Jack A. Schalken

HISTOLOGICAL GRADE HETEROGENEITY IN MULTIFOCAL PROSTATE CANCER. BIOLOGICAL AND CLINICAL IMPLICATIONS

In order to understand the clinical and biological implications of prostate cancer multifocality and heterogeneity, we investigated their occurrence in relation to variables such as tumour volume, local invasion, and biopsy findings. In a series of 61 completely sectioned whole‐mount radical prostatectomy specimens with clinical stage T2 prostate cancer, we mapped histological grade heterogeneity and tumour multifocality. We also evaluated 55 prostate biopsy cases to assess the accuracy of pre‐operative grading. Among all of the prostates, only 28 per cent had a single tumour and in 16 per cent one histological grade of cancer was evident. Extracapsular invasion was not restricted to the largest tumour in each case, but also occurred in tumours of relatively small volume and low histological grade. Variability of histological grade was directly proportional to tumour volume. Both grade heterogeneity and tumour multifocality of the prostatectomy specimen showed no significant relationship to the grade accuracy of biopsies. Biopsy grading error proved greatest among small, well‐differentiated tumours. Whole‐mount sectioning of prostatectomy specimens in patients with clinically localized adenocarcinoma demonstrates that grade heterogeneity is most closely related to tumour volume; that the largest (index) tumour lesion may not be representative of the pathological stage; and that grading error in prostate needle biopsies can be only partly explained by grade heterogeneity or tumour multifocality.

Canine prostate carcinoma: epidemiological evidence of an increased risk in castrated dogs.

The present retrospective study investigated the frequency of prostate carcinoma (PCA) among prostate abnormalities in dogs and determined whether castration influences the incidence of PCA in dogs. During the years 1993-1998, 15,363 male dogs were admitted to the Utrecht University Clinic of Companion Animals, and of these dogs 225 were diagnosed with prostatic disease. In addition, another 206 male dogs were diagnosed as having prostatic disease based on cytologic examination of aspiration biopsies submitted by referring veterinarians. Benign prostatic hyperplasia was diagnosed in 246 dogs (57.1%), prostatitis in 83 dogs (19.3%), and PCA in 56 dogs (13%). Dogs with PCA were significantly older (mean age=9.9 years) than dogs with other prostatic diseases (mean age=8.4 years). The Bouvier des Flandres breed had an increased risk (odds ratio (OR)=8.44; 95% CI 4.38-16.1) of having PCA. Castration (26/56) increased the risk (OR=4.34; 95% CI 2.48-7.62) of PCA. The mean age at diagnosis of PCA in castrated dogs and in intact male dogs was not significantly different. The interval between castration and onset of prostatic problems was highly variable, suggesting that castration does not initiate the development of PCA in the dog, but it does favour tumor progression.

Relation between aberrant α‐catenin expression and loss of E‐cadherin function in prostate cancer

It is now well documented that E‐cadherin expression correlates inversely with tumor grade in various carcinomas including prostate cancer. We also demonstrated a statistically significant correlation between decreased E‐cadherin expression and progression‐free period in early stage patients treated by radical prostatectomy and decreased survival in patients with advanced stage disease. We now study the relationship between E cadherin and α‐catenin expression, because in prostate cancer cell lines, mutational inactivation of the α‐catenin gene can be the cause of the impaired E‐cadherin function. Twenty patients treated by radical prostatectomy and 32 advanced stage patients were evaluated immunohistochemically for E‐cadherin and α‐catenin expression. The results were related to tumor grade and disease progression. Four patients in the radical prostatectomy group had aberrant E‐cadherin and α‐catenin expression and showed disease progression. The other 16 patients were free of progression and had normal E‐cadherin and α‐catenin expression. In the advanced stage group, 4 of 13 patients with normal E‐cadherin staining showed aberrant α‐catenin expression and 2 patients (50%) progressed, compared with only 22% progression in patients with both normal E‐cadherin and α‐catenin expression. The other 19 patients with aberrant E‐cadherin and α‐catenin staining had the poorest prognosis. Our results suggest that loss of α‐catenin expression could be one of the mechanisms responsible for the loss of E‐cadherin–mediated cell‐cell adhesion in human prostate cancer and might in some cases provide prognostic information. Int. J. Cancer 74:374–377, 1997.

Molecular analysis of multifocal prostate cancer lesions.

