Characterization of a new surface epitope specific for human epithelial cells defined by a monoclonal antibody and application to tumor diagnosis.

In an attempt to characterize the antigens attached to cells of a line established from a human squamous cell carcinoma of the tongue (CAL 27), BALB/c mice were immunized with whole CAL 27 cells; hybridomas were then produced using spleen cells of the animals and cells of an NS1 syngeneic myeloma. A hybridoma secreting a monoclonal antibody was obtained (CALAM 27); CALAM 27 was directed against an epitope attached to the CAL 27 cells. CALAM 27, IgG2a, reacted with a membrane antigen specific to all epithelial cells. After immunoprecipitation, this antigen corresponded to two bands (Mr 22,000 and 54,000). Reactivity disappeared when the tissue was embedded in paraffin but was conserved after fixation with acetone or methanol. This antigen was conserved for both benign and malignant epithelial cell pathologies. The action of CALAM 27 was tested on 80 samples of pleural effusions, ascites, and cerebrospinal fluid samples; after conventional cytological examinations, CALAM 27 failed to recognize either reactive mesothelial cells or meningothelial cells. In addition, the cell structure recognized by CALAM 27 is not found on certain lymphoid tissue cells. CALAM 27 also failed to react with small cell carcinoma of the lung. Its strictly epithelial specificity therefore permits its use for the diagnosis of micrometastases of carcinoma in ascites and cerebrospinal fluid, in pleural effusions, and in bone marrow. CALAM 27 may also prove useful in confirming diagnosis of pathologies suspected to be of epithelial origin.

Immunophenotyping and metastatic localization of breast cancer.

Monoclonal antibody SM 92 is involved in the immunophenotype of gastrointestinal and liver cells, SM 43 in ovarian cells, and SM 13 in lung cells. Based on a study of 61 breast adenocarcinoma patients, we found that tumors reacting with SM 92 appear associated with liver metastases, SM 43 with ovarian metastases, and SM 13 with lung metastases. These associations are highly significant. They lend some support to the concept that tumor cells that metastasize tend to go to sites where cells normally have the same surface antigens.

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.

Epidermal growth factor receptor and labeling index are independent prognostic factors in glial tumor outcome.

The aim of this study was to perform a multivariate analysis including clinical and biological prognostic factors on glial tumor outcome. Seventy-nine patients were analyzed (48 men and 31 women; mean age = 56 years, range = 16-77 years): 7 had a benign glial tumor (grades 1 and 2), 21 had an anaplastic glial tumor (grade 3), and 51 had a glioblastoma (grade 4). Median follow-up was 17.9 months for patients who survived (50 patients died). Biopsies were obtained at time of diagnosis (complete tumor resection in 62 patients and stereotaxic biopsies in 17 patients). Epidermal growth factor receptor (EGFR) was measured by a binding assay, and labeling index (LI) was measured by tritiated thymidine incorporation. EGFR varied from 4 to 73,110 fmol/mg protein (mean = 3912 fmol/mg protein; median = 374 fmol/mg protein; n = 79). LI varied between 0.1 and 16.5% (mean = 6.2%; median = 5.2%; n = 40). Log10 EGFR was significantly and positively correlated with patient age. LI was significantly different according to tumor histology. Univariate Cox analysis (end point was cancer death) showed that age (P = 0.027), log10 EGFR (P = 0.025), and LI (P = 0.0019) were significant continuous variables, the survival being shortened when the covariable increased; tumor resection (P = 0.015, relative risk = 0.45) and histology (P = 0.0009) were significant categorical factors. A multivariate Cox analysis (forward selection) including age, histology, tumor resection, log10 EGFR, and LI revealed that log10 EGFR, LI, and tumor resection were the only independent significant predictors of survival. This multivariate approach reveals that the clinical prognostic factors of glial tumors, namely age and tumor histology, disappear, to the benefit of intrinsic characteristics of the tumor, i.e., EGFR expression and LI, suggesting that coupled EGFR and LI determination could be a useful tool for better evaluation of glial tumor outcome.

