Pierre Chauvel

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.

The labelling index: a prognostic factor in head and neck carcinoma☆

The thymidine labelling index (LI), representing the percentage of cells in the DNA-synthesis phase, was measured in vitro prior to therapy in 87 patients with squamous cell carcinoma of the head and neck, who were treated between 1977 and 1982. The LI was not related to patient age, site of the tumour, clinical stage or histological grade. Overall survival was 44.5%. Univariate analysis demonstrated that survival was affected by the following factors: (1) age: patients older than 55 had a better outcome (p = 0.03); (2) site of the tumour (p = 0.005): laryngeal tumours had the best survival; (3) clinical stage (p = 0.05). Histological grade did not influence the survival (p = 0.41). Patients having a tumour LI higher than 15.5% (mean + 1 S.D.) had a significantly lower survival than patients with lower tumour LI (p = 0.008). A multivariate analysis using the Cox model showed that clinical stage and LI kept their prognostic impact with regard to survival. Finally, survival after relapse was lower in patients with a high tumour LI. These results demonstrate that a high tumour proliferation rate is an additional factor influencing the disease outcome in head and neck carcinoma. Patients with bad prognosis defined by this parameter could be offered a more energetic treatment.

Response to chemotherapy as justification for modification of the therapeutic strategy for pharyngolaryngeal carcinomas

From September 1983 to September 1987, 238 patients with squamous cell carcinoma of the upper aerodigestive tract were given 3 cycles of chemotherapy [cisplatinum (cis‐DDP), 100 mg/m2 on day 1; 5‐fluorouracil (5‐FU), 1,000 mg/m2 on days 2‐6] before any local treatment. Eighty‐one of these patients had pharyngolaryngeal cancer. Of the 45 of 50 laryngeal cancers and 26 of 31 hypopharyngeal cancers suitable for evaluation, complete responses (CR) were obtained in 51.1% and 53.8%, respectively. These response rates led to changes in the postchemotherapy protocols. For CRs, mutilating surgical protocols were replaced by definitive radiotherapy: one local recurrence has been observed among the 9 laryngeal cancers, and 3 of 10 hypopharyngeal cancer patients, who had an initial indication of total laryngectomy or total pharyngolaryngectomy replaced by radiotherapy. In the group of CR, survival rates at 2 years were 93% and 69%, respectively, for the larynx and hypopharynx vs 65.6% and 40% for non‐CR patients. The possibility of conservative treatment sparing vocal function with a high degree of reliability would in itself appear to be justification for induction chemotherapy in pharyngolaryngeal cancers, even though its long‐term effects remain controversial.

Pharmacokinetics of cisplatin given at a daily low dose as a radiosensitiser

SummaryA total of 25 patients with inoperable cervical cancer were treated by daily radiotherapy (2 Gy); sensitisation was obtained by administration of 5 mg cisplatin 30 min before each irradiation session. The total cumulative dose of cisplatin varied between 50 and 150 mg. A complete kinetic profile (0–24 h) of platinum (Pt) was established after the first dose and at the end of treatment for 22 patients. Pt was quantified by atomic absorption spectrophotometry using Zeeman-effect background correction for trace analysis. The total Pt AUC0–24 h increased from 1.53±0.77 to 7±3.55 μg·h·ml−1 between the start and the end of treatment (P<0.001). Ultrafilterable Pt (Pt UF) rose from 0.079±0.038 to 0.138±0.095 μg·h·ml−1 (P<0.01). Elimination half-lives were unchanged for total Pt but rose for Pt UF; these kinetic modifications in Pt UF did not correlate with any significant change in individual serum creatinine levels. No clear correlation was found between the cumulative cisplatin dose and tumor levels measured in 13 patients, and the tumor cisplatin dose did not correlate with response to treatment. Patients with hematological toxicity were characterised by an increase in their residual Pt UF level during treatment. Overall, our findings strengthen the notion of Pt UF kinetic variability during repeated treatment.

The RBE of Fast Neutrons for in Vitro Inactivation of Human Tumour Cells Determined by the Ratio of Mean Inactivation Doses

In an effort to clarify the relationship between sensitivity of human tumour cells to low-LET and to fast neutron irradiation, 10 human tumour cell lines were exposed to cobalt gamma-rays and to 60 MeV (p -> Be+) neutron beam. The data were pooled with results of 31 human tumour cell lines previously published. The analysis of date using the linear-quadratic model indicated that not only alpha values increased after neutron irradiation, but so did beta values too, although to a lesser extent. The mean inactivation dose (MID) was derived for each cell line from the linear-quadratic parameters after low-LET and high-LET exposure. MID values following neutron irradiation were closely correlated to those after gamma-ray irradiation. In these 41 cell lines, the extreme values of RBE derived by the ratio of MID varied by a factor of 3 among the cell lines. RBE was positively correlated to photon MID, meaning that intrinsically radiation resistant tumour cells have a higher neutron RBE, on average. Similar findings were observed if alpha ratios were used instead of MID ratios. In addition, the RBE/dose variations were more marked in cells with the higher RBE. Taken together, these data suggest that, although considerable variations exist among human tumour cell lines, intrinsically radioresistant cells are relatively more sensitized when exposed to high LET beams than radioresponsive tumours. An intrinsic gain factor may thus be expected in irradiating radiation resistant tumours with fast neutrons, in addition to the hypoxic or kinetic gain factors. Because the quadratic component is still present after neutron irradiation, we suggest using MID ratio as a reference RBE when comparing survival curves of cells exposed to radiations of different qualities.

