[Renal insufficiency due to light chain multiple myeloma]. / Nierinsufficiëntie door neerslag van lichte ketens bij multipel myeloom.

In 3 female patients, aged 65, 83 and 76 years, with severe renal failure, light chain multiple myeloma was diagnosed, following a substantial delay on the part of the doctors concerned. Either the diagnosis had not suspected or the serum proteins had been misinterpreted. After a while, the first two patients declined further treatment with chemotherapy and haemodialysis, and subsequently died. The third patient attained a creatinine clearance of 20 ml/min and was subsequently treated for the multiple myeloma in the outpatients department. The absence of a paraprotein peak in the serum does not exclude the possibility of a multiple myeloma. In the case of light chain disease, the gammaglobulin region is, in fact, often empty. Treatment of multiple myeloma consists of a rapid rehydration and forced diuresis; the usefulness of plasmapheresis has not been demonstrated.

Androgen receptor status in localized and locally progressive hormone refractory human prostate cancer.

Heterogeneity in human androgen receptor (hAR) expression in prostate cancer is considered to be implicated in tumor progression. hAR expression was therefore studied immunohistochemically in localized and locally progressive, hormone refractory (HR) prostate cancer. Because altered functional activity of the hAR may be due to changes in the structural integrity of the hAR gene, exons 2 to 8 of the hAR gene were assessed for mutations by single-strand conformation polymorphism (SSCP) analysis and exon 1 was analyzed for the size of the CAG repeat. The hormone binding capacity, a prerequisite for ligand-regulated receptor function, was determined by a ligand binding assay. Coexpression of the hAR and prostate-specific antigen (PSA) was studied by a sequential double immunoenzymatic staining to verify whether PSA expression is a parameter of hAR function. Almost all human prostatic carcinomas revealed heterogeneous hAR expression, regardless of tumor differentiation and progression. Putative predominance of hAR-negative tumor areas in HR prostate cancer was not observed. No hAR gene mutations or major changes in the CAG repeat were found in the 18 HR carcinomas or in the 9 control samples. Moreover, all selected hAR-expressing cancers were able to bind the synthetic androgen methyltrienolone (R1881). Immunoenzymatic double staining revealed even PSA expression in hAR-negative tumor areas. PSA immunohistochemistry in human prostatic carcinomas therefore is of no use in determining hAR functional activity. Thus, most prostatic carcinomas, even when progressed to a state of hormone insensitivity, contain a structurally intact hAR gene, heterogeneously expressed with retained androgen binding capacity.

Do neuroendocrine cells in human prostate cancer express androgen receptor?

The presence of androgen receptors (AR) in neuroendocrine cells was investigated in benign tissue of 10 prostatectomy specimens, in 12 prostatic adenocarcinomas with focal neuroendocrine differentiation and in 1 case of a pure neuroendocrine small cell carcinoma of the prostate. Neuroendocrine cells were defined by their reactivity with an antibody to chromogranin A. Monoclonal antibody F39.4 directed against the amino-terminal domain of the AR molecule was used to detect AR. AR and chromogranin A were simultaneously visualized with a double immunofluorescence technique. The results indicate that chromogranin positive cells in both benign and malignant prostatic tissue lack detectable expression of AR. No effect of endocrine therapy was noted. These results are in agreement with the hypothesis that prostatic neuroendocrine tumour cells represent an androgen insensitive cell population, which incidentally may expand to replace the androgen-sensitive tumour cell population during androgen ablation therapy.

Regulation of androgen receptor expression in the human heterotransplantable prostate carcinoma PC-82.

