Quantitative immunohistochemical detection of the molecular expression patterns in proliferative inflammatory atrophy

The proliferative inflammatory atrophy (PIA) is considered as a possible precursor of prostate intraepithelial neoplasia (PIN) or prostate cancer (PCa). In this study we assessed quantitatively the expression of AMACR, p63, COX-2, GST and iNOS in serial paraffin-embedded tissue sections obtained after radical prostatectomy of PCa patients (n = 30). The applicability of these markers to distinguish PIA, PIN and PCa was evaluated. We also compared the immunohistochemical expression profiles of AMACR, COX-2 and GST in the luminal and basal cells in lesions of PIN arisen in PIA or PIA alone. Two different patterns of COX-2 expression according to the p63 status of the basal cells were found. This observation gives us grounds to hypothesize that the diverse COX-2 patterns resulted from an initial basal cell damage which subsequently propagated to its luminal secretory cells progeny.     

Stem/progenitor and intermediate cell types and the origin of human prostate cancer

Theories of cell lineage in human prostatic epithelium, based on protein expression, propose that basal and luminal cells: 1) are either independently capable of self-renewal or 2) arise from stem cells expressing a full spectrum of proteins (p63, cytokeratins CK5/14, CK8/18, and glutathione-S-transferase-pi [GST-pi]) similar to cells of the embryonic urogenital sinus (UGS). Such embryonic-like stem cells are thought to give rise to mature basal cells and secretory luminal cells. By single cell cloning of an immortalized, normal human prostate-derived, non-tumorigenic RWPE-1 cell line, we isolated and characterized two epithelial cell types, WPE-stem and WPE-int. WPE-stem cells show: i) strong, sixfold greater nuclear expression of p63; ii) nearly twofold greater expression of CK14; iii) threefold less CK18, and iv) low androgen receptor (AR) expression as compared with WPE-int cells. WPE-stem cells are androgen-independent for growth and survival. WPE-int cells express very low p63 and CK5/14, and high CK18. WPE-int cells are androgen-independent for growth and survival but are highly responsive as shown by androgen induction of AR and prostate specific antigen (PSA). Compared with WPE-int cells, WPE-stem cells are smaller and show more rapid growth. WPE-stem cells can grow in an anchorage-independent manner in agar with 4.5-fold greater cloning efficiency and as free floating "prostaspheres" in liquid medium; and express over 40-fold higher matrix metalloproteinase-2 activity. These results indicate that WPE-stem cells express several features characteristic of stem/progenitor cells present in the UGS and in adult prostatic epithelium. In contrast, WPE-int cells have an intermediate, committed phenotype on the pathway to luminal cell differentiation. We propose that in normal prostatic epithelium, cells exist at many stages in a continuum of differentiation progressing from stem cells to definitive basal and luminal cells. Establishment and characterization of clones of human prostatic epithelial cells provide novel models for determining cell lineages, the origin of prostate cancer, and for developing new strategies for tumor prevention and treatment.     

Application of alpha-methylacyl coenzyme A racemase immunohistochemistry in the diagnosis of prostate cancer: a review

Alpha-methylacyl coenzyme A racemase (AMACR) is a recently discovered enzyme protein that has been shown to be increased at both the mRNA and protein levels in prostatic adenocarcinoma as compared with normal prostatic tissues. Since its discovery, AMACR has gained wide acceptance for use in the diagnosis of prostatic adenocarcinoma in conjunction with morphology and immunohistochemical staining for basal cell markers. Numerous studies have consistently shown high sensitivity and specificity of AMACR for prostate cancer. This review focuses on AMACR expression in prostate cancer and its morphologic variants, high grade prostatic intraepithelial neoplasia, adenosis and benign conditions of the prostate. In addition, we discuss AMACR expression in other tumors. We also focus on the utility and technical aspects of the now-popular "triple stain" immunohistochemical antibody cocktail, consisting of antibodies to high-molecular-weight keratin, p63 and AMACR. Finally, we emphasize diagnostic pitfalls in the application of AMACR to small, atypical foci of glands seen on prostate needle core biopsy and project future diagnostic as well as clinical applications for the protein.     

