Expression of human prostate-specific glandular kallikrein protein (hK2) in the breast cancer cell line T47-D.

Human glandular kallikrein (hK2) protein, like prostate-specific antigen (PSA), is produced mainly in prostatic epithelium. It may be useful as a new diagnostic indicator for prostate cancer. Recently, a number of hK2-specific monoclonal antibodies have been developed that enable us to detect hK2 protein in human prostate tissue, seminal fluid, and sera. Whether hK2 can be expressed, like PSA, in nonprostatic cells is not known. In this study, we have characterized the presence of hK2 in an androgen-responsive breast cancer cell line T47-D at both the protein and mRNA levels with an immunoassay, Western blot analysis, Northern blot analysis, and the reverse transcription-PCR. Using a sensitive immunoassay with monoclonal antibodies to hK2, we found that T47-D cells could be induced with androgens, mineralocorticoids, glucocorticoids, and progestins to produce significantly more hK2 than PSA. Estrogens failed to mimic the effect of the other steroids, blocking instead the stimulatory effect of androgens. Androgen induction of hK2 in T47-D cells was dose dependent. More interestingly, we found that the hK2 in androgen-induced T47-D cell spent media appears to be the pro-form of hK2 rather than mature hK2. Our study demonstrates that hK2, a serine protease thought to be found only in prostate-related tissues and fluids, is also produced in a breast cancer cell line T47-D after steroid stimulation. This finding suggests that hK2 may have a potential role in breast cancer as well as prostatic cancer and will be the impetus for further studies of hK2 distribution and function.

Association of fibrinogen with quantity of coronary artery calcification measured by electron beam computed tomography.

Increased plasma fibrinogen concentration is an independent risk factor for cardiovascular disease. Fibrinogen is the main coagulation protein in plasma, a determinant of blood viscosity, and can act as a cofactor for platelet aggregation. In this study of middle-aged men and women, we examined the association between plasma fibrinogen concentration and coronary artery calcification (CAC), a marker of preclinical coronary atherosclerosis. Two hundred twenty-eight participants were selected from the community-based Epidemiology of Coronary Artery Calcification Study, in which CAC was measured noninvasively by electron beam computed tomography. One hundred fourteen participants (57 men) were selected because they had high quantities of CAC; the remaining 114 participants (57 men) were selected because they had no detectable CAC. Logistic regression models were used to investigate the association between plasma fibrinogen concentration and high quantity of CAC. In men, an increase of 1 standard deviation in fibrinogen concentration was associated with a statistically significant odds ratio of 1.6 (95% CI 1.1 to 2.5) for a high quantity of CAC. In women, the corresponding odds ratio was 2.5 (95% CI 1.6 to 4.1). Inferences from sex-specific bivariate logistic models for odds ratios adjusted individually for each coronary risk factor and C-reactive protein were similar to those from the univariate models. In women, there was also a significant interaction between fibrinogen concentration and age. According to the models, younger women with high plasma fibrinogen were more likely to have high quantities of CAC than were younger women with low plasma fibrinogen. The strength of this association was diminished in older women.

Significance of neuron-specific enolase levels before and during therapy for small cell lung cancer.

The level of serum neuron-specific enolase (NSE) has been implicated as a prognostic factor for patients with small cell lung cancer (SCLC). A prospective evaluation was undertaken to assess the prognostic significance of pretreatment NSE and treatment-induced minimum NSE values in patients with SCLC. Patients from two Phase III North Central Cancer Treatment Group trials [one for patients with extensive stage SCLC and one for patients with limited stage SCLC] were asked to enter this laboratory correlational trial. Both trials included treatment with four to six cycles of etoposide and cisplatin, and 121 patients (71 extensive stage SCLC and 50 limited stage SCLC) were entered into the present study of NSE. Pretreatment NSE values and treatment-induced minimum NSE values were independent predictors of time to progression and survival in multivariate analysis. Hazard rate modeling allowed the formulation of specific relationships of NSE to time to progression and survival. Pretreatment NSE levels inversely correlated with time to progression and survival in these patients with SCLC. Pretreatment NSE accounted for 28% of the variance in survival. Both pretreatment NSE and treatment-induced minimum NSE were independent prognostic predictors of time to progression and survival.