Micronucleus induction in 10 human tumour cells after high- and low-dose radiation

A number of data measuring survival of animal or human cells to low LET ionizing radiation have demonstrated that these cells may be hypersensitive to doses below 1 Gy, possibly due to the absence of an inducible repair mechanism, which is observed at higher doses. The production of micronuclei (MN) in cells exposed to ionizing radiation reflects genotoxic damage. Moreover, the micronucleus assay is sensitive to low radiation doses. We have exposed 10 human tumour cell lines to doses ranging between 0.12 and 4 Gy. Using cytochalasin B to block the cells in a binucleate phase, we have scored the fraction of binucleate cells (BNC) expressing MN, as well as the number of MN per BNC, as a function of gamma-ray dose. Experimental points were fitted with a binomial equation. Doses from 1 to 4 Gy were fitted separately from those below 1 Gy, and the initial slopes after both fits were compared. Taken together, the initial slopes of MN induction after low-dose (LD) irradiation were not different from those after high-dose (HD) irradiation. Only in one cell line was a significant increase in MN production detected after LD irradiation. This cell line had the shallowest linear term after HD irradiation. It appeared that the likeliness of expressing hypersensitivity at LD was correlated with the quadratic term of MN induction at HD, which does not contradict an inducible repair hypothesis. However, the failure of observation of a significant hypersensitivity at LD for nine cell lines, and the high variability of response at LD suggests that this occasional effect may be influenced by other factors as well.

Micronucleus induction and reproductive death in a human cell line exposed to low-energy argon beam

Little is known about the action of charged particles of very high linear energy transfer (LET) on human cells and, in particular, the relationship between DNA damage and reproductive death. The aim of this study was to measure the biological efficiency of a low-energy argon beam (E=7.1 MeV/nucleon, LET= 1590 keV/µm) produced at GSI, Darmstadt, on a human melanoma cell line (CAL4) established in our Institute. Two different methods were used: the micronucleus (MN) test and the colonyforming assay. The MN test, using the cytochalasin-block method, is a measure of genotoxic damage. MN are scored in binucleate cells (BNC) and are formed from acentric fragments or whole chromosomes that have not been incorporated into daughter nuclei at mitosis. The colonyforming assay quantifies reproductive death. Parallel experiments were run with cobalt gamma-rays for comparison. After Co irradiation, the MN-free BNC dose-response curve coincided with that of the loss of colony-forming ability, suggesting the potential of the former as a predictive test of cell killing. After Ar irradiation, there was a dissociation between the two effects, especially at high doses: cell death was greater than the frequency of BNC with MN. The inactivation cross-section was 74 µm2; it was 39 µm2 for MN yield. Therefore, the relative biological effectiveness (RBE) was higher for cell killing than for MN yield (0.8 and 0.5, respectively, at a Co dose of 3 Gy). The total MN count in BNC followed the same pattern of response as the fraction of BNC with MN. However, multiple (>2) MN in BNC were more frequently observed after low-dose Ar irradiation than after gamma-ray exposure (RBE>1). Moreover, the frequency of multiple MN induction exceeded that expected from a Poisson distribution attribution at all dose levels of Ar irradiation. These results indicate that (1) cell killing after 7.1-MeV Ar beam irradiation is less effective than after Co irradiation at an equivalent average energy deposition; (2) unlike gamma-rays, Ar particles are more efficient at cell killing than in producing MN; (3) the frequent scoring of multiple MN suggests the production of multiple damage sites, even at low fluences of Ar particles.

Radiotherapy of inoperable lung cancer

Evaluation of loco-regional results obtained by radiotherapy for 31 patients with inoperable epidermoid lung cancer revealed objective remission (over 50%) in only 25% of patients. These results emphasize the limited effectiveness of radiotherapy in such cases and point out the need for increased research in radiotherapy techniques if survival rates are to be improved.

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 institutions central network. SISGRAD is in charge of surveillance of the radiotherapy treatments given by the Centers three radiotherapy units (1400 patients per year); it is also used for administrative purposes in the Department and physically connects all of the Departments 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 SISGRADs role in the prevention and detection of errors in treatment prescription and delivery.

A multicentric study of the GETTEC (Groupe d’Études des Tumeurs de la Tête et du Cou) about 408 clinically complete responders after Neo-Adjuvant chemotherapy for head and neck carcinomas

Clinically Complete Response (CCR) occuring after induction chemotherapy has often been considered as a good prognosis indicator. The routine use of CDDP-5-FU giving a relatively large amount of CCR, the GETTEC, a francophone study group, promoted in 1985 a multicentric study (ERCCACI: Enregistrement des Regressions Cliniques Completes Apres Chimiotherapie d’Induction) recording all these CCR in order to assess its value, the endpoints being survival, local and regional control, metastasis and second primaries. Retrospective inclusions were allowed and a total number of 408 cases were included by 8 institutions from april 1981 to may 1991: almost a half of the patients has been included by the Centre Antoine-Lacassagne (215) in Nice, followed by the Centre Oscar-Lambret (55) in Lille, Institut Gustave-Roussy (36) in Villejuif, Fondation Bergonie (31) in Bordeaux, Hopital Michallon (23) in Grenoble, Institut Jules-Bordet (20) in Brussels, Institut Curie (18) in Paris, Hopital de Rangueil (10) in Toulouse.