed that concentrations of beta-ara A between 200 and 1000 microM induced an increase in the linear component while the quadratic component underwent no systematic change. The sensitizing enhancement ratio (SER) measured from the Ds ratios, varied greatly from one line to another. For example, at a concentration of 500 microM, the extreme values of Ds ratios were 1.5 and 2.6. The radiosensitization is greater, the higher the radiosensitivity of the cell line studied during exponential growth. The results do not favor the use of beta-ara A in the treatment of intrinsically radioresistant human tumors.