Characterization of the BRCA1-like immunoreactivity of human seminal plasma.

OBJECTIVES: The subcellular localization of the breast cancer susceptibility gene product BRCA1 has been controversial. Discrepant results have been reported during the past 3 years, partially because of the unavailability of highly specific reagents for BRCA1 protein. Our objective was to characterize the BRCA1-like immunoreactivity that is detected in human seminal plasma by using monoclonal and polyclonal antibodies that are supposedly specific for BRCA1 protein. METHODS: We used immunologic, chromatographic, and protein sequencing techniques to detect the immunoreactivity of BRCA1 in seminal plasma and to purify and partially identify the immunoreactive species. RESULTS: We present data indicating that two BRCA1 antibodies, SG-11 and D-20, which were thought to be free of cross-reactivities, strongly interact with proteins present in human seminal plasma. This cross-reactivity is detectable even at seminal plasma dilutions as high as 10(6)-fold, and it is effectively blocked by peptides that capture the binding site of either SG-11 or D-20 antibodies. Purification and characterization of the immunoreactive compound revealed that this consists of a macromolecular complex that contains semenogelins. The D-20 polyclonal antibody was found to cross-react with purified semenogelins I and II; the SG-11 monoclonal antibody appeared to recognize a component of the macromolecular complex that was not semenogelin. CONCLUSIONS: Our data demonstrate that the BRCA1 antibodies SG-11 and D-20 strongly interact with seminal plasma proteins and are not highly specific for BRCA1 protein. It is thus suggested that BRCA1 antibodies should be used with caution until reagents free of interference are developed and evaluated. In light of the very high cross-reactivity of the two antibodies with seminal plasma proteins, we recommend that new BRCA1 antibodies should be examined for cross-reactivity with seminal plasma proteins to verify specificity.

Maternal serum prostate-specific antigen and Down syndrome in the first and second trimesters of pregnancy. International Prenatal Screening Research Group.

It has been suggested that high levels of maternal serum prostate-specific (PSA) may be associated with fetal Down syndrome. We retrieved stored blood samples from 102 singleton Down syndrome pregnancies at 8-14 weeks' gestation and 99 at 15-22 weeks' gestation, together with samples from five unaffected singleton control pregnancies matched for gestational age. PSA was measured using an ultrasensitive assay. Contrary to earlier reports, PSA levels were similar in affected and unaffected pregnancies in both the first and second trimester of pregnancy; 1.1 and 0.9 multiple of the normal median, respectively, in affected pregnancies. There was no indication that PSA would be a useful screening marker.

Development of an ultrasensitive immunoassay for human glandular kallikrein with no cross-reactivity from prostate-specific antigen.

BACKGROUND: Studies demonstrating that human glandular kallikrein (hK2) is increased in prostate cancer patients have prompted speculation that this marker may of use in addition to prostate-specific antigen (PSA). METHODS: An ultrasensitive hK2 sandwich immunoassay was developed, and its detection limit, cross-reactivity, analytical recovery, precision, and linearity of dilution were evaluated. hK2 was measured in seminal plasma and sera from healthy males, females, and prostatectomized patients. RESULTS: Our assay has an excellent detection limit (6 ng/L) and precision (>90%). Recovery studies indicated that hK2 binds to serum protease inhibitors. All sera from healthy males had measurable hK2 concentrations (median, 402 ng/L). Almost all female sera had undetectable hK2. Serum hK2 and PSA in males correlated positively (r = 0.44), but hK2 was present at concentrations approximately 2. 5-fold lower than PSA. The PSA/hK2 ratio in male sera was 0.1-34, with a median of 2.6. In seminal plasma, this ratio was 100-500. More than 94% of immunoreactive hK2 in serum was in the free form ( approximately 30 kDa); traces of hK2 complexed to alpha1-antichymotrypsin were present. CONCLUSIONS: The limit of detection of the method for hK2 measurement described here ( approximately 20-fold lower than any other reported assay for hK2) allows the generation of new clinical information. When combined with a previously described method for PSA measurement that has no cross-reactivity from hK2, this methods allows the relative proportions of hK2 and PSA in biological fluids to be measured.

