Two new human tumor cell lines derived from squamous cell carcinomas of the tongue: establishment, characterization and response to cytotoxic treatment.

Two new permanent cell lines derived from squamous cell carcinomas of the tongue, CAL 27 and CAL 33, have been established in culture. Both cell lines were isolated in standard culture media without epidermal growth factor or fibroblast feeder layer to avoid obtaining clones of more differentiated cells. Analysis of the morphology, ultrastructure, karyotype and immunohistochemical properties of these two cell lines demonstrated that they are both well characterized, uncontaminated by HeLa cells, and do in fact correspond to transformed epithelial cells that have conserved certain characteristics of the original Malpighian epithelium. CAL 27 and CAL 33 have relatively long doubling times (35 and 43 h respectively). Their response to 14 drugs used for cancer chemotherapy was evaluated by a short term assay based on tritiated thymidine incorporation after exposure to the drugs. CAL 27 was more resistant than CAL 33 in all cases but one. Although cytogenetic examination revealed both lines to be malignant, neither CAL 27 nor CAL 33 produced colonies in soft agar; both lines were tumorigenic after inoculation into nude mice. This study clearly demonstrates the diversity of cancers of a given histologic form, in agreement with the diversity noted previously in vivo. Isolated without the use of any selection criteria, these cell lines constitute appropriate models for the study of human tumors.

Development and use of a computer system in a radiotherapy department: SISGRAD.

SISGRAD, the interactive computer system of the Antoine-Lacassagne Cancer Center Radiotherapy Department, has been operational since January 1982. It completes the computerized dosimetry system installed several years earlier and is fully integrated with the institution's central network. SISGRAD is in charge of surveillance of the radiotherapy treatments given by the Center's three radiotherapy units (1400 patients per year); it is also used for administrative purposes in the Department and physically connects all of the Department's operating stations. SISGRAD consists of a series of microcomputers connected to a common mass memory; each microcomputer is used as an intelligent console. SISGRAD was developed to guarantee that the treatments comply with prescriptions, to supply extemporaneous dosimetric data, to improve administrative work, and to supply banks with data for statistical analysis and research. SISGRAD actively intervenes to guarantee treatment quality and helps to improve therapy-related security factors. The present text describes the results of clinical use over a 4-year period. The consequences of integration of the system within the Department are analyzed, with special emphasis being placed on SISGRAD's role in the prevention and detection of errors in treatment prescription and delivery.

CT measurement of lung density: the role of patient position and value for total body irradiation.

To obtain more precise data on pulmonary doses in preparation for total body irradiation, the lung density of patients was systematically analyzed in treatment positions using data obtained by computed tomography (CT). With the patient supine, the lung density was not significantly different for the right and left lungs. In contrast, considerable differences were noted between the two lungs in lateral decubitus positions owing to variations in ventilation and perfusion. The relative electron density of lung was also found to decrease with age, dropping to pe = 0.160 at 71 years.

The DNA labelling index: a prognostic factor in node-negative breast cancer.

The DNA labelling index (LI), representing the fraction of S-phase cells, was studied in 76 patients operated on for breast cancer from 1975 to 1979. No patient had lymph node involvement following axillary dissection (N-), and no adjuvant medical treatment was given. Patients were classified in one of two groups according to the median LI. Patient distribution by age, tumour size, and receptor status was identical in both groups. A higher frequency of grade 3 tumours was noted in the group with an LI above the median value. The cell proliferation rate was an important discriminative factor for metastatic potential. The probability of survival at 8 years for patients with a high LI was significantly lower than that of patients with low LI (36% versus 100%; p less than 0.001). Relapse-free survival at 8 years was respectively 56% and 83% (p less than 0.02). At 2 years, these values were 75% and 100%, indicating the early occurrence of metastases. By contrast, the LI had no prognostic value concerning loco-regional disease recurrence or survival after the appearance of metastasis. No relationship was found in this study between survival and other prognostic factors, namely tumour size, histological grade, or hormone receptor level. The LI currently appears to be the best prognostic factor for N- breast cancer. High risk patients identified by this method could thus be offered adjuvant medical treatment.

