Ovarian stimulation for in vitro fertilization using pure follicle-stimulating hormone with and without gonadotropin-releasing hormone agonist in high-responder patients.

There is a distinct pattern of response to gonadotropin stimulation in some patients marked by high peak estradiol (E2) levels, multifollicular ovarian response, and elevated basal luteinizing hormone (LH)/follicle-stimulating hormone (FSH) ratios. We reviewed the stimulation profiles of five such high-responder patients who failed to conceive during in vitro fertilization with ovarian stimulation using pure FSH. All patients had baseline LH/FSH greater than 1.5 and peak E2 greater than 800 pg/ml. One cycle was canceled prior to hCG administration because of marked ovarian response (E2 greater than 2500 pg/ml, multiple small follicles). In a subsequent cycle, all patients were pretreated with the gonadotropin releasing-hormone agonist (GnRHa) leuprolide acetate for 10-14 days prior to initiation of FSH for ovarian stimulation. Leuprolide was continued until the day of hCG administration. During cycles using GnRHa, there was a statistically significant decrease (P less than 0.05) in serum FSH on day 3 (less than 5 vs 8.3 mIU/ml), serum E2 on day 3 (14.6 vs 34.6 pg/ml), and peak serum E2 (1197.6 vs 1923.0 pg/ml). Patients during cycles with GnRHa had a greater number of preovulatory (8.6 vs 3.0) and total (12.4 vs 6.0) oocytes retrieved (P less than 0.05). The fertilization rate of preovulatory oocytes was also higher during cycles using GnRHa (83 vs 64%). Two pregnancies occurred in the cycles pretreated with GnRHa. These preliminary data indicate that in high-responder patients, a combination of GnRHa and pure FSH results in lower E2 levels during the stimulation cycle and a greater number of total and mature oocytes retrieved and fertilized.

Human prostate tissue antigens defined by murine monoclonal antibodies.

BALB/c mice were hyperimmunized against a membrane preparation derived from a pool of transurethral resection specimens which included three benign prostatic hyperplasia and one prostate adenocarcinoma tissue samples. The activated lymphocytes were fused with the NS-1 mouse myeloma cell line, and supernatants from immunogen-reactive hybridomas were screened for antibody binding activity using a solid-phase radioimmunoassay against the Calu-1 human lung adenocarcinoma cell line and several membrane preparations derived from various normal human tissues. Hybridoma cultures secreting antibodies which did not appear cross-reactive were doubly cloned by limiting dilution and screened against a large panel of membrane preparations derived from normal prostate, benign prostatic hyperplasia, and prostate adenocarcinoma tissues as well as samples obtained from a variety of normal human tissues. The monoclonal antibodies were also evaluated against 24 normal, virally transformed, and malignant human cell lines. Two monoclonal antibodies were isolated which demonstrated a restricted binding activity to prostate antigens and were not widely cross-reactive with nonprostate normal tissues or cell lines. These antibodies were designated TURP-27 (IgG3, k) and TURP-73 (IgG2a, k). Both of these monoclonal antibodies were reactive against formalin-fixed, paraffin-embedded tissues in the immunoperoxidase assay and were subsequently tested against a variety of normal, hyperplastic, and malignant human tissues. These studies indicated that TURP-27 may be directed against a new prostate organ-associated marker and that TURP-73 is directed against an antigen expressed on prostate and a limited number of other tissues.

Antiprostate carcinoma monoclonal antibody (D83.21) cross reacts with a membrane antigen expressed on cytomegalovirus-transformed human fibroblasts.

