Joan Morote

Prognostic value of immunohistochemical expression of the c-erbB-2 oncoprotein in metastasic prostate cancer.

It is well established that the activation of proto‐oncogenes could trigger uncontrolled cell growth and cancer development. Although this correlation has already been evidenced in several human tumors, no conclusive studies have related oncogene activation with the development of prostatic neoplasia. Nevertheless, some reports suggest that c‐erbB‐2, which is a prognostic marker in breast cancer, could be implicated in the development of prostatic adenocarcinoma. We have studied the expression of the c‐erbB‐2 oncoprotein in primary prostatic tissue in a series of 70 patients with metastasic disease, by means of immunohistochemistry. The NCL‐B 11 anti‐c‐erbB‐2 monoclonal antibody was used, and the immunoreactivity was quantified by image analysis. The overall rate of prostatic‐tissue sections presenting positive c‐erbB‐2 immunostaining was 64.3%. No significant relation was observed between histological grade and c‐erbB‐2 over‐expression or severity of the disease, based on the extent of metastases. The average specific survival in patients with c‐erbB‐2 over‐expression was 33 months, while it was 54 in patients with c‐erbB‐2 negativity; p < 0.034. These results, as well as the logistic‐regression analysis, suggest that expression of c‐erbB‐2 oncoprotein would be considered as an independent prognostic factor of metastatic prostate cancer. Moreover, it could discriminate between the prognosis of patients with Gleason score 2 to 7 and those with score 8 to 10. Our results suggest that the expression of c‐erbB‐2 oncoprotein in primary prostatic tissue could have a prognostic value in patients with metastatic prostate cancer. Int. J. Cancer (Pred. Oncol.) 84:421–425, 1999.

PTOV1, a novel protein overexpressed in prostate cancer containing a new class of protein homology blocks

In a search for molecular markers of progression in prostate cancer by means of differential display, we have identified a new gene, which we have designated PTOV1. Semiquantitative RT–PCR has established that nine out of 11 tumors overexpress PTOV1 at levels significantly higher than benign prostatic hyperplasia or normal prostate tissue. The human PTOV1 protein consists almost entirely of two repeated blocks of homology of 151 and 147 amino acids, joined by a short linker peptide, and is encoded by a 12-exon gene localized in chromosome 19q13.3. A Drosophila melanogaster PTOV1 homolog also contains two tandemly arranged PTOV blocks. A second gene, PTOV2, was identified in humans and Drosophila, coding for proteins with a single PTOV homology block and unrelated amino- and carboxyl-terminal extensions. A 1.8-Kb PTOV1 transcript was detected abundantly in normal human brain, heart, skeletal muscle, kidney and liver, and at low levels in normal prostate. Immunocytochemical analysis and expression of chimeric GFP-PTOV1 proteins in cultured cells showed a predominantly perinuclear localization of PTOV1. In normal prostate tissue and in prostate adenomas, PTOV1 was undetectable or expressed at low levels, whereas nine out of 11 prostate adenocarcinomas showed a strong immunoreactivity, with a focal distribution in areas of carcinoma and prostatic intraepithelial neoplasia. Therefore, PTOV1 is a previously unknown gene, overexpressed in early and late stages of prostate cancer. The PTOV homology block represents a new class of conserved sequence blocks present in human, rodent and fly proteins.

The reproducibility and predictive value on outcome of renal biopsies from expanded criteria donors

Reproducibility and predictive value on outcome are the main criteria to evaluate the utility of histological scores. Here we analyze the reproducibility of donor biopsy assessment by different on-call pathologists and the retrospective evaluation by a single renal pathologist blinded to clinical outcomes. We also evaluate the predictive value on graft outcome of both evaluations. A biopsy was performed in donors with any of the following: age≥55 years, hypertension, diabetes, creatinine>1.5 mg/dl, or stroke. Glomerulosclerosis, interstitial fibrosis, tubular atrophy, intimal thickening, and arteriolar hyalinosis evaluated according to the Banff criteria were added to obtain a chronic score. Biopsies were classified as mild (≥3), intermediate (4-5), or advanced (6-7) damage, and unacceptable (≥8) for transplantation of 127 kidneys biopsied. Weighted κ value between both readings was 0.41 (95% CI: 0.28-0.54). Evaluation of biopsies by the renal pathologist was significantly and independently associated with estimated 12-month glomerular filtration rate and a significant composite outcome variable, including death-censored graft survival and time to reach an estimated glomerular filtration rate<30 ml/min per 1.73 m2. Thus, there was no association between readings of on-call pathologists and outcome. The lack of association between histological scores obtained by the on-call pathologists and graft outcome suggests that a specific training on renal pathology is recommended to optimize the use of kidneys retrieved from expanded criteria donors.