Expression of vascular endothelial growth factor-b in human astrocytoma.

Growth of human malignant gliomas is stringently dependent on an angiogenic process that probably involves vascular endothelial growth factor (VEGF). Expressions of mRNA coding for the different forms of VEGF were analyzed in surgical specimens from human astrocytomas. Low levels of placental growth factor (PGF) and VEGFC mRNA were observed in polymerase chain reaction, but not in Northern blot experiments. VEGF mRNA was found in some but not all grade and grade IV astrocytomas. VEGFB mRNA was observed in all tissue samples analyzed irrespective of the tumor grade. A new splice variant of VEGFB (VEGFB155) that lacks exons 5 and 6 is described. Expressions of VEGF mRNA in cultured glioblastomas cells were upregulated by hypoxia, but the sensitivity of the cells to hypoxia was reduced as compared with normal rat astrocytes. VEGF expression was depressed by dexamethasone. Expressions of VEGFB mRNA were affected neither by hypoxia nor by dexamethasone. The results indicate a coexpression of VEGF mRNA and VEGFB mRNA in human astrocytomas. Expression of VEGFB is markedly different from that of VEGF. Possible roles of VEGFB as a cofactor for hypoxia-induced angiogenesis in human astrocytomas are discussed.

Effects of prolonged exposure to cisplatin on cytotoxicity and intracellular drug concentration.

The present study was designed to analyse the cytotoxic effect of cisplatin in vitro as a function of various exposure times (up to 120 h), keeping constant the parameter C x T (product of the drug concentration per time). Intracellular drug concentrations were measured in parallel following analysis of cisplatin influx and efflux characteristics. A head and neck cancer cell line was selected to represent the spectrum of cisplatin antitumour activity. The IC50 values (micrograms/ml) for 1, 2, 11 and 121 h were, respectively 4.51, 2.73, 0.27 and 0.151. Reduction of the IC50 was clearly not linearly related to prolongation of the cisplatin exposure time. The kinetics of cisplatin incorporation into CAL 27 cells was investigated as a function of different cisplatin concentrations. A plateau was reached after 16 h of contact. For the extracellular cisplatin concentrations of 1, 2.5, 5 and 10 micrograms/ml, the average intracellular Pt concentrations at the plateau were, respectively (ng/10(6) cells): [mean (S.D.)] 12.8 (0.98), 31.11 (5.12), 71.38 (6.03) and 136.7 (16.5). Intracellular Pt concentrations were linearly related to the extracellular drug concentration (r = 0.99). The drug left the cells following a two-slope kinetics pattern with an alpha half-life of 1.29 h and a beta half-life of 94.4 h. The cytotoxic effect for a given C x T clearly differed for the different cisplatin exposure times. The longest exposure time (121 h) gave the least pronounced cytotoxicity. The intracellular Pt concentrations were linearly related to the C x T values. Cisplatin levels were much lower after the 121 h exposure. These data may prove valuable in establishing a rationale which can aid in selection of optimal modes of clinical cisplatin administration.

Inherent radiosensitivity and split-dose recovery in plateau-phase cultures of 10 human tumour cell lines.

The radiation response of 10 human tumour cell lines, eight of them established in our Institute, was analysed. Single dose acute survival curves were constructed and fitted with the linear-quadratic (LQ) model. The mean inactivation dose (D) was also calculated, together with D(o) and n. In order to measure both split-dose recovery and delayed plating recovery of plateau-phase cells, confluent cultures were subjected to two doses of 2 Gy 24 h apart, and plated 24 h later, simulating clinical fractionation. Survival of these cells (S2 x 2) was compared to that following 4 Gy given to cells plated at low density and an overall recovery factor (RF) was derived, including both types of recovery. S2 x 2 and RF were compared to the intrinsic radiosensitivity parameters. The three melanoma cell lines differed in radiation response, and the three squamous cell carcinoma cell lines were rather radioresistant. The neuroblastoma cell line was highly sensitive, yet it expressed the highest beta and the highest RF. Using a non-parametric correlation test, S2 x 2 was found to be related to D, whereas RF was not related to the radiosensitivity parameters. However, the two cell lines with the lowest D and the lowest S2 expressed the highest RF. These results suggest that radiosensitive cell lines may have a considerable capacity to recover if confluent cultures are exposed to fractionated irradiation. The overall recovery factor (RF) used here is proposed as a useful measure of cellular recovery.