Virological and epidemiological studies have implicated human cytomegalovirus (HCMV) as a possible etiological agent of prostate cancer. Because of the suspected associations, this laboratory tested the reactivity of a prostate-associated monoclonal antibody with HCMV-transformed cells. This mouse monoclonal antibody, D83.21, reacts with a membrane antigen on prostate and bladder tumor cells and does not bind to a variety of other malignant or normal cells. The results of this study indicated that the prostate-associated antibody bound to a membrane antigen on HCMV-transformed cells as detected by radioimmunoassay, immunofluorescence, and complemented-dependent cytotoxicity. This cross-reactivity appeared to be specific for HCMV-transformed cells and did not react with HCMV-infected cells or those transformed by other viruses. Antibody affinity chromatography, used to isolate the D83.21-reactive protein, revealed two peptides of 60 and 28 kd on both prostate tumor and HCMV-transformed cells. The results suggest that D83.21 reacts with a common cell surface protein expressed on HCMV-transformed cells and urogenital tumors.

Immunohistochemical localization of prostate carcinoma-associated antigens.

The immunoperoxidase technique was used to study the localization and distribution of antigens reactive with two monoclonal antibodies, D83.21 and P6.2, produced against cultured prostate tumor cells, in formalin-fixed, paraffin-embedded histological sections of human tissues. Monoclonal D83.21 reacted with 11 of 19 (58%) primary prostate carcinomas and 1 of 6 (17%) metastatic tumors, whereas monoclonal P6.2 reacted with 14 of 19 (68%) primary and 4 of 6 (67%) metastatic prostate tumors. Neither antibody reacted with five primary prostate tumors and one metastatic prostate tumor. In some tumor cells, the antigens recognized by these monoclonals were localized in either the cytoplasm or cell membrane, while in other tumor cells, both diffuse cytoplasmic and membrane or focal staining patterns were observed. In addition to the variable staining patterns, antigenic heterogeneity was also noted within most prostate tumors examined. Two types of staining variability were observed: (a) tumor cells in one area of the tissue section stained positive, but in another area they did not react with the antibody; and (b) both stained and unstained tumor cells were adjacent to each other. These results would suggest that a panel of monoclonals will be required to detect the different subpopulations of prostate tumor cells. Neither antibody reacted with 6 normal or 12 benign prostate tissues, nor any of a variety of other normal human tissues except for staining of the proximal tubules of normal kidneys. The antigen detected by P6.2 demonstrated a wider tissue distribution being found on bladder, breast, lung, and pancreatic tumors, whereas the antigen recognized by D83.21 was restricted to prostate and bladder carcinomas. These antibodies may have clinical applicability for the identification of prostate tumor cells in biopsy specimens and for immunohistopathological classification of prostate carcinomas.

Monoclonal antibodies to human prostate and bladder tumor-associated antigens.

Monoclonal antibodies to human prostate adenocarcinoma membrane antigens were produced by fusion of P3X63/Ag8 mouse myeloma cells with spleen cells from BALB/c mice immunized against the prostate cancer cell line DU145. The hybrids were screened for antibody production using glutaraldehyde-fixed cells in a solid-phase radioimmunoassay. Antibody-binding specificity was also checked by quantitative adsorption, membrane immunofluorescence, and complement-dependent cytotoxicity assays. A hybridoma clone (83.21) was isolated that secreted antibodies which preferentially bound to several prostate and bladder cancer cell lines but did not bind to a variety of other normal and malignant human cell lines. This antibody also reacted with a cytomegalovirus-transformed human embryonic lung cell line but not to normal human embryonic lung cells. Quantitative adsorption studies demonstrated that the 83.21 monoclonal antibody was strongly reactive to membrane preparations from human prostate adenocarcinoma tissue and a liver metastasis of prostate carcinoma. Little or no binding activity was observed against two other prostate carcinomas, bening prostatic hyperplasia, normal prostate, or normal liver. Binding studies indicate that the 83.21 monoclonal antibody does not bind to alpha-fetoprotein, carcinoembryonic antigen, prostatic acid phosphatase, human leukocyte antigen, beta 2-microglobulin, HLA-Dr antigens, fibronectin, or prostate antigen. The data indicate that we have isolated a monoclonal antibody that binds to an antigen(s) expressed by several urogenital carcinoma cell lines as well as human prostate tumor tissue and that the antibody is not directed against well-known human tumor cell markers.

