Western blot assay for prostate-specific membrane antigen in serum of prostate cancer patients.

There is a need for the development of new diagnostic tools for the early detection of prostate cancer. A candidate molecule for a new screening test is a prostate-specific membrane antigen (PSM) recognized by the monoclonal antibody 7E11.C5. We carried out studies aimed at identifying PSM in the serum of normal and benign prostatic hyperplasia (BPH) donors and patients with adenocarcinoma of the prostate, in order to judge whether the development of a serum assay using this marker was feasible. By Western blotting, we found significant levels of PSM in serum samples from prostatic cancer patients, in the seminal fluid of pooled normal donors, in BPH patients, and in normal male sera. Similar to prostate-specific antigen (PSA), PSM was present in seminal plasma in higher concentrations than in serum, and PSM levels in prostatic cancer patients were significantly higher than in normal controls. These data suggest that the development of an assay utilizing the PSM and new monoclonal antibodies directed against the antigen, could provide a feasible test for prostatic cancers.

Immunoscintigraphy of prostatic cancer: preliminary results with 111In-labeled monoclonal antibody 7E11-C5.3 (CYT-356).

A phase 1 study was conducted with the investigational immunoscintigraphic agent, 111In-CYT-356, a radiolabeled, site-specific immunoconjugate of monoclonal antibody 7E11-C5.3, in 40 patients with prostatic carcinoma and known distant metastases. Each patient received a single intravenous infusion of CYT-356 (dose range, 0.1-5 mg) radiolabeled with approximately 5 mCi of 111In. None of the patients experienced adverse reactions. One patient who received a 5-mg dose developed antibodies to the CYT-356 immunoconjugate. 111In-CYT-356 immunoscintigraphy detected bony metastases in 21 of 38 patients (55%), including 12 of 14 (86%) receiving concomitant hormonal therapy, and soft tissue lesions in four of six patients (67%). Antibody imaging detected occult lesions in the bony pelvis and lumbar spine, which were confirmed by follow-up imaging tests, in one patient. Higher CYT-356 doses may clear the blood pool more slowly. These results suggest that 111In-CYT-356 can be safely administered to patients with prostatic carcinoma and that further clinical investigation of this agent is warranted.

Prospective new developments in laboratory research and clinical trials in prostatic cancer.

Laboratory research continues to develop new methods supporting clinicians in the management of prostatic cancer. Routine measurements of serum prostate-specific antigen (PSA) levels improve diagnosis, provide insight into tumor burden, and sharpen prognostication. New organ-specific antigenic targets are identified for monoclonal antibody-directed diagnostic and radiotherapeutic reagents. Such new immunospecific conjugates are already under evaluation in clinical trials. Cell culture techniques carry the promise of noninvasive early detection and prognostic evaluation of prostatic malignancy in preclinical stages. Molecular mechanisms responsible for androgen regulation of proliferation and malignancy of prostatic cancer are becoming open for scientific inquiry.

The human prostatic carcinoma cell line LNCaP and its derivatives. An overview.

The FGC (fast growing colony) line, a derivative of the LNCaP cell line shares all the main characteristics, including its androgen dependence, described for the original LNCaP cultures. A number of sublines originated from the FGC line which were characterized with respect to their response to steroid-depleted serum and to the synthetic androgen, R1881. After subcloning the FGC line a series of clones was isolated with distinct patterns of androgen-responsiveness. Among the sublines and clones studied, the FGC, FGC-JB and FGC clone-9 were androgen-dependent, whereas subline LNO, R and presumably also FGC clone-22 were androgen-independent. Distinct morphological differences were observed between the cells of the various sublines and between clone-9 and 22. The LNCaP cell line, its descending sublines and clonal derivatives provide a suitable in vitro model for studying different aspects of androgen-responsiveness of human prostate cancer.

Cytogenetic characterization of several androgen responsive and unresponsive sublines of the human prostatic carcinoma cell line LNCaP.

The cytogenetic evolution of the prostatic adenocarcinoma cell line LNCaP was investigated during long term in vitro culture. Study of five different sublines demonstrated that the original karyotype was well preserved in all sublines, with respect to the chromosome number as well as to the primary markers. All sublines showed additional, subline specific secondary marker chromosomes. Comparison of these markers in androgen responsive and nonresponsive sublines showed rearrangement of the short arm of chromosome 8 in both unresponsive sublines. The breakpoints were in 8p21 and 8p23, respectively, resulting in deletion of the 8p23----pter region in both sublines. In contrast, the hormone responsive sublines did not show any aberrations in chromosome 8. Review of published karyotypes of patients and cell lines seems to support our finding of partial deletion of 8p in androgen unresponsive prostate tumor cells.

Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patients.