To analyse the origin of multifocal prostate cancer lesions, radical prostatectomy specimens from 17 patients were examined. As a marker of genetic lineage, the allelotype based on 33 microsatellite loci was compared between the different tumours present in a given case. Some results provide evidence suggestive of a clonal origin of multiple tumours in a subset of the prostates. In five cases, for example, comparison of multifocal tumour lesions within a given case revealed at least two concordant changes in allelic imbalance (AI) sequence dosages at different loci. In addition, considerable heterogeneity of allelotype was found within and among tumour foci of a given case. In five of the six tumours analysed for intratumour heterogeneity, for example, more than five discordant AI changes were found in one tumour region but not in the other. Conclusions regarding the clonality of such heterogeneous lesions are difficult to draw. A high frequency of AI changes in four lesions exhibiting prostatic intraepithelial neoplasia (mean 6·5 changes per lesion, range 3–6) was found, compared with eight primary tumours present in the same cases (mean 5·8 changes per lesion, range 3–6). The interpretation of AI associated with clinically detected prostate cancer remains a highly complex issue. The fact that no clear evidence was obtained for either a clonal or a non‐clonal origin of multiple lesions in a given prostate indicates that several different mechanisms are likely to operate in establishing the allelotype and that additional evidence from unique mutations or selective gene inactivation may be necessary to obtain definitive results. Copyright

Models for studying benign prostatic hyperplasia

Benign prostatic hyperplasia (BPH) is one of the most common diseases affecting aging man. Attempts have been made to clarify the etiology and pathogenesis and, to that end, experimental models have been developed. To date, in vitro and in vivo models have been used, depending on the concept of the study. Spontaneous animal models are limited to the chimpanzee and the dog. Ethical and financial factors restrict the applicability of these models. The hormonal-induced canine BPH model is a good alternative that closely resembles human BPH in many aspects. The experimental models currently used for studying BPH are reviewed.

Morphogenic and tumorigenic potentials of the mammary growth hormone/growth hormone receptor system.

Due to the characteristics of the luteal phase of the ovarian cycle in the dog, which spans a prolonged time period, this species is a suitable model to study the role of progestins in both normal morphogenic and abnormal tumorigenic processes in the mammary gland. It has been convincingly shown that progestins, including endogenous progesterone, induce the synthesis of growth hormone (GH) in the normal and the tumorous canine mammary gland. The growth hormone receptor (GHR) is also expressed in normal and tumorous canine mammary tissues and in this concise overview we highlight recent advances in our understanding of the significance of the GH/GHR system for mammary gland (patho)biology. In an attempt to unravel the cellular and molecular mechanisms associated with the GH/GHR system, we were able to show that both GH and GHR are differentially expressed in normal canine mammary tissues. Maximum expression of both GH and GHR occurs during the proliferation phase of the tissue, which links the progestin-induced mammary GH synthesis to the progestin-associated proliferation of epithelial cells in the mammary gland. Expression of the GH/GHR system is also present in most canine mammary tumors, albeit that GHR expression may be downregulated in undifferentiated mammary carcinomas. Upon GH stimulation of the GHR-positive CMT-U335 canine mammary tumor cell line, the transcription factors STAT5A and STAT5B become phosphorylated on their tyrosine residues, which is likely to reflect the significance of mammary GH in vivo. Molecular analysis of the canine mammary GHR transcripts by RT-PCR provided evidence for normal and alternative processing of the GHR primary transcript encoding the full-length plasma membrane GHR and at least four putative GH binding proteins (GHBPs), respectively. The translation products from the alternatively spliced GHR transcripts indicate an intact N-terminal ligand binding domain and an unique C-terminal portion, lacking the transmembrane domain and cytoplasmic tail. Thus, these proteins are considered to be able to bind GH, but have lost their signaling potential. The exact biological role of these GHBPs remains to be established, but GHBPs may have a transport function in the endocrine route, regulate the level of biologically available GH locally, or dominant-negatively influence the full-length plasma membrane GHR. In dog mammary cancer specimens strongly reduced levels of alternatively spliced GHR transcripts were found compared to the non-malignant mammary tissue. Notably, expression of both GH and GHR in mammary cancer cells is not restricted to dogs. Recent experiments generated evidence for GH and GHR expression in human breast cancer cells, and also in human prostate cancer cells, which represents another highly prevalent hormone-sensitive human malignancy. In agreement with our findings in the dog, the expression of the hGH-N gene in human mammary cancer cells seemed to correlate positively with their progesterone receptor status, which warrants, in our opinion, a reconsideration of the role of progestins in breast cancer of women. In human prostate cancer cells four different hGH-N transcripts were detected, which encode classical 22 kDa GH and GH-related proteins. Consistent with the findings on the canine GHR, different GHR transcripts in human mammary cancer cells and prostate cancer cells were detected encoding the full-length plasma membrane GHR and putative GHBPs.