Prognostic value of plasma prostate specific antigen after megestrol acetate treatment in patients with metastatic breast carcinoma.

BACKGROUND: Prostate specific antigen (PSA) is an established tumor marker of prostate adenocarcinoma that recently also was found in breast tumors. Minute amounts of PSA are found in female plasma. It is known from cell culture studies that PSA expression can be up-regulated by androgens and progestins but not estrogens. In this study, the authors examined whether plasma PSA in women with breast carcinoma changes after the therapeutic administration of the progestin megestrol acetate (MA) and whether these changes have any prognostic value. METHODS: Plasma PSA was measured by a highly sensitive immunofluorometric procedure that can measure within 1 ng/L of PSA. Serial plasma levels from women with metastatic breast carcinoma who received either MA (N = 52) or other treatments (N = 24) were evaluated. PSA changes in plasma were correlated with patient outcomes. RESULTS: The study found that approximately 50% of the patients receiving MA had a significant increase in their plasma PSA concentration after the treatment and that this increase was rapid (starting within 1 week) and dose-dependent. PSA levels declined when treatment was withdrawn. Further comparisons with similar groups of patients receiving tamoxifen or doxorubicin have shown that the plasma PSA increases are specific to the MA treatment. The plasma PSA increases reflect the stimulation of the tumor by MA to produce PSA and the secretion of PSA into the general circulation. There is a statistically significant association between the plasma PSA changes after MA treatment and overall patient survival; patients with increased plasma PSA have an approximate threefold increase in their relative risk of death during the follow-up period. Multivariate analysis has shown that the increased risk of death in this group is associated, at least in part, with the frequent presence of distant metastasis. CONCLUSIONS: The measurement of plasma PSA after treatment with MA allows for patient classification into two groups. The group that did not demonstrate any changes in their plasma PSA level after MA treatment (approximately 50% of the patients) had a significantly better prognosis. The group that did demonstrate an increase in their plasma PSA level after MA treatment represented a subset of patients who may benefit more from MA withdrawal and the initiation of alternative regimens. However, these data need further confirmation with a larger pool of patients.

Quantification of pepsinogen C and prostaglandin D synthase in breast cyst fluid and their potential utility for cyst type classification.

OBJECTIVE: To quantify pepsinogen C (PEPC) and prostaglandin D synthase (PGDS) in breast cyst fluid and examine if these two parameters can be used for breast cyst type classification. DESIGN AND METHODS: We quantified PEPC and PGDS in 92 and 50 breast cyst fluids, respectively, using previously established immunofluorometric procedures. We then examined if the levels of PEPC or PGDS correlate with the type of cyst or with other clinicopathological variables. RESULTS: Quantitative analysis of the breast cyst fluids indicated that PEPC is present in all cyst fluids at various concentrations ranging from 3 to 31,000 ng/mL. PGDS positivity was confined to 30% of the cyst fluids. PEPC and PGDS levels were correlated with the breast cyst fluid cation ratio and were associated with the type of the cyst. Increased PEPC levels in breast cyst fluids were significantly correlated with a > or = 1.5 K+/Na+ ratio and were associated with the secretory/apocrine type of cyst (Type I) (p = 0.011). Immunoreactive PGDS levels were highly correlated with a low cation ratio and were associated with the transudative/flattened type of breast cyst (Type II) (p = 0.0003). A weak association was observed between PEPC levels in breast cyst fluid and menopausal status (p = 0.093). No significant associations were observed for either PEPC or PGDS concentration in breast cyst fluid and number of cysts, recurrence of the disease, family history of breast cancer, number of children, abortion, and breast feeding. CONCLUSIONS: Quantification of PEPC and PGDS in breast cyst fluid may be useful in the subclassification of cyst type in patients with gross cystic disease.

Seminal plasma biochemical markers and their association with semen analysis findings.