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.

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.

Characterization of cal39, a new human cell-line derived from a vulvar squamous-cell carcinoma.

We have obtained a permanent cell line from a squamous cell carcinoma of the vulva. These cells, named CAL39, exhibited morphological, ultrastructural, and immunochemical characteristics of epithelial cells. They were tumorigenic in nude mice. Our observations indicate that the behavior of the cell line in the nude mouse and in culture is similar to that of the original cancer from which it was derived. We have compared these cells with A431 which are currently the best studied model cells of the same tumor origin. Like A431, CAL39 had an elevated number of high affinity EGF receptors, and showed an amplification of DNA sequences at 11q13. We have compared the cytogenetic features of the amplification units in the two cell lines. Unexpectedly, an HSR was present, in both cases, on a marker chromosome which was a derivative of chromosome 11. This result addresses the issue of the in situ amplification of chromosomal DNA.

Non-specifically labelled cells that simulate bone marrow metastases in patients with non-metastatic breast cancer.

AIMS: To determine whether the presence of disseminated bone marrow tumour cells at diagnosis is a prognostic factor for breast cancer patients at high risk of recurrence or bone metastasis, and to assess their presence as a criterion for evaluation of the potential benefits of adjuvant chemotherapy. METHODS: Multiple bone marrow aspirates from 72 breast cancer patients free from metastasis were obtained during surgery at the time of diagnosis and were tested immunologically by alkaline phosphatase antialkaline phosphatase technique with a panel of three antiepithelial monoclonal antibodies (MoAb) KL1, EMA, and HMFG2. RESULTS: In nine of 72 patients, with each MoAb tested, numerous strongly positive cells always isolated were observed. However, it was demonstrated that these cells were non-specifically labelled and could be found in normal controls. CONCLUSIONS: There was no evidence of marrow tumour cells in 72 operable breast cancer patients. It is suggested that published results may be greatly overestimated and that non-specific labelling may be undetected. More specific MoAb should be found and a correlation with molecular biology should be performed if this criterion is to be considered as a prognostic factor.

Cytogenetics of malignant gliomas: I. The autosomes with reference to rearrangements.

Autosomal chromosome abnormalities are far from always detectable and, when detected, far from fully consistent in malignant gliomas. In 15 of 41 malignant gliomas, we found autosomal chromosome aberrations ranging from solitary trisomy to a wildly abnormal polyploid complement. The sequence of chromosome events appears to proceed from the normal to the near-diploid state (via structural and numerical changes) to near-tetraploidy (via polyploidization), and finally toward near-triploidy (via chromosome loss and additional rearrangements). Characteristic chromosome changes of trisomy 7 and monosomy 10 were repeatedly found, usually together in the same cell clones. In only one case was trisomy 7 an isolated change. We observed structural rearrangements of chromosomes 7 and 10 which may be of some use in mapping specific genes duplicated or deleted by the whole-chromosome changes of chromosomes 7 and 10. Nonrandom structural changes of other autosomes, including chromosomes 1, 5, and 11, fit with the model of malignant glioma as a process involving multiple genes. An unusual concentration of breakpoints in 12q13, juxtaposing it to at least five other regions, reflects the presence of genetic information in 12q13 important to the development of malignant gliomas.

Cytogenetics of malignant gliomas. II. The sex chromosomes with reference to X isodisomy and the role of numerical X/Y changes.

