T.H. van der Kwast

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.

Ultrasound-guided fine needle aspiration cytology of axillary lymph nodes in breast cancer patients. A preoperative staging procedure

Currently, axillary lymph node dissection is increasingly being replaced by the sentinel node procedure. This method is time-consuming and the full immunohistochemical evaluation is usually only first known postoperatively. This study was designed to evaluate the accuracy of preoperative ultrasound-guided fine needle aspirations (FNAs) for the detection of non-palpable lymph node metastases in primary breast cancer patients. We evaluated the material of 183 ultrasound-guided FNAs of non-palpable axillary lymph nodes of primary breast cancer patients. The cytological results were compared with the final histological diagnosis. Ultrasound-guided FNA detected metastases in 44% (37/85) of histologically node-positive patients, in 20% of the total patient population studied. These pecentages are likely to be higher when women with palpable nodes are included. Cytologically false-negative and false-positive nodes were seen in 28 (15%) and three cases (1.6%), respectively. Interestingly 25% (n=7) of the false-negative nodes, revealed micrometastases on postoperative histology. The sensitivity was 57%, the specificity 96%. We conclude that ultrasound-guided FNA of the axillary lymph nodes is an effective procedure that should be included in the preoperative staging of all primary breast cancer patients. Whether lymph nodes are palpable or not, it will save considerable operating time by selecting those who need a complete axillary lymph node dissection at primary surgery and would save a significant number of sentinel lymph node dissections (SLNDs).

The clinical significance of androgen receptors in breast cancer and their relation to histological and cell biological parameters

To analyse the clinical significance of the presence of androgen receptors (AR) in breast carcinomas, clinical and histological parameters of 153 primary breast carcinomas (median follow-up 46 months) were examined. Oestrogen (ER) and progesterone receptor (PR) levels were determined in cytosol preparations using enzyme immunoassay assays and in cryostat sections by immunohistochemistry. AR and Ki-67 levels were only determined immunohistochemically. Data were analysed by uni- and multivariate models. 94/153 (61%) breast carcinomas were ER+ PR+ AR+, while 14 cases were only positive for AR. All grade III tumours (n = 17) were steroid receptor negative and 14 (76%) of these cases demonstrated high Ki-67 values suggestive of more aggressive behaviour. Strikingly, 14 ductal carcinomas negative for ER and PR were positive for AR. In univariate analysis, AR as well as ER, tumour size, lymph node status, grade and Ki-67 proved to be significant prognostic factors for disease-free survival (DFS). Multivariate analysis, however, showed lymph node status, tumour size and ER status to be the only independent prognostic factors for DFS within this model. We conclude that simple histological and cell biological parameters, including AR, can be used to select high- and low-risk patients at the time of primary surgery and can provide valuable information on treatment options.

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.

The PTEN gene in locally progressive prostate cancer is preferentially inactivated by bi-allelic gene deletion

PTEN is frequently inactivated during the development of many cancers, including prostate cancer, and both bi‐allelic and mono‐allelic PTEN inactivation may contribute to tumorigenesis. PTEN mutations in clinical cancer specimens can easily be recorded but mono‐ or bi‐allelic gene deletions are often difficult to assess. We performed a comprehensive study to detect PTEN inactivation in 40 locally progressive clinical prostate cancer specimens obtained by transurethral resection of the prostate, utilizing a variety of complementary technical approaches. The methods to detect PTEN deletion included allelotype analysis, dual‐colour FISH and array‐based CGH. We also applied a novel semi‐quantitative approach, assessing the PTEN‐WT (wild‐type): PTEN‐Ψ (pseudogene) ratio (WPR). Structural analysis of PTEN was performed by single‐strand conformational polymorphism (PCR‐SSCP) and sequencing. PTEN protein expression was assessed by immunohistochemistry. Our data predict complete PTEN inactivation in 12 samples (30%), nine of these by bi‐allelic deletion. Loss of one PTEN copy was also detected by several methodologies but the number could not be accurately assessed. Immunohistochemistry indicated the absence of PTEN protein in 15 samples, and heterogeneous expression of the protein in eight tumours. Taken together, these data show that bi‐allelic deletion is a major mechanism of PTEN inactivation in locally progressive prostate cancer. Copyright

Immunohistochemical detection of the androgen receptor with monoclonal antibody F39.4 in routinely processed, paraffin-embedded human tissues after microwave pre-treatment.

We describe the immunohistochemical detection of the human androgen receptor (AR) in routinely processed, paraffin-embedded tissue with the monoclonal antibody (MAb) F39.4. Deparaffinized sections were heated in a microwave oven for antigen retrieval. A panel of human male- and female-derived tissues was investigated. We observed a nuclear staining pattern consistent with previous results on frozen sections. Moreover, we studied the possibility of detecting AR in prolonged formalin-fixed tissue and in paraffin-embedded archival material. After prolonged fixation times or long-term storage of paraffin-embedded tissue, the staining intensity for the AR did not deteriorate. Blocking experiments with the specific synthetic peptides demonstrated the specificity of this technique. We conclude that this method is specific, allows retrospective AR studies, and offers optimally preserved morphology.