In vivo effects of androgen withdrawal and substitution on human androgen receptor (hAR) expression were evaluated in the androgen-dependent human prostatic carcinoma tumor line PC-82. By application of several antibodies reactive with different epitopes of the hAR molecule, hAR protein expression was studied in tumor transplants by immunohistochemistry and immunoblotting. hAR messenger RNA (mRNA) levels were quantitated in PC-82 tumor tissue with a S1-nuclease protection assay. Most PC-82 tumor cells (> 97%) from testosterone-supplemented mice displayed nuclear hAR protein expression immunohistochemically. The almost complete reduction of nuclear hAR immunoreactivity within 5 days after androgen withdrawal (< 10%) was restored after androgen substitution within 1 day. The immunochemical data were confirmed by Western blot analysis. In contrast, no significant changes were observed in hAR mRNA content of PC-82 cells after 5 days of androgen withdrawal. Correlating hAR expression with proliferative activity of PC-82 tumor tissue during endocrine manipulation, a rapid, castration-induced decline of the percentage of bromodeoxyuridine-labeled cells accompanied the loss of hAR. Androgen substitution in castrated male mice restored the proliferative activity. However, this increase of proliferative activity lagged at least 24 h behind the normalization of the hAR protein level. In contrast to the steroid receptor down-regulation by homologous ligands observed in other experimental models, our data support the concept of hAR up-regulation by androgen. Since the hAR mRNA content of PC-82 tumor tissue was hardly affected by castration, expression of the hAR in PC-82 is thought to be modulated by translational and/or posttranslational mechanisms.

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.

Androgen receptors in endocrine-therapy-resistant human prostate cancer.

Despite the initial androgen-dependent growth of most human prostate cancers, eventually all prostate cancers become androgen-independent at varying intervals after androgen ablation or anti-androgen therapy. In order to gain more insight into the role of the androgen receptor (AR) in this process, AR and prostate-specific antigen (PA) expression was evaluated immunohistochemically in prostatic tumour tissues from patients who developed urinary flow obstruction between 4 and 107 months after onset of treatment. AR expression was evaluated with a monoclonal antibody (MAb) specific for the N-terminal domain of the human AR. To substantiate the progressive tumour growth, proliferative activity was assessed immunohistochemically by staining with MAb Ki-67. Ki-67-defined tumour-growth fractions varied from 0.8-64.7%. In 13 of the 17 examined tumours over 80% of the tumour cells were AR-positive, 3 tumours showed a considerable heterogeneity in AR expression and in 1 tumour almost all tumour cells seemed to be AR-negative. Two-thirds of the examined tumours contained variable proportions of PA-positive tumour areas. These observations contrast with the view that androgen ablation induces a preferential outgrowth of receptor-negative tumour cells.

Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multi-assay performance following the synthetic peptide strategy.

The human androgen receptor (hAR) is an important regulatory protein particularly in male sexual differentiation. The investigation of hAR functionality has been hampered by the lack of AR specific monoclonal antibodies recognizing the functional domains of the receptor. Therefore production of high affinity mono-specific polyclonal (PAbs) and monoclonal antibodies (MAbs) directed to the hAR was initiated following the synthetic peptide (SP) strategy. Five hAR specific peptides were selected on the basis of their predicted antigenic properties avoiding homology with other steroid hormone receptors. Peptide specific polyclonal antisera were obtained following selected immunization protocols. Mono-specific polyclonal antibody responses were elicited to all peptides in mice and rabbits. Crossreactivity of the peptide specific antisera with the native hAR in various biochemical assays was observed with two out of five peptides. Peptide SP61 (hAR residues 301-320) was used for the generation site-directed MAbs specific for the hAR. Specificity for the hAR was established by immunoprecipitation, immune-complex density gradient centrifugation and immunohistochemistry on human prostate tissue sections. The multi-assay performance of the selected high affinity antibodies proved the usefulness of the straight forward peptide approach and opens a wide field of possible biochemical and physiological investigations into questions related to androgen action.

Characterization of polyclonal antibodies against the N-terminal domain of the human androgen receptor.

Antibodies against the N-terminal domain of the human androgen receptor (hAR) were prepared by two different approaches. Firstly, rabbits were immunized with a beta-galactosidase-hAR (amino acids (aa) 174-353) fusion protein. Secondly, two synthetic peptides corresponding to potentially antigenic sites located within this fragment (aa 201-222 and 301-320) were used as immunogens. The obtained antisera contained high titer anti-hAR antibodies as was established with several independent methods (e.g. sucrose gradient centrifugation, immunoprecipitation, Western blotting). The two anti-peptide antisera specifically stained nuclei of glandular epithelial cells in frozen sections of human prostate tissue. Progesterone, estradiol and glucocorticoid receptors were not immunoprecipitated with these antisera. The specific hAR antibodies provide new tools for the characterization of this steroid receptor as well as for diagnostic purposes in pathology of the human prostate and androgen resistance.