Increased glyceraldehyde-3-phosphate dehydrogenase expression in renal cell carcinoma identified by RNA-based, arbitrarily primed polymerase chain reaction

Renal cell carcinoma (RCC) comprises 85% of renal tumors and displays a great capacity to metastasize. The lack of diagnostic and prognostic markers complicates its early detection and in the majority of cases metastases are present at the time of diagnosis.

Identification of multipotent mesenchymal stromal cells in the reactive stroma of a prostate cancer xenograft by side population analysis.

Cancer stem cells are a distinct cellular population that is believed to be responsible for tumor initiation and maintenance. Recent data suggest that solid tumors also contain another type of stem cells, the mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs), which contribute to the formation of tumor-associated stroma. The Hoechst 33342 efflux assay has proved useful to identify a rare cellular fraction, named Side Population (SP), enriched in cells with stem-like properties. Using this assay, we identified SP cells in a prostate cancer xenograft containing human prostate cancer cells and mouse stromal cells. The SP isolation, subculture and sequential sorting allowed the generation of single-cell-derived clones of murine origin that were recognized as MSC by their morphology, plastic adherence, proliferative potential, adipogenic and osteogenic differentiation ability and immunophenotype (CD45(-), CD81(+) and Sca-1(+)). We also demonstrated that SP clonal cells secrete transforming growth factor beta1 (TGF-beta1) and that their inhibition reduces proliferation and accelerates differentiation. These results reveal the existence of SP cells in the stroma of a cancer xenograft, and provide evidence supporting their MSC nature and the role of TGF-beta1 in maintaining their proliferation and undifferentiated status. Our data also reveal the usefulness of the SP assay to identify and isolate MSC cells from carcinomas.

Over-expression of epidermal growth factor receptor and c-erbB2/neu but not of int-2 genes in benign prostatic hyperplasia by means of semi-quantitative PCR.

It is well established that activation of cellular genes may trigger uncontrolled cell growth and cancer development. Previous reports suggest that the epidermal growth factor receptor (EGFr), c‐erbB2/neu and int‐2, fibroblast growth factor‐3, may be implicated in the development of benign prostatic hyperplasia (BPH). Using the polymerase chain reaction technique, we have assessed the amplification and expression of these molecular markers in 30 prostate samples from patients with BPH as well as from 5 normal donors. We detected mRNA over‐expression of EGFr and c‐erbB2/neu in 36% and 63%, respectively, of the BPH samples, but no gene amplification was found. No amplification or over‐expression of int‐2 was detected in any of the samples analyzed, suggesting that int‐2 is not involved in BPH. Our results thus suggest a role for EGFr and c‐erbB2/neu but not for int‐2 in the development of BPH. Int. J. Cancer 76:464–467, 1998.© 1998 Wiley‐Liss, Inc.

Estrogen receptor beta displays cell cycle-dependent expression and regulates the G1 phase through a non-genomic mechanism in prostate carcinoma cells

Background: It is well known that estrogens regulate cell cycle progression, but the specific contributions and mechanisms of action of the estrogen receptor beta (ERβ) remain elusive. Methods: We have analyzed the levels of ERβ1 and ERβ2 throughout the cell cycle, as well as the mechanisms of action and the consequences of the over-expression of ERβ1 in the human prostate cancer LNCaP cell line. Results: Both ERβ1 mRNA and protein expression increased from the G1 to the S phase and decreased before entering the G2/M phase, whereas ERβ2 levels decreased during the S phase and increased in the G2/M phase. ERβ1 protein was detected in both the nuclear and non-nuclear fractions, and ERβ2 was found exclusively in the nucleus. Regarding the mechanisms of action, endogenous ERβ was able to activate transcription via ERE during the S phase in a ligand-dependent manner, whereas no changes in AP1 and NFκB transactivation were observed after exposure to estradiol or the specific inhibitor ICI 182,780. Over-expression of either wild type ERβ1 or ERβ1 mutated in the DNA-binding domain caused an arrest in early G1. This arrest was accompanied by the interaction of over-expressed ERβ1 with c-Jun N-terminal protein kinase 1 (JNK1) and a decrease in c-Jun phosphorylation and cyclin D1 expression. The administration of ICI impeded the JNK1–ERβ1 interaction, increased c-Jun phosphorylation and cyclin D1 expression and allowed the cells to progress to late G1, where they became arrested. Conclusions: Our results demonstrate that, in LNCaP prostate cancer cells, both ERβ isoforms are differentially expressed during the cell cycle and that ERβ regulates the G1 phase by a non-genomic mechanism.