Colorectal cancer detection in the practice setting. Impact of fecal blood testing.

To assess the contribution of fecal blood testing to cancer detection in a clinical practice setting, we studied records from 160 patients with both a new tissue diagnosis of colorectal adenocarcinoma and a preceding stool blood test (HemoQuant, Mayo Medical Laboratories, Rochester, Minn) determination. In this group, 71% had suggestive colorectal symptoms (particularly stool changes, overt bleeding, and abdominal pain) or anemia at presentation, and 29% were asymptomatic. Fecal blood levels remained normal in more than 40% of both symptomatic and asymptomatic patients. In only 26 patients (16% overall) was an abnormal fecal blood level the sole heralding feature, but this subset of patients had a more favorable stage. Fecal blood levels were higher with advanced, larger, and more proximal tumors and with stools collected before purgation. We conclude that, in the practice setting, fecal blood level elevation alone is an uncommon but important manner of colorectal cancer presentation, most cancers present with symptoms, and fecal blood levels are often normal in both symptomatic and asymptomatic patients.

Predictive properties of serum-prostate-specific antigen testing in a community-based setting.

BACKGROUND: Most studies that have described the sensitivity and specificity of prostate-specific antigen (PSA) as a screening test have been conducted in urology practice settings or in media-based screening programs. The control patients from these settings may have a higher prevalence of urologic disorders that increase serum PSA levels than that of the general population in which screening efforts might take place, leading to biased estimates of sensitivity and specificity. OBJECTIVE: To determine the sensitivity and specificity of serum PSA levels for the early detection of prostate cancer in a population-based setting. PATIENTS AND METHODS: This population-based case-control study was conducted in Olmsted County, Minnesota, where the Rochester Epidemiology Project could identify all incident cases of prostate cancer through passive surveillance of medical care provided to local residents. Case patients were all 177 men (age range, 50-79 years) who were newly diagnosed as having prostate cancer from 1990 through 1992 and had a prediagnostic serum PSA determination (90% of all incident cases). Control patients were randomly selected from the Olmsted County population and had undergone a clinical examination to exclude prostate cancer. RESULTS: The median (25th and 75th percentiles) of serum PSA levels was 9.4 ng/mL (5.4 and 18.6 ng/mL, respectively) for case patients and 1.2 ng/mL (0.7 and 2.1 ng/mL, respectively) for control patients (P < .001). When sensitivity was plotted against 1-specificity, the area under the receiver operating characteristic curve was 0.94 (SE, 0.01). The predictive power declined somewhat with age, with areas under the curve of 0.96, 0.94, and 0.90 for men in their 50s, 60s, and 70s, respectively. When cases were restricted to the 155 men with clinically localized disease, the area under the curve was essentially unchanged (0.94; SE, 0.01) and still much greater than the estimates of 0.75 that were reported from urology practice- and media-based settings. CONCLUSIONS: In a community-based setting, serum PSA levels provide better discrimination between men with and without clinically localized prostate cancer than has been observed in studies that were conducted in urologic practices. These results suggest that previous decision analyses may have underestimated the predictive value of PSA for the detection of prostate cancer in a primary care or community-wide screening program.

Effects of changing diagnostic criteria on the risk of developing diabetes.

OBJECTIVE: The American Diabetes Association (ADA) has recommended that the fasting plasma glucose (FPG) level used to diagnose diabetes be changed from 7.8 mmol/l (the level recommended by the National Diabetes Data Group [NDDG] in 1979) to 7.0 mmol/l. We examined the impact of this change on rates of progression to overt diabetes from different levels of FPG. RESEARCH DESIGN AND METHODS: Using the laboratory database of Mayo Clinic, we assembled a cohort of 8,098 nondiabetic Olmsted County residents 40 years of age or older on 1 July 1983. Subjects were followed for a median of 9 years. RESULTS: Among 7,567 individuals with follow-up FPG data, 778 (10.3%) progressed to ADA diabetes and 513 (6.8%; P < 0.0001) progressed to NDDG diabetes. The risk of developing ADA diabetes was 7, 19, and 39% for individuals with initial FPG values in the ranges of <5.6, 5.6-6.0, and 6.1-6.9 mmol/l, respectively. For progression to NDDG diabetes, the respective risks were 3, 11, and 25%. A clear gradient of risk was observed within the "normal" range of FPG (<5.6 mmol/l). Among the 793 individuals who developed ADA diabetes, 222 (29%) developed NDDG diabetes simultaneously and 291 (37%) developed NDDG diabetes later. In all FPG subgroups, progression to ADA diabetes occurred approximately 7 years sooner than progression to NDDG diabetes. CONCLUSIONS: The baseline level of FPG is a major predictor of an individual's risk of developing diabetes. The proposed change in the diagnostic criteria for diabetes will lead to earlier diagnosis among individuals who are destined to develop the disease.

