Hermann Rogatsch

Immunohistochemical localization of interleukin‐6 and its receptor in benign, premalignant and malignant prostate tissue

Interleukin‐6 (IL‐6) is a pleiotropic cytokine that interacts with its receptor in prostate cells, thus regulating proliferative response and differentiation. It also activates the human androgen receptor in prostate cancer cell lines. In order to assess the significance of these findings in vivo, the expression of key elements of the IL‐6 signalling pathway, IL‐6 and its receptor, was investigated in tissue samples obtained on radical prostatectomy from prostate cancer patients. IL‐6 immunohistochemistry was performed on 17 frozen prostate cancer specimens. IL‐6 receptor immunostaining was evaluated in 21 paraffin‐embedded prostate tumour specimens. In both groups, adjacent areas of high‐grade prostatic intraepithelial neoplasia and benign tissue were also investigated. In benign prostatic epithelium, IL‐6 was localized predominantly in basal cells, whereas in prostate cancer tissues more IL‐6‐positive glandular cells were identified. No IL‐6 expression was detected in stromal cells on immunohistochemistry, although IL‐6 protein was measured in the supernatants obtained from cultured stromal cells by ELISA. IL‐6 receptor was expressed in benign prostatic tissue in both epithelial and stromal cells. Furthermore, IL‐6 receptor expression was observed in all tumour specimens investigated and the majority of Gleason patterns analysed had more than 50% of cells showing a positive reaction. IL‐6 and IL‐6 receptor expression patterns in high‐grade prostatic intraepithelial neoplasia lesions were similar to those observed in tumour tissues. Taken together, the results of the present study imply that there are paracrine and autocrine IL‐6 loops in benign and neoplastic prostate. Copyright

Inhibition of LNCaP prostate tumor growth in vivo by an antisense oligonucleotide directed against the human androgen receptor

We have shown recently that a 15-mer phosphorothioate oligodeoxynucleotide (ODNas750/15) that hybridizes to the (CAG)n polyglutamine region of mRNA encoding human androgen receptor (AR) inhibits the expression of AR in LNCaP prostate cancer cells in vitro. This AR downregulation was accompanied by significant cell growth inhibition and reduced PSA secretion. In the present study we investigated the effects of this antisense AR ODN on prostate tumor growth in vivo using a mouse xenograft model. Via subcutaneously implanted diffusion pumps, either ODNas750/15 or a scrambled control sequence ODNsr750/15 was continuously administered into LNCaP tumor–bearing male nude mice for 7 weeks. Compared with untreated control animals, treatment with ODNas750/15 resulted in significant tumor growth inhibition. Retardation of tumor growth was also significant in castrated mice, whereas the scrambled control ODN did not exert any effects. No side effects such as loss of body weight were observed at any time of treatment. ODN treatment was well tolerated and, in contrast to castration, did not induce shrinkage of mouse prostates. Both AR expression in the tumor and PSA levels in mouse serum correlated with tumor size. However, we failed to demonstrate a correlation between tumor retardation and Ki-67 antigen expression and the number of apoptotic cells, respectively. Testing of antisense-treated LNCaP cells revealed that expression levels of other proteins that contain shorter polyglutamine sequence stretches such as HDAC2, TFIID, and c-jun were not affected. The present study demonstrates that downregulation of AR with antisense ODNas750/15 causes prostate tumor growth inhibition. These results further point out the important role of the AR in prostate tumors and support further testing of AR downregulation for treatment of prostate cancer.