Sex chromosomal monosomy with total loss of an X or Y is frequently observed in malignant gliomas. Beyond that, not much is known about the behavior of the sex chromosomes in these tumors. We noted loss of the X from 3 of 13 gliomas from women (23%) compared to loss of the Y from 16 of 28 gliomas from men (57%). There were two structural rearrangements of the Y (an inversion and a translocation with chromosome 4). Most unexpectedly, clones with sex chromosome reversal were encountered in 3 cases. These XX clones in gliomas from men are perforce the consequence of Y loss coupled with X isodisomy, a nonrandom sequence of sex chromosome changes. We examined the company kept by numerical X and Y changes in clones and found that clones with numerical sex chromosome changes had fewer autosomal abnormalities, reflecting a distinct tendency to clonal separation of sex chromosome from autosomal abnormalities. We conclude that the sex chromosome changes are not a necessary part of the neoplastic process in malignant gliomas but that they must be of biologic significance to the brain since they are highly nonrandom in frequency, type, and sequence in brain cells.

Sequence-dependent cytotoxic effects of the combination of a new nitrosourea, fotemustine, with 5-fluorouracil plus folinic acid.

The present study was designed to analyse the cytotoxic effects of the combination of fotemustine with 5-fluorouracil (5-FU) plus folinic acid (FA). Two human tumor cell lines were used; one line was derived from colon cancer (WIDR) and the other, from a non-small-cell lung cancer (CAL 12). Cytotoxic effects were assessed using the MTT (tetrazolium bromide) semi-automated test in 96-well incubation plates. The effects of various drug combinations were evaluated by the isobologram method. The drug combinations tested included fotemustine concentrations of 20, 30, 40, 50 and 70 micrograms/ml, 5-FU concentrations of 5, 15 and 30 micrograms/ml, and a constant FA concentration of 10(-5) M. A total of 180 different experimental conditions were tested. When cells were exposed to fotemustine prior to treatment with 5-FU, the final cytotoxic effects on both cell lines were additive or synergistic in the majority of cases (P less than 0.001). The 5-FU concentration was a determinant factor that modified the effects of the drug combination from antagonism (at low 5-FU concentrations) to synergism (high 5-FU concentrations; P less than 0.001). The addition of FA (10(-5) M) resulted in a significant shift towards synergistic associations in both cell lines. Administration of 5-FU prior to treatment with fotemustine caused marked antagonism, which 10(-5) M FA could not significantly shift towards simple additivity.

In vitro chemosensitivity testing of Fotemustine (S 10036), a new antitumor nitrosourea.

Fotemustine (S 10036) is a new anti-tumor nitrosourea characterized by a phosphonoalanine carrier group coupled to the nitrosourea moiety, which potentially increases the cellular penetration of the drug. Using human tumor cell lines, the activity of S 10036 was compared with that of the more established nitrosoureas BCNU and CCNU. Growth-inhibiting effects were evaluated by the [3H]-thymidine incorporation test. In a panel of 12 human cancer cell lines [melanoma (4), ovary (2), head and neck (3), lung (1), bladder (1), breast (1)], the dose-response curves of S 10036 (0-100 microM) were similar to those obtained with equimolar concentrations of BCNU and CCNU; they indicated a moderately more marked effect for two and an equal effect for six melanoma cell lines with S 10036 as compared with BCNU. Moderate but significant synergistic combinations were obtained when S 10036 (0-80 microM) and CDDP (0-100 microM) or DTIC (250-6,500 microM) were combined in melanoma cell lines. In conclusion, the new nitrosourea S 10036 shows promising activity, particularly against human melanoma cell lines.

Screening of human bladder carcinomas for the presence of Ha-ras codon 12 mutation.

Contradictory results were obtained from previous studies aiming at defining the frequency of Ha-ras codon 12 mutations in bladder tumors. Differences in the sensitivities of the methods used could account for this discrepancy. In this study, we reevaluated the frequency of Ha-ras codon 12 mutations in a series of 87 human bladder tumors using a combination of two different methods. The first was derived from the protocol of Ooi et al and consisted in a one-step allele-specific polymerase chain reaction using mismatched primers in two separate PCR. This method is very rapid and highly sensitive, detecting the presence of minor populations (less than 10%) of mutant alleles. The second strategy consisted in screening all tumors using natural restriction fragment length polymorphism (RFLP) analysis. The two methods were in complete concordance and enabled us to show that only one out of 87 primary bladder carcinomas (1%) exhibited the mutation, in accordance with previous studies. These results strongly suggest that, even if minor cell populations overexpress codon 12 Ha-ras mutation, the analysis of this mutation cannot be used to screen potentially invasive transitional cell tumors of the bladder.