Calcium regulation of parathyroid and C cell function in familial benign hypercalcemia.

The roles of parathyroid hormone (PTH) and calcitonin (CT) in the pathogenesis of familial benign hypercalcemia (FBH, or hypocalciuric hypercalcemia) are uncertain. Thus we performed studies in 26 patients with FBH, 12 patients with primary hyperparathyroidism (HPT), and 20 normal volunteers, to answer these questions: are plasma levels of intact or biologically active PTH frequently elevated in FBH? Is plasma intact PTH nonsuppressible during calcium infusion? Is there blunting of the C cell CT response to calcium infusion as occurs in primary HPT? We used three methods for measurement of PTH: a mid region-specific radioimmunoassay (iPTH, antiserum GP-1M), an extraction-concentration bioassay (bioPTH, stimulation of cAMP generation in osteoblastlike cells), and a two-site immunoradiometric assay (IRMA) for intact PTH. PTH levels were significantly elevated in primary HPT by all three methods, but mean PTH was normal in FBH and 85-92% of values overlapped the normal range. During 5 minute calcium infusions (2 mg Ca2+ per kg) iPTH values fell little, but bioPTH and intact PTH fell sharply in all three groups. Mean calcium-induced decreases of intact and bioPTH were indistinguishable from normal in FBH, but PTH levels generally remained elevated at 5 minutes in primary HPT. In FBH basal and postinfusion CT levels were normal. The data show that, in the majority of patients with FBH, PTH concentrations and bioactivity in blood are within the normal range and are suppressed rapidly to very low levels with further increases of calcium. The data suggest that the abnormality of parathyroid function in FBH differs from that in primary HPT.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactive effects of triiodothyronine and androgens on prostate cell growth and gene expression.

T3 plays an important role in the regulation of cell growth and differentiation. In this study, we show the interactive effects of T3 and androgens on the growth response and expression of the prostate-specific genes, PSA (prostate-specific antigen) and hK2 (human glandular kallikrein), in the human prostate cancer cell line, LNCaP. T3 alone showed pronounced growth enhancement in a dose-dependent fashion. However, in the presence of androgens, higher concentrations of T3 were required to produce additional proliferative effects. T3, androgens, or a combination of the two up-regulated PSA protein production in a dose-dependent fashion, but T3 had little stimulatory effect on hK2 protein expression, regardless of the presence or absence of androgens. Using gene transfer assays, T3 alone showed no effect on transcriptional activation of a reporter gene mediated by the PSA or hK2 enhancer/promoters. T3 potentiated the androgen-mediated transcription of the PSA gene but not that of the hK2 gene. A previous study suggested that the T3 effect on PSA protein expression was caused by an up-regulation of the androgen receptor (AR) protein by T3. Our results contradict these. Although AR expression was increased by T3 alone, Western blot analysis showed that the total cellular AR level was not further increased by T3 in the presence of androgens, in comparison with cells stimulated by androgens alone. Both Western blot analysis and a gel DNA band shift assay revealed that nuclear AR was not increased by T3. This study suggests that transcription factor(s) other than the AR may mediate T3 enhancement of androgenic induction of PSA expression.

Assessment of sensitive thyrotropin assays for an expanded role in thyroid function testing: proposed criteria for analytic performance and clinical utility.