MAMMARY GROWTH HORMONE AND TUMORIGENESIS : LESSONS FROM THE DOG

The discovery in the early 1990s that progestin-induced growth hormone (GH) excess in the dog originates in the mammary gland can be seen as a hallmark in the research on the pathogenesis of mammary cancer in the dog. The local biosynthesis and release of GH may provide a highly proliferative environment in the mammary gland, which contributes to the development and/or progression of mammary tumours. Before final goals such as prevention of tumour formation or inhibition of tumour promotion can be achieved it is of eminent importance to elucidate the mechanism of progesterone-induced mammary GH production and the mechanism of local autocrine/paracrine action of GH. These local GH effects may be achieved through direct growth stimulating effects of GH as well as by indirect effects mediated by the stimulation of the biosynthesis of insulin-like growth factor-I (IGF-I). The biological effects of the IGFs largely depend on the presence of IGF binding proteins (IGFBPs) which may both enhance or inhibit the activity of the IGFs. This review concentrates on recent advances in the understanding of the local mammary GH-IGF axis and the lessons which can be drawn from the dog for mammary cancer research in other species.

Expression and Molecular Characterization of the Growth Hormone Receptor in Canine Mammary Tissue and Mammary Tumors

GH synthesis has been documented in canine mammary tissue and mammary tumors. In the present report, the characteristics of the GH receptor (GHR) are studied in these tissues. First, using immunohistochemistry, GHR was found to be present throughout normal and tumorous mammary tissues, being localized in epithelial and myoepithelial/spindle cell components and in the activated fibroblasts of desmoplastic tumor stroma. GHR expression seemed to be downregulated only in terminally differentiated alveoli in normal tissue. GHR immunoreactivity in particular mammary (adeno)carcinomas was heterogenous. Second, the canine GHR was characterized at the molecular level. Northern blot analysis revealed a major GHR transcript of approximately 4.2 kb. The coding sequence of the canine GHR shows extensive homology with the GHR of several species. Seminested RTPCR (using primers annealing in exons 4 ‐5, exon 6, and exon 9) generated, next to the primary product, four different products in mammary tissues and the canine mammary tumor cell line CMTU335, which seemed to be alternative GHR transcripts. These alternative GHR transcripts were characterized by exon 8 skipping, exon 7 skipping, and use of alternative splice donor and acceptor sites. Especially, the transcript that is missing exon 8 may encode a GH binding protein. In most malignant mammary samples, only the primary transcript was present; and alternative transcripts could not be detected. The absence of alternative GHR transcripts in mammary carcinomas, and thus putative inhibitors of GH-induced signal transduction, may contribute to enhanced sensitivity of malignant tumors to GH. (Endocrinology 140: 5907‐5914, 1999)

Cell kinetics and differentiation after hormonal‐induced prostatic hyperplasia in the dog

Our aim was to characterize the immunophenotypical changes in canine prostate epithelium after hormonal‐induced benign prostatic hyperplasia (BPH).

Homozygous deletions of p16INK4 occur frequently in bilharziasis-associated bladder cancer

We have studied p16INK4 mutation (by PCR‐SSCP) and deletion (by Southern blotting and/or multiplex PCR) in a series of 47 bilharziasis‐associated tumors from Egypt and compared the results with those obtained on a series of 17 established bladder cell lines and non‐bilharziasis‐associated bladder cancers from the Netherlands. In the cell lines we found 9 homozygous deletions and 1 mutation (59% of p16INK4 alterations in cell lines), whereas in cases from the Netherlands deletions were found in 4 of 22 samples. No mutations were detected in the 46 samples screened. Interestingly, in bilharziasis‐associated bladder cancer, deletions were present in 23 samples and mutations in a further 2 cases (53% of p16INK4 alteration in bilharziasis‐associated bladder cancer). No correlation was found between p16INK4 alteration and histopathological data. Likewise, the same frequency of alteration was found in tumors with different differentiation patterns (squamous, transitional or adenocarcinoma). Three conclusions can be drawn from our findings: (i) p16INK4 alterations are more frequent in cell lines than in primary tumors; (ii) in primary bladder tumors (bilharziasis‐associated or not), p16INK4 deletions are much more frequent than p16INK4 mutations; (iii) p16INK4 alterations are more frequent in bilharziasis‐associated bladder tumors than in other bladder tumors. This high frequency of deletion is not related to a specific histological type but to the specific etiology of these tumors.