Prostate Cancer Screening in Tyrol, Austria: Experience and Results

Background: This article summarizes the experience and results of different prostate carcinoma screening projects using total prostate-specific antigen (PSA) and percent free PSA as the initial test. Methods: The twelve projects studied included: (1) a mass screening study using PSA as the initial test in 21,079 volunteers; (2) an investigation of the usefulness of normal and age-referenced PSA cut-offs in 1,618 men; (3) a PSA-based screening study of 2,272 asymptomatic blood donors; (4) an investigation of the evidence and significance of transition zone carcinoma in 340 men with negative digital rectal examination findings; (5) determination of percent free PSA in one retrospective and two prospective studies to determine the appropriate cutpoints for percent free PSA; (6) evaluation of the diagnostic benefit of PSA transition zone density in 308 screening volunteers; (7) a study of the impact of PSA-based screening on the percentage of incidental prostate carcinoma in 1,543 men undergoing transurethral resection of the prostate; (8) an evaluation of the changes in total PSA and pathologic stages in radical prostatectomy over 5 years in a PSA-based mass screening program; (9) a study evaluating the probability of having prostate cancer given the patient’s age, total PSA and digital rectal examination findings; (10) an evaluation of the correlation between preoperative predictors and pathologic features in radical prostatectomy specimens; (11) an investigation of the correlation of total PSA with pathologic stage and tumor volume in patients undergoing radical prostatectomy with low PSA cut-off level, and (12) a study whether age has an impact on the extension of prostate cancer. Results: (1) of the 21,079 volunteers, 1,618 (8%) had elevated PSA levels. Of these men, 778 (48%) underwent biopsies; 197 biopsies were positive for prostate carcinoma and 135 underwent radical prostatectomy. Ninety-five were found to be organ-confined. (2) A PSA cut-off of 2.5 ng/ml in men aged 45–49 years and of 3.5 ng/ml in men aged 50–59 years resulted in an 8% increase in the detection rate of organ-confined disease. (3) Of the 2,272 men, 284 had elevated PSA levels and prostate carcinoma was detected in 62 men. All patients underwent radical prostatectomy and histologic examination revealed organ-confined tumor in all but 8 men. (4) Ninety-eight of 340 men had biopsies positive for carcinoma; 28 of these patients (28.5%) had carcinoma that originated in the transition zone only. (5) In the retrospective study, receiver-operating characteristic curve analysis showed that by using a percent free PSA of 18% as a biopsy criterion, 37% of the negative biopsies could be eliminated although 94% of all carcinomas would still be detected. In the first prospective study, 106 of 158 men with elevated PSA levels <10.0 ng/ml were further evaluated and 37 prostate carcinomas were detected. By using a % free PSA of <22% as a biopsy criterion, 30% of the negative biopsies could be eliminated although 98% of the carcinomas would still be detected. In the second prospective study, 120 of 465 men with total PSA levels between 1.25 and 6.49 ng/ml and a % free PSA <18% were further evaluated and 27 (22.5%) were found to have prostate carcinomas. (6) Receiver-operating characteristic curve analysis for PSA transition zone density showed that by using a PSA transition zone density of >22 ng/ml/cm3 as a biopsy criterion, 24.4% of negative biopsies could be avoided without missing a single carcinoma. (7) In the prescreening era the incidence of T1a grade 1 and 2 carcinomas was 3.1% and the incidence of T1a grade 3 and T1b carcinoma was 2.3% whereas in the years after the establishment of PSA-based screening the incidence was 4.6 and 1.03% respectively. (8) The rate of organ-confined tumors increased from 28.7% in 1993 to 65.7% in 1997. (9) In this evaluation a new approach to proceed with a prostate biopsy based upon the individual risk of having prostate cancer rather than a single PSA cutpoint was developed. (10) High total PSA levels, PSA density and PSA transition zone density correlated significantly with high Gleason scores, capsular penetration, a high percentage of cancer in the prostatectomy specimen and a high cancer volume. (11) In this evaluation all of the 95 patients with PSA levels <3.99 ng/ml who underwent radical prostatectomy showed clinically significant, organ-confined prostate cancer with negative surgical margins. (12) The results of this evaluation suggest that older men have larger tumor volumes compared to younger men with the same PSA levels. Conclusions: These data suggest that PSA-based screening with low PSA cut-off values increase the detection rate of clinically significant, organ-confined and potentially curable prostate cancer. Percent free PSA and PSA transition zone density provide an additional diagnostic benefit over total PSA.

Accelerated in Vivo Growth of Prostate Tumors that Up-Regulate Interleukin-6 Is Associated with Reduced Retinoblastoma Protein Expression and Activation of the Mitogen-Activated Protein Kinase Pathway