Assays capable of detecting subnormal serum TSH concentrations can permit the prediction of response to TRH and the detection of hyperthyroidism. Five performance criteria for evaluating the eligibility of TSH assays for these new roles are proposed. Criterion 1 requires a less than 1% overlap between the variation of the lower normal value and the assay detection limit. Criteria 2 and 3 require subnormal basal TSH values in 95% of patients with subnormal TRH response, and detectable basal TSH values in 95% of patients with normal TRH responses. Criteria 4 and 5 require undetectable TSH values in 95% of hyperthyroid patients and detectable TSH values in 95% of clinically euthyroid subjects. Evaluation of 20 published studies using commercial reagents showed that only 5 assays met criterion 1; most reports did not provide adequate information for evaluation. We evaluated 2 sensitive assays by assessment of sera from 149 normal subjects and 893 patients. Substantial differences were found; only assay B met all performance criteria. With assay B, serum TSH levels were normal in 86%, increased in 7%, undetectable in 4%, and subnormal in 3% of 454 patients undergoing a screening T4 measurement. Sensitive TSH assays fulfilling our criteria may be useful as front-line thyroid function tests.

Thyroid peroxidase and thyroid microsomal autoantibodies.

The antithyroid microsomal antibodies found in the serum of patients with autoimmune thyroid disease are directed largely, if not entirely, against thyroid peroxidase (TPO). In this study we used a highly purified, well characterized, large tryptic fragment of porcine TPO (hereafter referred to as purified porcine TPO) to examine possible differences among microsomal antibodies in patients with autoimmune thyroid disease. Antibodies against this TPO preparation and also against a synthetic peptide corresponding to residues 780-793 of the deduced sequence of the native enzyme were compared with microsomal antibodies from patients in immunoblot experiments. The antiporcine TPO and antisynthetic peptide antibodies reacted with crude preparations of human TPO. Binding of serum microsomal antibodies to purified porcine TPO was also found. Purified porcine TPO shows two fragments after gel electrophoresis under reducing conditions: a 59K fragment corresponding to the amino end of the molecule, and two approximately 30K fragments corresponding to the carboxyl end. Using an immunoblot procedure with purified porcine TPO as the antigen, we found that at least two epitopes were involved in microsomal antibody production: one associated with the 59K fragment and the other with the approximately 30K fragment(s). The distribution of serum antibodies against these epitopes differed among the patients, indicating that these antibodies comprise a heterogeneous group. Serum from patients with autoimmune thyroid disease significantly inhibited human TPO activity, raising the possibility that microsomal antibodies may contribute to the impaired thyroid function that occurs in some patients with autoimmune thyroid disease.

Preparation of synthetic bovine parathyroid hormone fragment 1-34 for parenteral use in human studies.

There is growing interest in use of synthetic parathyroid hormone peptides such as the aminoterminal 1-34 fragment (PTH 1-34) in human studies, since bovine parathyroid extract is no longer commercially available. We found no data concerning how to sterilize and dilute the synthetic bovine PTH 1-34 (bPTH 1-34) to minimize adsorptive losses and maximize conservation of bioactivity. Therefore, we examined adsorptive losses of electrolytically-labelled, biologically-active bPTH 1-34 onto sterile filtration devices (Millex GV, Millipore Corp.) in solutions of 0.1 M acetic acid containing varying human serum albumin (HSA) concentrations (0.1-5.0%, w/v) and varying hormone concentrations (1-50 micrograms bPTH 1-34/ml). We also assessed preservation of bPTH 1-34 bioactivity (canine renal cortical plasma membranes) in diluted, sterile-filtered solutions refrigerated for 4 days. Adsorptive losses were inversely related to bPTH 1-34 concentrations, being least with 50 micrograms bPTH 1-34/ml, at all concentrations of HSA. Losses during filtration were essentially indistinguishable at HSA concentrations of 0.1 - 1.0%, but were, surprisingly, increased by 2.5 and 5.0% HSA. There were no important differences in adsorptive losses among five different lots of Millex-GV filters. Full bioactivity was preserved over 4 days of refrigeration at a bPTH 1-34 concentration of 20 micrograms/ml. The data suggest that bPTH 1-34 should be sterile-filtered at a concentration of greater than or equal to 20 micrograms/ml in 0.1M acetic acid containing 0.1 - 1.0% HSA. Such sterile solutions are stable at 4 degrees C for at least 4 days.

Studies with purified human thyroid peroxidase and thyroid microsomal autoantibodies.