Radiosensitization of human tumor cells by alpha-difluoromethylornithine.

The effect of polyamine depletion on the radiosensitivity of a human tumor cell line was investigated. CAL 18 A cells, derived from a breast carcinoma, were incubated with alpha-difluoromethylornithine (DFMO)--a specific and irreversible inhibitor of ornithine decarboxylase (ODC)--at a 1 mM or 10-mM concentration for either 1 hr or 24 hr and irradiated thereafter. Survival curves of exponentially growing cells revealed a moderate but significant enhancement of radiosensitivity as compared to untreated irradiated cells. Maximum radiosensitization was observed at a concentration of 10 mM after 1 hr incubation. Plateau-phase cells were used to study the effect of polyamine inhibition on repair of radiation-induced potentially lethal damage (PLD). DFMO enhanced the radiation response and significantly inhibited PLD repair in these cells. Measurement of ODC indicated that this enzyme was markedly inactivated upon brief incubation of CAL 18 A cells with DFMO, reflecting a depletion of polyamine synthesis. These results extend findings that have demonstrated enhancement of drug-induced cytotoxicity, and raise the possibility of clinical use of this substance for potentiation of radiation response.

Conservative treatment (chemotherapy/radiotherapy) of locally advanced breast cancer.

Once treated almost exclusively by radical surgery, locally advanced breast cancers (Stages III, IV; MO) were later treated solely by irradiation, but local results (cosmesis and recurrences) remained poor. Since 1977, we have used induction chemotherapy in an attempt to treat subclinical metastatic disease while allowing better quality mammary conservation in a greater number of patients. From 1977 to 1980, 25 patients (Stages III, IV; MO) were treated by a sequential association of chemotherapy (Adriamycin [doxorubicin], vincristine, cyclophosphamide, 5-fluorouracil; 3 cycles) followed by irradiation (45 Gy to the breast and nodal areas; 15-30 Gy mammary boost dose). Responses of 50% or more were observed in 86% of the breast lesions and in 80% of nodal lesions. This sequential treatment was always well tolerated. Local recurrences occurred in six patients (24%) who underwent mastectomy without any complications. The survival rate at 4 years is 55%.

In vitro study of the cell populations in two cases of Kaposi's sarcoma.

Despite numerous studies, the histogenesis of Kaposi's sarcoma remains unclear. In connection with the culture of two Kaposi's sarcomas, the morphological, ultrastructural, and immunological properties of the various cell types observed are discussed. Cloning in agar, loss of contact inhibition, and karyotyping were used to determine which cells had undergone malignant transformation. Findings for both cases revealed that endothelial cells had undergone neoplastic transformation. Fibroblastic cell lines were isolated from both sarcoma fragments; although their growth characteristics distinguished them from normal fibroblasts (increased growth and possibility of culture in soft agar), cytogenetic investigations on both lines confirmed that they were genetically normal, and occurred along with malignant cells as a accessory compartment within lesions. Endothelial cells appear to be the sole origin of Kaposi's sarcoma, and may release factors which alter fibroblastic growth.

Prospects for the application of fast neutrons in cancer therapy. Radiobiological bases and survey of the clinical data.