Establishment and characterization of a new cell line derived from a human chondrosarcoma.

A new human cell line, CAL 78, derived from a dedifferentiated chondrosarcoma of the muscle of the thigh has been established in culture. Fibroblastoid morphology, vimentin expression and lack of epithelial antigens are in agreement with mesodermic origin of these cells. The xenograft of CAL 78 cells in nude mouse showed the characteristics of hyaline cartilaginous differentiation. Cytogenetic changes were numerous and complex, all the metaphases were tetraploid and no alterations described by other authors have been found. CAL 78 constitutes an appropriate model to evaluate efficiency of a new therapy for chondrosarcomas. Moreover, this cell line may be used to study some stage of chondrocytic differentiation.

Effects of tamoxifen on potential doubling time of human breast cancer cell line determined by image cytometry of double fluorescent BrdU and DNA labeling.

Tamoxifen is extensively used for the treatment of human breast cancer. However, the mechanisms by which antiestrogens regulate the growth of estrogen receptor positive tumors have not been totally defined. A new methodology, using automated image analysis BIOCOM 500, was developed for determining potential doubling time (Tpot) of tumors. This new method was checked on three different human breast cancer cell lines (MCF-7, CAL 85-1, CAL 148) in comparison with flow cytometry and then applied to determine the effects of short-term tamoxifen treatment on Tpot of MCF-7 cells. Using the resulting bivariate contour plot of blue fluorescence (DNA content) versus green fluorescence (Bromodeoxyuridine content), a labeling index (LI) value of 0.39 +/- 0.05 and a Tpot value of 21 +/- 2.09 hours were determined for MCF-7 cells. As expected, data demonstrated that 72 hours of 1 microM tamoxifen treatment decreased the LI to 35% by increasing the proportion of G0/G1 cells. It increased the Tpot to 35% compared to untreated cells (Tpot = 31.8 + 4 hours) by a lengthening of G0/G1 phase without changing the length of S phase (Ts = 10.2 +/- 1 hours). At suprapharmacological concentrations (5, 10 microM), an approximately 50% increase in Tpot was observed without modification in Ts. These data suggested a specific cell cycle action of tamoxifen which was probably mediated by mechanisms other than estrogen inhibition, since these experiments were performed in estrogen-deprived medium. In addition, the automated imaging procedure appears to provide a rapid and quantitative approach to determine Tpot in fine needle biopsies which is useful for investigating alterations in cell growth after endocrine treatment or chemotherapy.

Labeling index and labeling distribution in the colonic crypts: a contribution to definition of patients at high risk for colorectal cancer.

The labeling index (LI), representing the percentage of S-phase cells, was measured in the healthy colorectal mucosa of subjects without a personal or familial history of neoplasm as well as in persons with a positive history, and was compared to the LI in benign or malignant lesions in a total of 128 subjects. In the healthy mucosa, LI was lowest in control subjects; it rose progressively in patients with a personal history of adenoma, patients with non colorectal cancer, and colorectal cancer patients respectively. There was a significant increase in LI from healthy mucosa to adenoma to carcinoma. The pattern of labeling in the crypts was studied in 56 subjects with different familial and personal histories. Limitation of labeling to the deepest part of the crypts was observed in control subjects and to a lesser extent in the healthy mucosa of patients with adenoma. An upward shift of the proliferative zone was observed in patients with a personal or familial history of cancer; adenomatous lesions had an inverse labeling distribution: labeling decreased from the luminal aspect towards the deep segments. High LI values and a shift of the proliferative zone towards the surface of the crypt may identify individuals at high risk for cancer of the colon and rectum.

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.