We have isolated highly purified thyroid peroxidase (TPO) from human thyroid tissue to study further the relationship between TPO and the thyroid microsomal antigen that elicits the production of microsomal autoantibodies in patients with autoimmune thyroid disease. Serum samples were obtained from 24 patients with suspected autoimmune thyroid disease, and from 7 normal subjects. Microsomal autoantibodies in the patient sera, as determined by the microsomal hemagglutination assay (MCHA), varied between 1:100 and 1:102,400. Antithyroglobulin antibodies, however, were very low (less than 1:100). Binding of serum autoantibodies to purified human TPO, as determined by enzyme-linked immunosorbent assay, correlated fairly well with MCHA titers (r = 0.72; P less than 0.001). An immunoblot procedure was developed to study the binding of serum antibodies to the major active fragment of TPO (93 kDa), after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Binding under both conditions correlated very well with MCHA titers (r = 0.80-0.84; P less than 0.001). Studies were performed to determine the inhibitory effect of patient serum on the enzymatic activity of purified human TPO. A marked inhibitory effect on guaiacol activity was observed when TPO was preincubated with as little as 10 microL high titer serum. There was a significant correlation (r = 0.47; P less than 0.01) between MCHA titer and inhibitory effect. The addition of 2 micrograms purified human TPO completely or almost completely inhibited the binding of serum antibodies to thyroid microsomes (enzyme-linked immunosorbent assay) in 10 of 11 patient sera with high MCHA titers (1:25,600 or greater).

Demonstration of biological activity of prolactin molecular weight variants in human sera.

Three immunoreactive forms of PRL, separated by Sephadex G-100 column chromatography, were identified in serum samples from 10 normal subjects and 7 patients with PRL-secreting pituitary tumors. Fractions eluted from the column were assayed for bioactivity by using a sensitive bioassay with the Nb2 cell line. Three molecular weight variants of PRL were identified in normal subjects. In samples from 9 of the 10 normal subjects, 80.1 +/- 3.6% (mean +/- SEM) of the total bioactivity eluted in a peak corresponding to a PRL monomer (peak III) with a mol wt of approximately 24,000, 18.3 +/- 3.7% eluted in a peak with a mol wt of approximately 56,000 (peak II), and 1.6 +/- 1.1% of the biological activity was in the void volume (peak I). In the 10th normal sample, 65% of the total bioactivity was in the void volume (peak I), 31% was in peak III, and 4% was in peak II. Samples from the patients had 3.6 +/- 0.7% of the total bioactivity in peak I, 9.3 +/- 1.0% in peak II, and 87.0 +/- 1.1% in peak III, percentages not significantly different from normal. For comparison with bioassay, RIA measurement of PRL was performed on all fractions of six samples (three normal subjects and three tumor patients). Good correlation was found between RIA and bioassay measurements under each of the three peaks identified. We conclude that 1) in sera from normal subjects, three molecular weight variants of PRL have biological activity; 2) in patients with PRL-secreting tumors, secretion of biologically active PRL molecular weight variants is not proportionately different from that in normal subjects; and 3) the results of the Nb2 PRL bioassay correlate well with PRL levels determined by RIA for each of three molecular weight variants identified.

Serum bioactive parathyroid hormone in hemodialysis patients.

In 41 hemodialysis patients with bone disease (histological diagnosis with histomorphometric confirmation of PTH activity) results from a serum immunoreactive PTH (iPTH) assay correlated with results from a new serum bioactive PTH (bio-PTH) assay (r = 0.84; P less than 0.001). The serum bio-PTH values correlated well with osteoclast numbers (r = 0.70; P less than 0.001), resorption surfaces (r = 0.55; P less than 0.001), and presence of marrow fibrosis (P less than 0.02). The serum iPTH values also correlated with osteoclast numbers (r = 0.61; P less than 0.001), resorption surfaces (r = 0.47; P less than 0.003), and presence of marrow fibrosis (P less than 0.02). Serum bio-PTH and iPTH values were higher in patients with severe hyperparathyroidism than in other patients. The assays were equally useful in identifying dialysis patients with severe hyperparathyroidism. Patients with osteomalacia or low turnover bone disease had low serum bio-PTH and/or iPTH values. Low bio-PTH values (less than or equal to 3.6 pmol/L) had a sensitivity and a specificity of 93% for osteomalacia or low turnover bone disease. Low bio-PTH values also were useful in identifying those patients with positive aluminum staining in bone. The serum bio-PTH assay was useful in identifying patients with osteomalacia, low turnover bone disease, or aluminum accumulation.