The rationale for introducing fast neutrons in therapy initially was a reduction in the Oxygen Enhancement Ratio (OER). The recent radiobiological developments indicate that, more generally, fast neutrons tend to reduce the difference in radiosensitivity between cell lines, or related to the degree of oxygenation (Oxygen Gain Factor), the position of the cells in the mitotic cycle (Kinetics Gain Factor), etc. The reduction of the differences in radiosensitivity brings a benefit for certain types of tumours (normally resistant to photons), but a disadvantage for other types of tumours (those which can currently be controlled by photon treatment). A review of the available clinical data is presented. The tumour types or sites for which a benefit has been observed are discussed: locally advanced tumours of the salivary glands, paranasal sinuses, some tumours of the head and neck area with metastatic lymph nodes, slowly growing, well differentiated soft tissue sarcomas, inoperable/recurrent melanomas, locally extended (C, D1) prostatic adenocarcinomas. Selection of the patients suitable for neutron therapy remains the main problem. Collaboration between neutron therapy centres is essential to accelerate the acquisition of sufficient clinical data needed in order to improve patient selection, as well as the optimum modality of application of fast neutrons.

The MEDICYC programme.

The major goal of MEDICYC, the medical cyclotron programme of the Antoine-Lacassagne Cancer Center (Nice, France) is radiotherapy based on protons and fast neutrons. This cyclotron has also been designed to meet the medical requirements for the Center's Nuclear Medicine Department. Technical specifications and anticipated applications are discussed. The construction of the machine is nearly finished and a first axially injected beam is scheduled for early 1987.

[Viability of human cancer cells in culture after exposure to diagnostic ultrasound]. / Viabilité de cellules cancéreuses humaines en culture après exposition aux ultrasons émis par un appareil de diagnostic.

Reports have appeared in the literature regarding biological damage to human cells following exposure to diagnostic ultrasound. We have examined the effects of diagnostic ultrasound (Sonel 400, C.G.R.) on human cell lines established in our laboratory. We report here that exposure to diagnostic ultrasound, at maximum exposure intensity and at exposure time as long as 60 minutes, produces no cell lysis as determined by vital dye exclusion ability, and as confirmed by electron microscopy. However, the exposure to ultrasound produced by an apparatus delivering an acoustic power higher than the diagnostic levels (Sonoscope-Alcatel) can cause the complete lysis of the same cells.

Establishment, characterization, chemosensitivity, and radiosensitivity of two different cell lines derived from a human breast cancer biopsy.

In vitro culture of a human breast cancer biopsy fragment gave rise to two permanent cell lines, CAL 18 A and CAL 18 B, which were differentiated by both morphological and ultrastructural analysis. The karyotypic and growth properties of these two cell lines also differed, providing further evidence of cell heterogeneity within a given tumor. Both cell lines lost their hormone receptors in vitro. CAL 18 A cells grew in agar and were tumorigenic after inoculation into nude mice; neither of these properties was observed in CAL 18 B cells. The chemosensitivity of 12 antineoplastic drugs was assessed by a short-term assay, using inhibition of tritiated thymidine incorporation by the cells after contact with the drugs as the end point. Only a few drugs were active at moderate concentrations. The overall responses of both cell lines were similar. The cell survival curves, established by the colony method following a single dose of radiation, were also very similar, despite the greater heterogeneity of CAL 18 B cells. The two cell lines appear to be interrelated, since CAL 18 B cells were occasionally observed to emerge from CAL 18 A clones, suggesting that malignant cell redifferentiation may occur spontaneously in vitro.

[Conservative treatment of cancer of the breast. Apropos of 108 cases]. / Traitement conservateur du cancer du sein. A propos de 108 cas.

Between 1975 and 1980, 108 cancers of the breast (T1 and small T2) were treated by associated conservative surgery and radiotherapy. Axillary glands were curetted in 92 patients; 17% showed lymph node invasion. Radiotherapy was by telecobalt: 45 grays to the whole breast and 60 grays to the tumor bed. Esthetic results were satisfactory. Intramammary recurrence was rare (6%) and was independent of site or size of tumor and of degree of lymph node involvement. Metastases not associated with local recurrence were very rarely observed (4% of cases) and were present only in patients without glandular involvement. The 5-year actuarial survival rate was 95%.

Conservative treatment of breast cancer: a report on 108 patients.