Stable clones of murine hybridomas 7E11-C5 and 9H10-A4 were obtained following immunization with LNCaP cells. The LNCaP cells were isolated from a human prostatic cancer (Ca). Both hybridomas secreted monoclonal antibodies (MoAb) of the IgG1 subclass which were reactive with the insoluble, cytoplasmic, membrane rich fractions of the immunogen. Neither MoAb reacted with the soluble cytosol of LNCaP cells nor with purified human prostatic acid phosphatase (PAP) nor prostate specific antigen (PSA). MoAb 9H10-A4 reactivity was very narrow and limited to the surfaces of LNCaP cells only. MoAb 7E11-C5 specificity was restricted to human prostatic epithelium, both normal and malignant. Except LNCaP, none of the 32 lines of human normal or neoplastic cells reacted with MoAb 7E11-C5. In a survey of frozen sections from 175 human specimens, positive indirect immunoperoxidase staining was limited to epithelium in all 11 specimens of localized and metastatic CaP, 7 benign prostatic hypertrophy (BPH) cases and 7 normal prostates. None of the 26 various nonprostatic tumors nor 120 out of 122 specimens from 28 different normal organs were reactive. Positive staining occurred in 2 out of 14 normal kidneys. Competitive binding with MoAb 7E11-C5 or its F(ab')2 fragments demonstrated the presence of circulating epitope 7E11-C5 in 20 out of 43 sera from CaP patients. Only 3 out of 66 sera from nonprostatic malignancies reacted. None of 30 normal blood donors sera nor 7 BPH sera were positive. Thus, highly significant (p less than 0.0001) association between diagnosed prostatic cancer and circulating molecules expressing the epitope reactive with MoAb 7E11-C5 was established. Significant probability (p less than 0.05) also suggested that patients with positive ELISA test are more likely to be in progression, than those who are negative. These results suggest that this apparently new antigenic marker may be of clinical potential in CaP.

Cell cultures derived from Wilms' tumour animal model.

Several cell cultures were established from a transplantable Wistar/Furth rat Wilms' tumour. Following cloning by the limiting dilutions method, three morphologically distinct types of cells were identified and preserved for further study of growth regulation in the nephroblastoma model.

Effect of methotrexate-monoclonal anti-prostatic acid phosphatase antibody conjugate on human prostate tumor.

Methotrexate (MTX) was conjugated to an immunoglobulin G1 (IgG1) monoclonal antibody specific for human prostatic acid phosphatase (PAP) by the active ester method. The molar ratio of MTX to IgG was 14. MTX-monoclonal antibody conjugate retained substantially the original PAP-binding inhibition activity of the monoclonal antibody. Both MTX-monoclonal antibody conjugate and an identically prepared MTX-normal mouse IgG conjugate preserved 90% of the original dihydrofolate reductase inhibitory activity of MTX. [3H]MTX conjugated to monoclonal anti-PAP antibody was significantly accumulated more in PAP-producing human prostate tumor LNCaP cells than its normal mouse IgG counterpart. No statistical difference was found between the uptake of [3H]MTX conjugated to monoclonal antibody and that of [3H]MTX conjugated to normal mouse IgG by control PAP nonproducing thyroid tumor cells (TT). The antitumor effect of the conjugate was evaluated in vitro by its inhibition on deoxy[6-3H]uridine incorporation into LNCaP cells. The inhibition by MTX-monoclonal antibody conjugate was significantly higher than that by MTX-normal mouse IgG conjugate at 8 micrograms of drug per ml, although it was significantly less than that by free MTX. However, an in vivo tumor and tissue distribution study of [3H]MTX and its conjugates revealed that, 5 days after i.v. administration, [3H]MTX conjugated to monoclonal antibody was preferentially accumulated in LNCaP prostate tumor. Tumor:blood ratios for [3H]MTX, [3H]MTX-monoclonal antibody conjugate, and [3H]MTX-normal mouse IgG conjugate were 1.47, 5.06, and 1.26, respectively. Preliminary results obtained from a pilot study with a small number of animals demonstrated that multiply injected MTX-monoclonal antibody conjugate retarded the growth of xenografted prostate tumor (LNCaP) as compared with the control groups, including free MTX which showed a shorter period of therapeutic effectiveness. This study suggests that MTX conjugated to monoclonal anti-PAP antibody could be a potential reagent for experimental immunochemotherapy of prostate tumor, should the initial in vivo data be extended and confirmed.

Potentiation of the cell growth inhibitory effect of beta interferon by mopidamole.

It was suggested that the antitumor effect of the interferons is based in part on their ability to stimulate increased cAMP production. We have explored the interaction of human fibroblastic beta interferon (HFIF) with a cAMP decomposition inhibitory pyrimido-pyrimidine derivative, Mopidamole (RA-233) in cultures of neoplastic and normal cell lines. Mopidamole potentiated the growth inhibitory effect of HFIF in cultures of ES-1 malignant melanoma cells, LNCaP prostatic carcinoma cells, RT-4 transitional carcinoma cells, HT-29 colon adenocarcinoma cells and in diploid fibroblast cells.