Interleukin-6 (IL-6) is a multifunctional cytokine that activates the signaling pathways of Janus kinases-signal transducers and activators of transcription (STAT) and/or mitogen-activated protein kinases (MAPK) in various tumors. Thus, it modulates cell growth and apoptosis. IL-6 levels are elevated in tissues and sera from prostate cancer patients and IL-6 receptor expression has been detected in prostate cancer cell lines and clinical specimens. Continuous exposure of prostate cancer cells to IL-6 might alter their responsiveness to this cytokine. To gain more insight into the function of IL-6 in prostate carcinoma, we have inoculated LNCaP-IL-6+ cells, generated after prolonged treatment with IL-6, into nude mice (total n = 16, two independent experiments). Controls included animals bearing LNCaP-IL-6- cells, passaged at the same time as LNCaP-IL-6+ cells without supplementation of IL-6. LNCaP-IL-6+ tumor volumes were larger than those of their counterparts at all time points. There were no signs of cachexia in any of the experimental animals and all mice were free of metastases. To better understand the mechanisms responsible for accelerated growth of LNCaP-IL-6+ tumors, we have investigated the expression of cell-cycle regulatory molecules by Western blot analysis. The levels of cyclin-dependent kinase 2 were elevated in LNCaP-IL-6+ cells. There was a strong down-regulation of cyclins D1 and E in the LNCaP-IL-6+ subline. The cell-cycle inhibitor p27 was expressed at a low level in LNCaP-IL-6+ cells and could not be up-regulated by addition of IL-6. Most notably, LNCaP-IL-6+ cells exhibited a reduced expression of the hypophosphorylated form of the retinoblastoma protein (pRb). Accelerated tumor growth in our model system was also associated with alterations in IL-6-signaling pathways. The ability of IL-6 to induce tyrosine phosphorylation of STAT3 was abolished in the LNCaP-IL-6+ subline. In contrast, the levels of the MAPK extracellular signal-regulated kinases 1/2 increased in cells generated after long-term IL-6 treatment. The inhibitor of MAPK kinase PD 98059 retarded the proliferation of LNCaP-IL-6+ but not that of control cells. In summary, we show in the present study that chronic exposure of prostate cancer cells to IL-6 facilitates tumor growth in vivo by abolishment of the growth control by pRb and activation of the MAPK signaling pathway. These findings could be relevant to understand the role of IL-6 in prostate cancer progression.

Frozen section analysis-guided organ-sparing approach in testicular tumors: Technique, feasibility, and long-term results

OBJECTIVES To evaluate retrospectively the indications, surgical technique, feasibility, and follow-up data of our frozen section analysis-guided organ-sparing approach to small testicular tumors. Removal of a solitary testis or bilateral orchiectomy for testicular neoplasm results in androgen deprivation and infertility. Moreover, removal of a testis for benign lesions is to be avoided. Organ-sparing surgery aims at preserving enough testicular parenchyma to maintain physiologic endocrine function and, if possible, fertility. METHODS Tumors of 25 mm or less in diameter were managed by an organ-sparing approach. Normal preoperative plasma levels of luteinizing hormone and testosterone were a prerequisite. After localization of the tumor by ultrasonography and accurate staging, organ-sparing surgery was performed under cold ischemia. Frozen section analyses of the tumor and tumor bed biopsies were obtained. In the case of malignant germ cell tumor with a normal contralateral testis, ablation of the tumor-bearing testis was performed. RESULTS A total of 32 organ-preserving procedures were performed in 30 patients without any complications. Local recurrence was observed in 1 patient who refused to undergo local radiotherapy for concomitant testicular intraepithelial neoplasia; repeat organ-sparing surgery was performed in this patient. After organ-sparing surgery, ablation of the remaining testis was performed in 1 patient because of positive margins on final histologic analysis and in another patient because of endocrine insufficiency. In all other patients, the serum testosterone levels remained within normal limits. No retroperitoneal, pulmonary, or other recurrences were encountered; all patients were free of disease at a mean follow-up of 46.3 months. CONCLUSIONS The organ-sparing frozen section analysis-guided approach to testicular masses represents a promising treatment alternative to orchiectomy in selected patients with bilateral malignant testicular tumors, tumors in a solitary testis, or unilateral or bilateral benign tumors. The technique is oncologically efficient, lifelong hormonal substitution can be prevented, and, in some patients, even fertility may be preserved, provided certain criteria concerning patient selection and surgical technique are observed.