Radioimmunoassay versus counterimmune electrophoresis for measurement of serum prostatic acid phosphatase.

Radioimmunoassay (RIA) kits obtained from commercial sources were evaluated and compared with a standard counterimmunoelectrophoretic (CIE) assay for the measurement of prostatic acid phosphatase (PAP) in serum. None of the radioimmunoassays was found to be more sensitive than the CIE assay in detecting elevated serum PAP. Both immunoassays were somewhat more effective clinically in measuring prostatic specific acid phosphatase than an enzyme colorimetric assay. The results obtained by CIE agreed with the results obtained by RIA in 96 per cent of the tests. The number of positive results in patients with confirmed prostate adenocarcinoma increased with disease progression. The low number of positive tests in localized adenocarcinoma (Stages A and B) suggests that neither the CIE nor any RIA procedure is useful for screening unselected populations for adenocarcinoma of the prostate.

Bone marrow and acid phosphatase by counterimmune electrophoresis: pre-treatment and post-treatment correlations.

The value of bone marrow acid phosphatase in the staging of prostatic cancer has been a controversial issue. A number of investigators have concluded that the enzymatic determinations of bone marrow acid phosphatase are inaccurate because of lack of specificity. The introduction of the immune methods for measuring acid phosphatase has revived interest in the role of immune bone marrow acid phosphatase in pre-treatment staging. Fifty-five patients underwent determination of simultaneous immune bone marrow and serum acid phosphatase before any treatment. While positive values did predict a risk for initial and subsequent metastasis they could not be used to dictate against definitive therapy. Positive bone marrow values were paralleled by positive serum values and provided no additional staging information.

Comparison of countercurrent immunoelectrophoretic assay with commercial radioimmunoassay kits for measuring prostatic acid phosphatase.

We evaluated and compared five commercial radioimmunoassay kits with a standard counter-immunoelectrophoretic assay for the measurement of prostatic acid phosphatase in serum. Four of the five radioimmunoassays performed as described by the supplier with respect to sensitivity, stability, precision, linearity, analytical recovery, and expected values for the normal male population. None of the radioimmunoassays was more clinically sensitive then the counter-immunoelectrophoretic assay for detecting increased prostatic acid phosphatase in serum. Results obtained by counter-immunoelectrophoretic assay agreed with results obtained by radioimmunoassay in 96% of the tests. The proportion of positive results in patients with confirmed prostatic adenocarcinoma increased with disease progression. The fewer positive tests in localized adenocarcinoma (Stages A and B) suggests that neither the counter-immunoelectrophoretic assay nor the radioimmunoassay procedures are useful for screening unselected populations for adenocarcinoma of the prostate. The high percentage of normal values found in those patients clinically free of disease after treatment is encouraging and supports the use of the prostatic acid phosphatase immunoassays in prospectively monitoring the treatment of prostatic cancer patients.

Leukocyte migration inhibition assay in patients with prostate adenocarcinoma.

The leukocyte migration inhibition (LMI) assay was used to determine the cell-mediated immune reactivity of prostate cancer patients to putative tumor antigens present in potassium chloride extracts of surgically removed prostate tumor tissue. Using an extract prepared from prostate tumor tissue, inhibition of leukocyte migration was found more frequently in prostate tumor patients (61%) than in patients with benign prostate hyperplasia (37%), patients with nonprostate cancers (26%), or normal donors (10%). Control extracts prepared from normal prostate tissue, benign prostate hyperplasia tissue, and unrelated tumor tissue were statistically less reactive in the LMI assay than the prostate tumor extract when reacted against leukocytes from prostate tumor patients. These results suggest that the LMI assay might be potentially useful for measuring the tumor-directed, cell-mediated immune responses in patients with prostate cancer.