OBJECTIVES: To examine the clinical value of six seminal plasma components in the evaluation of sperm quality and in the differential diagnosis of men with infertility. METHODS: We analyzed 202 seminal plasmas for prostate-specific antigen, glucose, pepsinogen C, insulin-like growth factor binding protein-3, prostaglandin D synthase (PGDS), and BRCA1-like immunoreactive protein (BRCA1-LIP) using quantitative immunofluorometric procedures. The semen donors were categorized in four clinical groups: normal, oligospermic, azoospermic, and vasectomy patients. We then evaluated whether any of these biochemical markers were associated with other parameters of sperm quality, including patient age, total cell concentration, percentage of motility, and percentage of normal morphology. RESULTS: We found that only PGDS concentration was significantly associated with other parameters of sperm quality. PGDS concentration correlated positively with total cell concentration (r = 0.55), percentage of motility (r = 0.31), and percentage of normal morphology (r = 0.31). Median PGDS concentration in seminal plasma decreased progressively from normal to oligospermic to azoospermic to vasectomy patients (P <0.001). There was no overlap between seminal plasma PGDS concentration of normal subjects versus vasectomy patients. The only other parameter that was moderately decreased in vasectomy patients was BRCA1-LIP. The source of PGDS in seminal plasma was determined with various techniques, including immunohistochemistry. This protein is produced and secreted by the Sertoli cells. CONCLUSIONS: Our findings suggest that PGDS concentration in seminal plasma correlates with other known indicators of semen quality and is a new marker of post-testicular obstruction. This biochemical parameter could be used to aid in the differential diagnosis of obstructive and nonobstructive azoospermia in men with infertility.

Highly elevated levels of prostaglandin D synthase in the serum of patients with renal failure.

OBJECTIVES: To investigate whether prostaglandin D (PGD) synthase levels differ in the serum of patients with or without renal dysfunction. PGD synthase or beta-trace protein is a major constituent (approximately 3% of total protein) of human cerebrospinal fluid (CSF). We previously reported that PGD synthase levels in serum are approximately 40- to 60-fold lower than those in CSF. METHODS: We measured the PGD synthase concentration in various sera with a highly sensitive and specific immunofluorometric assay along with the serum creatinine level. Analysis for PGD synthase and creatinine was performed in 30 sera from non-renal failure subjects, in 7 sera from patients treated with continuous ambulatory peritoneal dialysis, and in 34 sera that were before and after hemodialysis samples from 17 patients with renal failure. RESULTS: Elevated creatinine concentration was observed in patients with renal insufficiency, as expected (Mann-Whitney P < 0.0001; chi-square P < 0.0001 ). We found that serum PGD synthase concentration from patients with renal failure is significantly elevated compared with the serum PGD synthase concentration from non-renal failure subjects (Mann-Whitney P < 0.0001; chi-square P < 0.0001). Approximately a 35-fold increase of serum PGD synthase is observed for patients with renal failure compared with non-renal failure subjects. Serum PGD synthase concentration is not affected by hemodialysis in acute renal failure patients (Mann-Whitney P = 0.918), unlike serum creatinine levels, which were decreased significantly after hemodialysis (Mann-Whitney P = 0.0001). CONCLUSIONS: We conclude that renal impairment is highly associated with elevated serum PGD synthase levels. Measurement of PGD synthase in serum is a new biochemical marker of renal insufficiency.

Prostate specific antigen production by breast tumors after induction with oral contraceptives.

PIP: This study examined the levels of prostate specific antigen (PSA) in breast tumor tissue of patients who received progestin-containing oral contraceptives (OCs) 1 week prior to surgery. About 20 patients with primary breast cancer were enrolled in the study: 10 were not given any treatment before surgery and served as control subjects and 10 received OCs. Tumor tissue was excised during surgery and stored until extraction of PSA before biochemical analysis was performed. Post-surgical serum was obtained after 2 days. Tumor extract PSA levels were 80 ng/l in all samples tested. The exception was one patient who¿s tumor PSA level reached 1934 ng/l; this patient received OCs and had a serum PSA level of 26 ng/l, which decreased to 2 ng/l after tumor removal and OC discontinuation. Overall findings suggest that PSA levels are relatively low in all breast tumor extracts receptive of steroid hormone status, tumor stage or grade, lymph node status, patient age, or menopausal status.^ieng

Prostate-specific antigen induction by a steroid hormone in T47D cells growing in SCID mice.