CD30 expression in seminoma

In testicular germ cell tumors the CD30 antigen has been shown to be regularly expressed in embryonal carcinoma and was thus suggested as a marker for this particular neoplasm. Very recently, it has been proven that the monoclonal antibody Ber-H2 is suitable for the detection of this membrane antigen in paraffin sections. We conducted an immunohistochemical study to investigate the CD30 expression in a large series of different presentations of seminoma (ie, pure, mixed, and spermatocytic) because there is evidence from several sources that embryonal carcinoma is histogenetically closely related to, and probably derives from, seminoma. Sections from formalin-fixed, paraffin-embedded tissue from 38 cases of testicular seminomas were immunostained for the demonstration of the CD30 antigen using the monoclonal antibody Ber-H2, cytokeratins, and placental alkaline phosphatase following an indirect streptavidin-peroxidase regimen. In selected cases, immunostainings were performed on consecutive sections to investigate a possible colocalization of CD30 and cytokeratins in seminoma. Specific immunostaining for CD30 in seminoma cells could be detected in single minute foci in 4 of 21 cases of pure classic seminoma. Seminomatous components of mixed tumors showed CD30 positivity in single, but also multiple, foci in 7 of 14 cases. CD30 immunoreactivity in seminoma cells occurred with and without colocalized expression of cytokeratin. Spermatocytic seminoma (n = 3) as well as intratubular germ cell neoplasia in tumor adjacent parenchyma (n = 36) were negative in all cases investigated. We conclude that in testicular germ cell tumors, the expression of CD30 is not restricted to embryonal carcinoma but can also be found focally in seminoma, adding further evidence for a close relationship between these two tumors. The prevalence of CD30 expression in seminomatous components of mixed tumors, as well as the coexpression with cytokeratins, suggest that CD30 expression in seminomas might indicate their upcoming transformation to embryonal carcinoma. This conclusion coincides with a model featuring seminoma in a central role of germ cell tumor development.

The Cochaperone Bag-1L Enhances Androgen Receptor Action via Interaction with the NH2-Terminal Region of the Receptor

ABSTRACT Members of the Bag-1 family of cochaperones regulate diverse cellular processes including the action of steroid hormone receptors. The largest member of this family, Bag-1L, enhances the transactivation function of the androgen receptor. This occurs primarily through interaction with the NH2 and COOH termini of the receptor. At the NH2 terminus of the receptor, Bag-1L interacts with a region termed τ5. Bag-1M, a naturally occurring variant of Bag-1L that binds to τ5 but is defective in the COOH-terminal interaction, is less efficient in enhancing the transactivation function of the receptor. Surface plasmon resonance and transfection studies showed that the molecular chaperone Hsp70 contributes to the binding of Bag-1L to τ5 and to the regulation of the transactivation function of the androgen receptor. Chromatin immunoprecipitation studies demonstrated that the androgen receptor, Hsp70, and Bag-1L are all targeted to the androgen response elements of the gene that encodes prostate-specific antigen. These studies demonstrate the regulation of transcriptional activity of androgen receptor by a molecular chaperone-cochaperone complex.

The Transcriptional Co-Activator cAMP Response Element-Binding Protein-Binding Protein Is Expressed in Prostate Cancer and Enhances Androgen- and Anti-Androgen-Induced Androgen Receptor Function

Progression of human prostate cancer toward therapy resistance occurs in the presence of wild-type or mutated androgen receptors (ARs) that, in some cases, exhibit aberrant activation by various steroid hormones and anti-androgens. The AR associates with a number of co-activators that possess histone acetylase activity and act as bridging molecules to components of the transcription initiation complex. In previous reports, it was shown that the transcriptional co-activator CREB (cAMP response element-binding protein)-binding protein (CBP) enhances AR activity in a ligand-dependent manner. In the present study, we have investigated whether CBP modifies antagonist/agonist balance of the nonsteroidal anti-androgens hydroxyflutamide and bicalutamide. In prostate cancer DU-145 cells, which were transiently transfected with CBP cDNA, hydroxyflutamide enhanced AR activity to a greater extent than bicalutamide in the presence of either wild-type or the mutated AR 730 val-->met. In two sublines of LNCaP cells that contain the mutated AR 877 thr-->ala and overexpressed CBP, increase in AR activity was observed after treatment with hydroxyflutamide but not with bicalutamide. Anti-androgens did not influence AR expression in cells transfected with CBP cDNA, as judged by Western blot analysis. Endogenous CBP protein was detected by Western blot in nuclear extracts from the three prostate cancer cell lines, LNCaP, PC-3, and DU-145, all derived from therapy-resistant prostate cancer. In addition, CBP was expressed in both basal and secretory cells of benign prostate epithelium, high-grade prostate intraepithelial neoplasia, and prostate cancer clinical specimens, as evidenced by immunohistochemical staining. Taken together, our findings demonstrate the selective enhancement of agonistic action of the anti-androgen hydroxyflutamide by the transcriptional co-activator CBP, which is a new, potentially relevant mechanism contributing to the acquisition of therapy resistance in prostate cancer.