The prognostic significance of apoptosis-associated proteins BCL-2, BAX and BCL-X in clinical nephroblastoma

Apoptotic cell death represents an important mechanism for the precise regulation of cell numbers in normal tissues. Various apoptosis-associated regulatory proteins, such as Bcl-2, Bax and Bcl-X, may contribute to the rate of apoptosis in neoplasia. The present study was performed to evaluate the prognostic value of these molecules in a group of 61 Wilms’ tumours of chemotherapeutically pre-treated patients using an immunohistochemical approach. Generally, Bcl-2, Bax and for Bcl-XS/L were expressed in the blastemal and epithelial components of Wilms’ tumour. Immunoreactive blastema cells were found in 53%, 41% and 38% of tumours for Bcl-2, Bax and for Bcl-XS/L, respectively. An increased expression of Bcl-2 was observed in the blastemal component of increasing pathological stages. In contrast, a gradual decline of Bax expression was observed in the blastemal component of tumours with increasing pathological stages. Also blastemal Bcl-XS/L expression decreased with stage. Univariate analysis showed that blastemal Bcl-2 expression and the Bcl-2/Bax ratio were indicative for clinical progression, whereas epithelial staining was of no prognostic value. Multivariate analysis showed that blastemal Bcl-2 expression is an independent prognostic marker for clinical progression besides stage. These findings demonstrate that alterations of the Bcl-2/Bax balance may influence the clinical outcome of Wilms’ tumour patients by deregulation of programmed cell death.

Variations in activin receptor, inhibin/activin subunit and follistatin mRNAs in human prostate tumour tissues.

The possible role of activin in the regulation of malignant prostatic growth was studied using RNAase protection assays of activin receptors, inhibin/activin subunits and follistatin mRNAs in the human prostatic carcinoma cell lines LNCaP-FGC, -R and -LNO, in human prostatic carcinoma xenografts and in human prostatic tissue. Activin receptor types IA (ActRIA), IB (ActRIB), IIA (ActRIIA) and IIB (ActRIIB) mRNAs were generally expressed in prostate pithelial cells, with significantly lower levels of ActRIB mRNA in prostate tumour aterial when compared to non-malignant tissue (P< 0.05; Mann–Whitney U -test). Inhibin/activin βA- and βB-subunit mRNA expression was also found in prostate tissue. Androgen-independent xenografts expressed significantly lower amounts of βB-subunit mRNA when compared to androgen-dependent xenografts (P< 0.05). While βB-subunit mRNA was expressed by LNCaP-FGC and -LNO cells, virtually no expression was found in the androgen-independent LNCaP-R line. Inhibin α-subunit mRNA levels were low or undetectable in all samples investigated. Follistatin mRNA was undetectable in LNCaP-sublines, while low levels were found in prostatic tissues. In androgen-independent LNCaP-R cells, activin inhibited cell growth in a dose-dependent manner. These results suggest that prostate tumour progression is accompanied by a decrease of the inhibitory effect of locally produced activin by either a decrease in the expression of activin βB-subunit mRNA or by a decrease of ActRIB mRNA levels.

Prostatic intraepithelial neoplasia and endocrine manipulation

Prostatic intraepithelial neoplasia (PIN) is the most common precursor lesion of prostatic adenocarcinoma. In 50- to 70-year-old participants of a randomized screening program for prostate cancer (Rotterdam section of the ERSPC) the frequency of high-grade PIN as an isolated finding in sextant prostatic needle biopsies was estimated to be about 1%. As yet, data in literature on the impact of androgen deprivation on PIN lesions are limited, showing discrepant outcomes. In part this may be the consequence of the application of different criteria for the identification of PIN under conditions of androgen deprivation. Foci of PIN could be distinguished in the majority of radical prostatectomy specimens of men treated for 3 or 6 months with combined endocrine therapy. Endocrine manipulation led to architectural changes (remodelling) in residual PIN which were more pronounced at 6 months of endocrine therapy. This is consistent with a prolonged effect of androgen deprivation on this precursor lesion. The presence of MIB-1 immunopositive nuclei in PIN lesions suggests that they still have the potential to expand after cessation of therapy.

Epidermal participation in post-burn hypertrophic scar development

Abstract The reconstruction of epidermal architecture over time in normotrophic and hypertrophic scars in untransplanted, spontaneously healed partial-thickness burns has scarcely been studied, unlike the regeneration of epidermal grafts used to cover burn wounds and the regeneration of the dermis during hypertrophic scarring. The expression of markers of epidermal proliferation, differentiation and activation in normotrophic and hypertrophic scars in spontaneously healed partial-thickness burns was assessed and compared with the expression of these markers in normal control skin of healthy persons to determine whether hypertrophic scarring is associated with abnormalities in the phenotype of keratinocytes. Punch biopsies were taken both of partial-thickness burns after re-epithelialisation and of matched unburned skin. At 4 and 7 months post-burn, biopsies were taken of normotrophic and hypertrophic scars that had developed in these wounds. The biopsies were analysed using immunostaining for markers of keratinocyte proliferation, differentiation and activation (keratins 5, 10, 16 and 17, filaggrin, transglutaminase and CD36). We observed a higher expression of markers for proliferation, differentiation and activation in the epidermis of scars at 1 month post-burn than in normal control skin of healthy persons. There was a striking difference between normotrophic and hypertrophic scars at 4 months post-burn. Keratinocytes in hypertrophic scars displayed a higher level of proliferation, differentiation and activation than did normotrophic scars. At 7 months post-burn all keratinocyte proliferation and differentiation markers showed normal expression, but the activation marker CD36 remained upregulated in both normotrophic and hypertrophic scars. Surprisingly, in matched unburned skin of burn patients, a state of hyperactivation was observed at 1 month. Our results suggest that keratinocytes may be involved in the pathogenesis of hypertrophic scarring.