Immunolocalization of Androgen Receptors, Estrogen α Receptors, and Estrogen β Receptors in Experimentally Induced Canine Prostatic Hyperplasia

Benign prostatic hyperplasia (BPH) is an age-dependent prostatic disease affecting male humans and dogs. In dogs, the combined administration of estrogens and androgens synergistically increases prostate weight, and continued treatment leads to the development of glandular hyperplasia. The aim of the present study was to examine the immunohistochemical expression of androgen receptor (AR), estrogen receptor alpha (ER alpha), and estrogen receptor beta (ER beta) in the different cell types of the prostate gland in an experimental model. Five male beagle dogs were castrated and treated with 25 mg of 5 alpha-androstane-3 alpha and 17beta-diol and 0.25 mg 17beta-estradiol for 30 weeks. Prostate specimens were surgically obtained every 45 days (experimental stages M0 to M6: 0, 12, 18, 24, 30, and 36 weeks from the beginning of the hormonal treatment). The control group consisted of 3 noncastrated dogs treated with a vehicle, from which specimens were only taken at the time points M0, M1, M4, and M6. Immunohistochemical data revealed high AR and ER alpha expression in the epithelial and stromal cell nuclei of all the experimental and control specimens. Weak staining of the cytoplasm was observed only in epithelial cells. The suspension of hormone treatment led to a significant reduction in the expression of both receptors. On the contrary, ER beta was expressed only in epithelial cell nuclei, with no significant differences in the percentages of stained nuclei between control and hormonally treated or atrophic prostates. Results indicate that AR, ER alpha, and ER beta are differently expressed in canine prostate tissue and that they show specific expression patterns in response to the hormonal induction of BPH.

Preoperative Prediction of Pathologically Insignificant Prostate Cancer in Radical Prostatectomy Specimens: The Role of Prostate Volume and the Number of Positive Cores

Introduction: To determine clinical and biopsy features with predictive capacity to identify pathologically insignificant prostate cancer (pIPCa). Material and Methods: pIPCa was defined as cancer volume <0.5 cm3 and a Gleason score (GS) of ≤6 in radical prostatectomy (RP) specimens. Clinical and biopsy parameters were studied as predictors of pIPCa and validated by applying them to d’Amico’s low-risk cases: T1c-T2a, prostate-specific antigen (PSA) <10 and biopsy GS ≤6. Appropriate cut-offs were selected. Results: 280 patients were evaluated; 11.8% (33) had pIPCa, increasing to 23% in low-risk cases. In patients fulfilling d’Amico’s low-risk criteria, variables significantly different in pIPCa were: volume, number of positive cylinders (NPC), percentage of positive cylinders (%PC), percentage of the most affected cylinder (%MAC) and uni/bilaterality. In these cases, volume and the NPC increased as independent variables on logistic regression and when adding a volume threshold of 45 cm3 and 1 positive core, specificity reached 95.8%. Conclusions: The incidence of pIPCa in RP specimens is relevant, especially in low-risk cases. Prostate volume and NPC are independent predictors of pIPCa. We propose a simple predictive model by adding the features of 1 positive core and volume ≧45 cm3 to d’Amico’s criteria. This allows to preoperatively distinguish between patients that most probably would benefit from radical treatment and patients that might be offered active surveillance.

Ultrastructural Changes in Prostate Cells During Hormone-induced Canine Prostatic Hyperplasia

Benign prostatic hyperplasia is a prevalent disease that has received relatively little attention in spite of its morbidity and remarkable social impact. There are few animal models of prostatic hyperplasia. The dog is the only species, along with humans, in which prostatic hyperplasia develops spontaneously and almost universally with age. The aim of the present study has been to compare the ultrastructural findings in a model of experimentally induced canine prostatic hyperplasia with those of the spontaneously developed changes in untreated dogs. An experimental group of 5 male beagle dogs were castrated and treated with combined steroids (3 weekly doses for over 30 weeks). Prostate samples were surgically obtained every 42 days (experimental stages 0 through 6). The control group consisted of 3 noncastrated dogs that were treated with vehicle and in which samples were taken only at stages 0, 1, 4, and 6. Changes in the control groups were similar but of lower intensity compared to those of the experimental groups. In luminal cells, crowding with papillary projections, prominent, branching microvilli, and abundant, often compartmentalized granules were observed. The most striking change was the previously unreported finding of caveolae in basal cells. They were mostly located in the basal aspect of basal cells and were more prominent in the experimental group and in advanced stages of treatment. These ultrastructural findings have not been previously reported in canine or human prostatic hyperplasia and merit further research. The model of experimentally induced canine prostatic hyperplasia provides an adequate setting for the understanding of this disease.