Relationship of serum sex steroid levels and bone turnover markers with bone mineral density in men and women: a key role for bioavailable estrogen.

Estrogen (E) deficiency associated with the menopause is the major cause of bone loss in aging women. However, men also lose significant amounts of bone with age, but they do not have the equivalent of menopause, and serum total testosterone (T) and E levels decline only marginally with age in men. Thus, it has been difficult to attribute bone loss in aging men to either T or E deficiency. Here, we show in a population-based, age-stratified sample of 346 men, aged 23-90 yr, that serum total T and E (estradiol plus estrone) levels decreased over the life span by 30% and 12%, respectively, but bioavailable (or nonsex hormone-binding globulin-bound) T and E levels decreased by 64% and 47%, respectively. In these men and in a parallel cohort of 304 women, aged 21-94 yr, serum PTH increased 84% and 64% over the life span, and urinary N-telopeptide of type I collagen (NTx) excretion, a bone resorption marker, increased 77% and 80% between age 50-85 yr in the men and women, respectively. By univariate analyses, serum bioavailable T and E levels correlated positively with bone mineral density (BMD) at the total body, spine, proximal femur, and distal radius and negatively with urinary NTx excretion in men and women. Urinary NTx excretion was also negatively associated with BMD in both sexes. By multivariate analyses, however, serum bioavailable E level was the consistent independent predictor of BMD in both men and postmenopausal women. Thus, bioavailable E levels decline significantly with age and are important predictors of BMD in men as well as women. These studies suggest that in contrast to traditional belief, age-related bone loss may be the result of E deficiency not just in postmenopausal women, but also in men.

Biochemical testing of thyroid function.

Various published guidelines recommending serum thyrotropin (TSH)-first thyroid testing are outlined. The entities called "subclinical hypothyroidism" and "subclinical hyperthyroidism" are defined on the basis of abnormal TSH concentrations and normal values of other biochemical thyroid tests. The controversies about follow-up and treatment of these disorders are discussed. The laboratory experience of Mayo Clinic Rochester in using TSH-first thyroid testing and the subsequent implementation of a thyroid test ordering cascade are presented. Finally, recommendations are given for further optimizing laboratory testing for thyroid disorders.

Primary aldosteronism. Diagnostic evaluation.

The regulation of mineralocorticoid secretion and the pathophysiology of primary aldosteronism are reviewed. For conceptual and practical purposes, the diagnostic evaluation of primary aldosteronism is discussed as two series of studies. The first series involves the studies necessary to confirm the diagnosis. The second series of studies guides the therapeutic approach by distinguishing unilateral from bilateral adrenal disease.

Thyroid dysfunction.

This article reviews currently available thyroid function tests and considers the utility of these tests in the investigation of suspected thyroid dysfunction. Potential thyroid screening tests as well as those secondary tests useful in establishing or excluding thyroid dysfunction are evaluated. Current controversies regarding free thyroid hormone and sensitive TSH assays are considered, and algorithms are provided for diagnosing both hypothyroidism and thyrotoxicosis.

Hypercalcemia.

Hypercalcemia is a relatively common clinical finding; prevalence rates are 1.4 to 3.0 per cent in hospitalized and general clinical populations. Malignancy is the major cause of hypercalcemia in hospital patients, whereas primary hyperparathyroidism (HPT) is the major cause in ambulatory patients. In both hospitalized and ambulatory patients, however, there are many other causes of hypercalcemia, and numerous procedures have been proposed to aid in the differential diagnosis. Unfortunately, no single test is truly diagnostic. The work-up for hypercalcemia requires an integrated knowledge of the strengths and weaknesses of the various procedures as well as an understanding of the various clinical presentations associated with hypercalcemia.

Diagnostic evaluation of pheochromocytoma.

Progress in the diagnostic evaluation of pheochromocytoma has taken place in biochemical studies and localization techniques. Measurement of fractionated catecholamines and their metabolites has been subjected to sensitivity and specificity assessment. Magnetic resonance imaging and isotopic imaging have led to much better localization of extra-adrenal tumors. Flow cytometry of the DNA of the tumor cells very likely indicates the malignant nature of the tumor.