Epstein-Barr virus-associated multicentric leiomyosarcoma in an adult patient after heart transplantation: case report and review of the literature.

Epstein-Barr virus (EBV)-associated smooth muscle tumors following solid organ transplantation are extremely rare, with only 12 cases reported in the literature thus far. The exact pathogenetic role of EBV infection in the oncogenesis of these soft tissue tumors in immunodeficient patients and the biologic behavior of such tumors is still unclear. We report a 26-year-old man in whom multiple smooth muscle tumors developed 36 to 51 months after heart transplantation. All tumors, two synchronous liver nodules, two subsequently occurring paravertebral tumors, and a single tumor in a vein at the left ankle were surgically resected. The tumor tissue was processed for routine histology and immunohistochemical (IHC) stains. Additionally, competitive polymerase-chain-reaction (PCR), reverse-transcriptase PCR (RT-PCR), as well as in situ hybridization (ISH) were used for EBV particle quantification and gene transcription analysis. The histologic features and immunohistochemical profiles were consistent with leiomyosarcoma in all tumor nodules. EBV infection was detected in >95% of tumor cell nuclei by EBER 1/2 ISH. Competitive PCR revealed 3105 EBV particles per milligram of tumor tissue. The EBV gene expression pattern analyzed by RT-PCR and IHC corresponded to the latency type III with specific expression of EBNA1, EBNA2, LMP1, and LMP2A genes. Under continuous antiviral therapy (famcyclovir) the patient currently shows no evidence of disease. Our data indicate that EBV infection plays a causal role in the development of smooth muscle tumors following organ transplantation. A latency type III, identical to EBV-associated posttransplant lymphoproliferative disorders, was identified and suggests a common pathogenetic mechanism in the development of these histogenetically distinct neoplasms. The fact that the patient currently shows no evidence of disease may be the result of the continuous administration of antiviral therapy because the soft tissue recurrences of the leiomyosarcoma occurred while the patient was not receiving antiviral prophylaxis.

Mature Dendritic Cells Induce T-Helper Type-1-Dominant Immune Responses in Patients with Metastatic Renal Cell Carcinoma

We performed a pilot study on a dendritic cell (DC)-based vaccine in 4 patients with advanced renal cell carcinoma. The vaccine consisted of cultured blood DCs loaded with autologous tumor cell lysate plus keyhole limpet hemocyanin (KLH) and matured with a combination of tumor necrosis factor α and prostaglandin E2. We describe the immune response against KLH induced by DC-based immunization in a patient undergoing an objective partial response and compare it with the responses observed in patients with either stable or progressive disease. The patient with the clinical response developed strong delayed-type hypersensitivity (DTH) against KLH after a single vaccination with antigen-loaded DCs, whereas the other patients failed to develop DTH reactivity even after repeated vaccinations. Antigenic stimulation of mononuclear cells (MNCs) induced proliferation and IFN-γ but not IL-4 production as well as expression of the chemokine receptor CXCR3 consistent with a T-helper (Th) type-1 bias. Exogenous IL-12 enhanced and exogenous IL-4 diminished IFN-γ production. In the 2 patients with stable disease two or more vaccinations were required to induce maximal MNC responses. In the patient with progressive disease MNC responses were hardly detectable. Anti-KLH antibodies appeared with different kinetics but could be detected in the serum of all patients. Isotype analysis revealed the presence of IgM, IgG1, IgG2 and IgG3 as well as IgA and complete absence of IgE. The patient with progressive disease also developed IgG4 antibodies indicative of a deviation towards Th2. Cultured blood DCs can be a potent vaccine for the antigen-specific immunization of patients with advanced kidney cancer. KLH serves as a tracer molecule which allows determination of the magnitude, kinetics and Th bias of the cellular and humoral immune response induced by DC-based immunization. The data also suggest that Th type-1-dominant immune responses involving DTH reaction are required for the induction of tumor regression.