A high-resolution study of chromosome changes in a human prostatic carcinoma cell line (LNCaP).

A high-resolution study of chromosomal rearrangements in a human prostatic cancer cell line, LNCaP, has been performed. The cytogenetic analysis revealed a pseudodiploid karyotype and the presence of seven marker chromosomes resulting from five aberrational events. The analysis of four clones derived from the original line showed a near-tetraploid chromosome number and the presence of the same seven markers observed in the original line. This is the first complete description of karyotypic rearrangements in a prostatic cancer cell line.

Antineoplastic effect of the pyrimido-pyrimidine derivative: RA 233.

A number of normal and neoplastic human cell lines in culture were studied by cell count and 3H thymidine incorporation for growth inhibitory effect by the pyrimido-pyrimidine derivative RA-233 (mopidamole). There was more inhibition when the drug was added to the culture in the lag phase than in the logarithmic growth phase. There was more inhibition (particularly at low doses) of the neoplastic cell lines than of the non-neoplastic cell lines.

LNCaP model of human prostatic carcinoma.

The LNCaP cell line was established from a metastatic lesion of human prostatic adenocarcinoma. The LNCaP cells grow readily in vitro (up to 8 x 10(5) cells/sq cm; doubling time, 60 hr), form clones in semisolid media, are highly resistant to human fibroblast interferon, and show an aneuploid (modal number, 76 to 91) human male karyotype with several marker chromosomes. The malignant properties of LNCaP cells are maintained. Athymic nude mice develop tumors at the injection site (volume-doubling time, 86 hr). Functional differentiation is preserved; both cultures and tumor produce acid phosphatase. High-affinity specific androgen receptors are present in the cytosol and nuclear fractions of cells in culture and in tumors. Estrogen receptors are demonstrable in the cytosol. The model is hormonally responsive. In vitro, 5 alpha-dihydrotestosterone modulates cell growth and stimulates acid phosphatase production. In vivo, the frequency of tumor development and the mean time of tumor appearance are significantly different for either sex. Male mice develop tumors earlier and at a greater frequency than do females. Hormonal manipulations show that, regardless of sex, the frequency of tumor development correlates with serum androgen levels. The rate of the tumor growth, however, is independent of the gender of hormonal status of the host.

Human pancreatic adenocarcinoma: in vitro and in vivo morphology of a new tumor line established from ascites.

A human pancreatic tumor cell line has been established from the ascites of a patient with histopathologically confirmed adenocarcinoma of the head of the pancreas and maintained for more than 12 months in the laboratory. Epithelioid tumor cell colonies, which resulted from primary tissue cultures of the ascitic cell component, were mechanically isolated by needle micromanipulation. Tumorigenicity was proven in athymic nude mice. Morphologically the pancreatic tumor epithelial cells grew to confluency with moderately tight adhesion to the culture plastic surface and with free-floating cells in the medium. Upon re-establishment of the tumoral xenograft in tissue culture, the epithelial cells retained their original morphology. Histologically the tumor grown in nude mice exhibited prototypic characteristics of the primary adenocarcinoma in the patient, producing abundant mucin and displaying a broad spectrum of glandular differentiation, which ranged from well to poorly differentiated adenocarcinomas with occasionally localized lymphocytic infiltrations. Furthermore, the tumor expressed carcinoembryonic antigen and human pancreas cancer associated antigen. This tumor line, designated AsPC-1, has been cultured for at least 10 passages in vitro and 3 in vivo. It represents a new model for human pancreatic cancer.

[Treatment of leukemias and lymphomas with interferons: III. Trial treatment of meningeal localizations of acute leukemia and non-Hodgkin's lymphomas with beta interferon administered by the intrathecal route]. / Traitement des leucémies et des lymphomes par les interférons: III. Essai de traitement des localisations méningées de la leucémie aiguë et des lymphomes non hodgkiniens par l'interféron bêta administré par voie intrathécale.

Six patients with pure meningeal relapse of acute lymphoblastic leukemia (ALL) (5 patients) or leukemic lymphosarcoma (LS) (non Hodgkin's lymphoma) (NHL) (1 patient) were treated with intrathecal (I.T.) human fibroblastic interferon (IF beta), one vial (1.3 million units) every other day or every day up to remission or failure. Tolerance was excellent in all six patients with no local or general side effects. 5 patients had no improvement of their meningeal blast infiltrations after 5 to 8 injections and were given IT chemotherapy. The sixth patient achieved a complete remission (CR) after 11 injections, and was maintained in CR for eleven months under systemic and IT chemotherapy. IF cannot be proposed as standard treatment for meningeal leukemia, but we may be able to select a population of patients in which it could be indicated. Its combination with methotrexate and arabinoside cytosine, the two agents used IT which are S-dependent, has to be studied for a possible potentiation. Moreover, its good tolerance would permit its use in severe meningeal viral diseases.