Previous studies revealed that prostate-specific antigen (PSA) is present in > 30% of human breast tumor cytosols. Survival analysis showed that patients with PSA-producing tumors have a reduced risk for relapse, suggesting PSA to be an independent favorable prognostic marker for a large subset of breast cancer patients. The present investigation established an in vivo model for the induction of PSA in human breast cancer tumors growing as xenografts in severe combined immunodeficient (SCID) mice. The human mammary cancer cell-line T47D was grown i.m. in female mice. When the tumor and leg diameter reached 10 mm, the mice were stimulated daily with norgestrel for either 5 or 7 days to produce PSA, and sacrificed on day 8. The prostate cancer cell-line LNCaP was grown in male mice and functioned as a positive control for PSA production. After T47D and LNCaP mice were sacrificed, a highly sensitive immunofluorometric assay was used to analyze the PSA concentration in the tumor, muscle, liver, and kidney cytosols. Norgestrel-stimulated T47D mice showed significantly more PSA in the tumors compared to tumors of the control mice. However, PSA levels in tumors of the stimulated mice were significantly lower than those in the LNCaP xenografts. No PSA levels above background were present in the blood and normal tissue of the norgestrel-stimulated or control T47D xenografts. This mouse model will be a valuable tool for investigating and screening new therapies for a subgroup of breast cancer patients who have significant PSA concentrations in their tumors.

Increased concentrations of prostate-specific antigen in maternal serum from pregnancies affected by fetal Down syndrome.

Down syndrome is one of the most common causes of mental retardation in the industrialized world. Prenatal serum screening to identify mothers at risk of carrying a fetus affected with Down syndrome is presently part of routine obstetrical care. Prostate-specific antigen (PSA) concentration was measured in stored second-trimester maternal serum samples from 19 pregnancies affected with fetal Down syndrome and in 95 samples from unaffected pregnancies, with each case matched to five controls for gestational age and duration of frozen sample storage. Concentrations of PSA in Down syndrome pregnancy were significantly higher (case median = 2.28 multiples of the median; P = 0.02) than in unaffected pregnancy. PSA concentrations were not significantly correlated with the current serum screening analytes, alpha-fetoprotein, unconjugated estriol, or human chorionic gonadotropin in either cases or controls. The increased maternal serum PSA concentrations in Down syndrome pregnancy and their relative independence from other markers suggest the possible utility of PSA as a prenatal screening marker for fetal Down syndrome.

Immunofluorometric assay of pepsinogen C and preliminary clinical applications.

We developed mouse monoclonal antibodies (Abs) against pepsinogen C with highly purified antigen isolated from gastric mucosa. The Abs were used to construct a two-site sandwich-type assay for pepsinogen C with time-resolved fluorometry as a detection technique. The assay has a detection limit of 0.1 microgram/L and is precise (within-run and day-to-day CVs < 11%). We used this assay to measure pepsinogen C in seminal plasma, breast cyst fluid, amniotic fluid, male and female serum, serum from patients with prostate cancer, urine, breast tumor cytosolic extracts, breast milk, and cerebrospinal fluid. Highest pepsinogen C concentrations were in seminal plasma, followed by breast cyst fluid and amniotic fluid. We found no correlation between prostate-specific antigen concentrations and concentrations of pepsinogen C in serum of prostate cancer patients, and concluded that this marker is not useful for either diagnosing or monitoring prostatic carcinoma. The availability of a highly sensitive, reliable, and convenient method for quantifying pepsinogen C will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including benign breast diseases, breast cancer, fertility, and pregnancy.

BRCA1 tumor suppressor gene product shares immunoreactive epitopes with a protein present in seminal plasma.

OBJECTIVES: To develop immunofluorometric procedures for measuring BRCA1 protein in various biological fluids and tissue extracts. DESIGN AND METHODS: Five commercially available monoclonal and polyclonal antibodies against BRCA1 were evaluated for developing competitive and non-competitive immunofluorometric procedures for BRCA1. Biotinylated and nonbiotinylated peptides were used to assess the specificity of the antibodies for blocking experiments and for the competitive immunoassay. Extensive studies to exclude cross-reactivity and non-specific effects in the non-competitive immunoassay were undertaken. Seminal plasmas as well as breast tumor extracts, amniotic fluids and cerebrospinal fluids were analyzed. RESULTS: We designed novel methods for measuring BRCA1 immunoreactivity. One configuration based on the "sandwich-type" immunoassay principle was used for further studies. We discovered that seminal plasma contains an immunoreactive protein which appears to possess the D-20 (aminoterminal) and C-20 (carboxyterminal) epitopes of BRCA1. Molecular weight identification using gel filtration chromatography has shown that the immunoreactive species has a molecular weight between 660 and 160 KDa. CONCLUSIONS: We identified for the first time a protein in seminal plasma that shares immunoreactive epitopes with the BRCA1 tumor suppressor protein. We are currently purifying this protein in order to examine if it is homologous or identical to BRCA1.