Potential localization of putative stem/progenitor cells in human bulbar conjunctival epithelium

Although the conjunctival fornix appears to contain the greatest proportion of stem cells, it is likely that pockets of conjunctival epithelial stem cells may also exist throughout the conjunctival epithelium. This study was to investigate the potential localization of putative stem/progenitor cells in the human bulbar conjunctival epithelium by evaluating 6 keratins and 13 molecules that have been previously proposed stem cell associated or differentiation markers. We found that cornea specific cytokeratin (CK) 3 was not expressed by the bulbar conjunctival epithelial cells. In contrast, CK4 and CK7 were expressed by the superficial cells of bulbar conjunctival epithelium. CK14 and CK15 were confined to the basal cell layer. CK19 was strongly expressed by all layers of the bulbar conjunctival epithelium. The expression patterns of molecular markers in the basal cells of human bulbar conjunctival epithelium were found to be similar to the corneal epithelium. Basal conjunctival epithelial cells strongly expressed stem cell associated markers, including ABCG2, p63, nerve growth factor (NGF) with its receptors tyrosine kinase receptor A (TrkA) and neurotrophin low‐affinity receptor p75NTR, glial cell‐derived neurotrophic factor (GDNF) with its receptor GDNF family receptor alpha 1 (GFRα‐1), integrin β1, α‐enolase, and epidermal growth factor receptor (EGFR). The differentiation associated markers nestin, E‐cadherin and involucrin were not expressed by these cells. These findings indicate that the basal cells of bulbar conjunctival epithelium shares a similar expression pattern of stem cell associated markers to the corneal epithelium, but has a unique pattern of differentiation associated cytokeratin expression. J. Cell. Physiol. 225: 180–185, 2010. © 2010 Wiley‐Liss, Inc.     

Cell differentiation lineage in the prostate

Prostatic epithelium consists mainly of luminal and basal cells, which are presumed to differentiate from common progenitor/stem cells. We hypothesize that progenitor/stem cells are highly concentrated in the embryonic urogenital sinus epithelium from which prostatic epithelial buds develop. We further hypothesize that these epithelial progenitor/stem cells are also present within the basal compartment of adult prostatic epithelium and that the spectrum of differentiation markers of embryonic and adult progenitor/stem cells will be similar. The present study demonstrates that the majority of cells in embryonic urogenital sinus epithelium and developing prostatic epithelium (rat, mouse, and human) co-expressed luminal cytokeratins 8 and 18 (CK8, CK18), the basal cell cytokeratins (CK14, CK5), p63, and the so-called transitional or intermediate cell markers, cytokeratin 19 (CK19) and glutathione-S-transferase-pi (GSTpi). The majority of luminal cells in adult rodent and human prostates only expressed luminal markers (CK8, CK18), while the basal epithelial cell compartment contained several distinct subpopulations. In the adult prostate, the predominant basal epithelial subpopulation expressed the classical basal cell markers (CK5, CK14, p63) as well as CK19 and GSTpi. However, a small fraction of adult prostatic basal epithelial cells co-expressed the full spectrum of basal and luminal epithelial cell markers (CK5, CK14, CK8, CK18, CK19, p63, GSTpi). This adult prostatic basal epithelial cell subpopulation, thus, exhibited a cell differentiation marker profile similar to that expressed in embryonic urogenital sinus epithelium. These rare adult prostatic basal epithelial cells are proposed to be the progenitor/stem cell population. Thus, we propose that at all stages (embryonic to adult) prostatic epithelial progenitor/stem cells maintain a differentiation marker profile similar to that of the original embryonic progenitor of the prostate, namely urogenital sinus epithelium. Adult progenitor/stem cells co-express both luminal cell, basal cell, and intermediate cell markers. These progenitor/stem cells differentiate into mature luminal cells by maintaining CK8 and CK18, and losing all other makers. Progenitor/stem cells also give rise to mature basal cells by maintaining CK5, CK14, p63, CK19, and GSTpi and losing K8 and K18. Thus, adult prostate basal and luminal cells are proposed to be derived from a common pleuripotent progenitor/stem cell in the basal compartment that maintains its embryonic profile of differentiation markers from embryonic to adult stages.     