Between 1975 and 1980, 108 breast cancers (T1 and small T2) were treated at the Centre Antoine-Lacassagne (Nice, France) by a combination of conservative surgery and irradiation. Ninety-two of these patients underwent axillary node dissection; 17% of them presented with nodal involvement. All irradiation was given by telecobalt: 45 Gy to the entire breast, 60 Gy to the site of the tumor. All cosmetic results were acceptable. Intramammary recurrences were rare (6%) and independent of the tumor site or size or of any nodal involvement. Solitary metastases (not associated with a local recurrence) were extremely rare (4%) and were observed in patients who had had no nodal involvement. The actuarial survival rate at 5 years is 90%.

Simultaneous micro measurement of steroid receptors in breast cancer.

The oestradiol (RE) and progesterone (RP) receptor levels were analyzed in 26 tumour fragments (200-500 mg) from breast cancer patients. After pulverization of tissue, one part was analyzed by the routine dextran-coated charcoal (DCC) method and the other by a micromethod as follows: (i) cytosol incubation using the DCC method but in the simultaneous presence of [3H]oestradiol and [3H]R5020 (ii) extraction of the steroids bound to the receptor by precipitation with ethanol/TCA (iii) high pressure liquid chromatography (HPLC) on a modular system, with a C185 microns column and an elution by gradient mixture methanol/water. The fractions were collected and the radioactivity counted. The separation of oestradiol from R 5020 was rapid and complete. In addition dexamethasone was separated by this system making possible triple measures of RE, RP and glucocorticoid receptors. A highly significant correlation was obtained between the 2 methods: RE = 0.996, P less than 0.001; RP r = 0.975, P less than 0.001, implying that the thresholds of positivity, i.e. for therapeutic decisions, remain unchanged. Simultaneous measurement of RE and RP in a single needle biopsy is possible with this micromethod.

Alpha 1 isoenzyme of alkaline phosphatases. Clinical importance and value for the detection of liver metastases.

Measurement of the alpha 1 (fast liver) fraction of alkaline phosphatases in the serum for 217 cancer patients, 92 patients with nonmalignant hepatic affections and 131 controls, revealed that the alpha 1 fraction offers better global value (94%), sensitivity (96%), and specificity (93%) than gamma GT or total alkaline phosphatase determinations for the detection of liver metastases during cancer. Initial data from study of the time of appearance of the alpha 1 fraction reveals that this fraction shows up earlier than rises in the gamma GT or total alkaline phosphatases. Results of a multiparametric study conducted on the alpha 1 fraction and various hepatic enzymatic tests (SGOT, SGPT, GLDH, ALP, gamma GT) indicate that the alpha 1 fraction used alone is better than any other test or combination of tests for biological detection of liver metastases. As concerns the influence of chemotherapy on the appearance of the alpha 1 fraction, the majority of the drugs used for anticancer chemotherapy do not seem to affect measurement of the alpha 1 ALP fraction. The alpha 1 fast liver fraction of alkaline phosphatases, detected by electrophoresis on cellulose acetate, can be considered one of the best known tests for the detection of liver metastases.

Establishment, characterization, and response to cytotoxic and radiation treatment of three human melanoma cell lines.

Three human melanoma cell lines were derived from tumor specimens and established in culture. CAL 1 originated from a bone marrow metastasis and CAL 4 and CAL 7 were derived from solid tumor fragments. CAL 1 and CAL 7 were cloned before establishment. Ultrastructural and chromosome analysis were carried out along with the response to nine chemotherapeutic agents at various concentrations. Survival curves after irradiation were also plotted. The uncloned cell line, CAL 4, displayed some differences from the other two cell lines as regards ploidy and response to chemotherapy. Greater spread of chromosome numbers were observed with this cell line, which contained both hypoploid and a hyperploid modal numbers. All three cell lines showed a relatively high extrapolation number after irradiation, suggesting that inherent cellular properties may be partly responsible for the clinical radioresistance of malignant melanomas.