Prostaglandin D synthase concentration in cerebrospinal fluid and serum of patients with neurological disorders.

Prostaglandin D synthase (PGD synthase) or beta-trace protein is a major constituent of human cerebrospinal fluid (CSF) representing-3% of the total CSF protein. We have recently developed a highly specific immunofluorometric assay for PGD synthase, which enabled us to quantify the presence of PGD synthase in fluids and tissues not associated with the CNS. In this report we provide quantitative data of the presence of PGD synthase in CSF and serum from 302 subjects with various neurological diseases and symptoms. PGD synthase levels in CSF are approximately 35-fold higher than those of serum, with a median concentration of 11,299 micrograms/L. A statistically significant association of PGD synthase concentration in CSF was observed with both patient age and gender. There was no correlation between PGD synthase concentration in serum and patient age or gender. To evaluate the clinical utility of PGD synthase in diagnosing neurological diseases, the distribution pattern of PGD synthase in CSF and serum was examined for each neuropathology of 268 patients whose diagnosis was known. No statistical difference was observed between PGD synthase concentration in the CSF (129 cases) or the serum (94 cases) of multiple sclerosis afflicted subjects in comparison to all other patients studied. The distribution pattern was also not different for PGD synthase levels in CSF of patients with HIV/AIDS related neuropathies, viral meningitis and fibromyalgia. We conclude that PGD synthase measurement presents no clinical utility in diagnosing neurological disorders in adulthood. PGD synthase may have a physiological and/or pathological role in the developing brain and in neurodegenerative diseases.

Prostate-specific antigen in female serum, a potential new marker of androgen excess.

Prostate-specific antigen (PSA) is present at very low concentrations in female serum, but it can now be measured with highly sensitive immunoassays. We have found that in female tissues the PSA gene is regulated by steroid hormones through the action of steroid hormone receptors. Thus, we examined whether female serum PSA is associated with hyperandrogenic states. Serum PSA levels were compared between 22 hirsute women with a Ferriman-Gallwey score higher than 8 and 50 women without hirsutism. The results show that PSA levels were higher in hirsute women in comparison with controls. In hirsute women, levels of PSA and 3 alpha-androstanediol glucuronide (3 alpha-AG), a specific metabolite of androgen action, showed a significant positive correlation, whereas PSA and 3 alpha-AG showed a significant negative correlation with patient age. Receiver operating characteristic (ROC) analysis revealed that 3 alpha-AG was a slightly better marker of androgen excess than PSA. We conclude that female serum PSA may be a new biochemical marker of androgen action in females.

Molecular forms of prostate-specific antigen in the serum of women with benign and malignant breast diseases.

Using a highly sensitive immunofluorometric procedure, we measured the total prostate-specific antigen (PSA) concentration in 632 sera obtained from female blood donors and women with idiopathic hirsutism, breast cancer or benign breast diseases. A total of 50 sera with total PSA > 15 ng l(-1) were fractionated by high-performance liquid chromatography (HPLC) in order to resolve the two immunoreactive molecular forms, i.e. free PSA (approximately 30 kDa) and PSA bound to alpha1-antichymotrypsin (PSA-ACT, 100 kDa). We found that breast cancer patients have presurgical serum total PSA levels similar to those of blood donors. Total serum PSA concentration decreases with age in women with idiopathic hirsutism, in cancer patients and in patients with benign breast diseases. The major molecular form of PSA in the serum of all normal and hirsute women (n = 15) is PSA bound to the proteinase inhibitor alpha1-antichymotrypsin. The major molecular form in 44% of presurgical cancer patient sera is free PSA. A total of 58% of benign breast disease patients also have in their serum mainly free PSA. We conclude that about half the patients with breast cancer or benign breast diseases have free PSA as the major molecular form in their serum, whereas patients without breast pathologies (normal blood donors, idiopathic hirsutism) have PSA bound to alpha1-antichymotrypsin as the major molecular form. The ratio of PSA/PSA-ACT may have value as a simple biochemical test for diagnosis of breast pathologies including breast cancer.

Prostate-specific antigen in amniotic fluid of normal and abnormal pregnancies.

OBJECTIVE: To examine if prostate-specific antigen (PSA) is present in amniotic fluid or maternal serum during pregnancy and if its presence is associated with fetal abnormalities. METHODS: Samples tested included amniotic fluids from 853 pregnant women for whom amniocentesis was performed; 312 nonpregnant women who donated blood; 259 pregnant women who donated blood at various gestational ages. Amniotic fluid or serum PSA was measured with an ultrasensitive time-resolved immunofluorometric procedure. 372 pregnancies were studied for the presence of genotypic or phenotypic fetal abnormalities. RESULTS: PSA was present in most amniotic fluids; the median PSA concentration increased from gestational week 11 to 22 and stabilized thereafter until delivery. The most prominent PSA concentration change occurred during gestational weeks 13-14. Pregnant women had significantly higher serum PSA concentrations than nonpregnant women; the pattern of serum PSA concentration change during pregnancy was similar to that of amniotic fluid; however, serum PSA concentrations were lower by a factor of 20-40. No association existed between amniotic fluid PSA and maternal age, gender of fetus, or length of abstinence of mother from sexual intercourse. After gestational week 15, fetuses with trisomy 21 or 18, anencephaly, or renal disorders were associated with low amniotic fluid PSA levels. CONCLUSION: Our data suggest that PSA may play a role in fetal development, especially at gestational ages between 13-20 weeks. The diagnostic usefulness of PSA in identifying fetal abnormalities remains to be determined.

Immunofluorometric assay of prostaglandin D synthase in human tissue extracts and fluids.

A two-site sandwich-type assay for human prostaglandin D (PGD) synthase (beta-trace) was developed with two monoclonal antibodies and using time-resolved fluorometry as the detection technique. The assay is precise (CVs < 10%), accurate, and highly specific for PGD synthase and has a detection limit of 0.05 microgram/L. Using this assay, we measured PGD synthase concentrations in serum, urine, amniotic fluid, cerebrospinal fluid (CSF), seminal plasma, breast cyst fluid, breast discharge fluid, breast milk, and breast tumor extracts. The highest concentrations were found in CSF. We identified proteolytic degradation of PGD synthase in amniotic fluid. Fetal tissues contained various amounts of the enzyme, with the highest values being found in brain and heart. In placental extracts, PGD synthase content was greatest at 11-28 weeks of gestation-in accordance with the concentrations measured in amniotic fluids for this gestational period. We conclude that PGD synthase is ubiquitous and is present in many fluids and tissues of adults and fetuses. This first quantitative and sensitive assay of PGD synthase should facilitate expansion of knowledge on this enzyme and possibly will have applications for diagnosis and monitoring of human diseases.

Prostaglandin D2 synthase: a component of human amniotic fluid and its association with fetal abnormalities.

Prostaglandin D2 (PGD2) synthase is responsible for PGD2 production in the brain. Western blot analysis of human amniotic fluid and probing with a polyclonal antibody against prostate-specific antigen (PSA) revealed a strong immunoreactive band with a molecular mass of 25 kDa. The immunoreactive species, which does not react with monoclonal anti-PSA antibodies, was purified to homogeneity from 1 L of amniotic fluid through successive cycles of gel filtration and ion-exchange chromatography. Amino acid sequence analysis (15 cycles) revealed that the protein was highly homologous or identical to PGD2 synthase. On semiquantitative analysis, PGD2 synthase concentration appears to increase dramatically during gestational weeks 12-25 and then declines slowly until term. PGD2 synthase concentration in amniotic fluid was altered in many abnormal pregnancies, most notably its decrease in trisomic fetuses and fetuses with renal abnormalities.