Anchorage-independent culture maintains prostate stem cells

Freshly isolated mouse prostate epithelial cells regenerate fully differentiated prostate tissue when combined with embryonic urogenital sinus mesenchyme and grafted in vivo. We show here that this regenerative capacity, which has been attributed to a small population of pleuripotential progenitor epithelial cells, is rapidly lost when the cells are placed in monolayer culture but can be maintained by culture in anchorage-independent conditions. Epithelial cells placed in anchorage-independent culture formed proliferating spheres that could be serially passaged and exhibited increased expression of putative stem cell markers as compared to cells grown in monolayer culture. Epithelial cells isolated from the fetal urogenital sinus, the newborn, and adult prostate formed spheres with similar efficiency, while cells isolated from the post-castration prostate exhibited significantly higher sphere-forming abilities. When passaged spheres were recombined with E17 rat urogenital sinus mesenchyme and grafted in vivo, they generated fully differentiated mouse prostate glandular epithelium containing both p63+ basal cells and p63− luminal cells and expressing a variety of prostate-specific and terminal differentiation markers.     

A statistical method for identifying differential gene-gene co-expression patterns

MOTIVATION: To understand cancer etiology, it is important to explore molecular changes in cellular processes from normal state to cancerous state. Because genes interact with each other during cellular processes, carcinogenesis related genes may form differential co-expression patterns with other genes in different cell states. In this study, we develop a statistical method for identifying differential gene-gene co-expression patterns in different cell states. RESULTS: For efficient pattern recognition, we extend the traditional F-statistic and obtain an Expected Conditional F-statistic (ECF-statistic), which incorporates statistical information of location and correlation. We also propose a statistical method for data transformation. Our approach is applied to a microarray gene expression dataset for prostate cancer study. For a gene of interest, our method can select other genes that have differential gene-gene co-expression patterns with this gene in different cell states. The 10 most frequently selected genes, include hepsin, GSTP1 and AMACR, which have recently been proposed to be associated with prostate carcinogenesis. However, genes GSTP1 and AMACR cannot be identified by studying differential gene expression alone. By using tumor suppressor genes TP53, PTEN and RB1, we identify seven genes that also include hepsin, GSTP1 and AMACR. We show that genes associated with cancer may have differential gene-gene expression patterns with many other genes in different cell states. By discovering such patterns, we may be able to identify carcinogenesis related genes.     

Defining Cell Lineages in the Prostate Epithelium

Understanding the stages of cell differentiation in the normal prostate epithelium isessential for the identification of the cell type(s) involved in prostatic carcinogenesis.Prostate glands are composed of three types of epithelial cells (i.e. basal, secretory andneuroendocrine) but the hierarchical relations among these cell types have been longcontroversial. We have recently developed a novel system to define prostate epithelialcell lineages in vivo. We find that, during normal prostate organogenesis, terminallydifferentiated secretory cells derive from p63-positive basal cells, which thusrepresent/include prostate stem cells. Future studies will determine if p63-positive basalcells retain stem cells capabilities in the adult prostate epithelium.     

GSTP1 CpG island hypermethylation as a molecular biomarker for prostate cancer

Somatic hypermethylation of CpG island sequences at GSTP1, the gene encoding the pi-class glutathione S-transferase, appears to be characteristic of human prostatic carcinogenesis. To consider the potential utility of this epigenetic alteration as a biomarker for prostate cancer, we present here a comprehensive review of the literature describing somatic GSTP1 changes in DNA from prostate cells and tissues. GSTP1 CpG island hypermethylation has been detected in prostate cancer DNA using a variety of assay techniques, including (i) Southern blot analysis (SB), after treatment with (5-m)C-sensitive restriction endonucleases, (ii) the polymerase chain reaction, following treatment with (5-m)C-sensitive restriction endonucleases (RE-PCR), (iii) bisulfite genomic sequencing (BGS), and (iv) bisulfite modification followed by the polymerase chain reaction, using primers selective for target sequences containing (5-m)C (MSP). In the majority of the case series so far reported, GSTP1 CpG island hypermethylation was present in DNA from at least 90% of prostate cancer cases. When analyses have been carefully conducted, GSTP1 CpG island hypermethylation has not been found in DNA from normal prostate tissues, or from benign prostatic hyperplasia (BPH) tissues, though GSTP1 CpG island hypermethylation changes have been detected in DNA from candidate prostate cancer precursor lesions proliferative inflammatory atrophy (PIA) and prostatic intraepithelial neoplasia (PIN). Using PCR methods, GSTP1 CpG island hypermethylation has also been detected in urine, ejaculate, and plasma from men with prostate cancer. GSTP1 CpG island hypermethylation, a somatic epigenetic alteration, appears poised to serve as a molecular biomarker useful for prostate cancer screening, detection, and diagnosis.     

Prostate basal cell lesions can be negative for basal cell keratins: a diagnostic pitfall

BACKGROUND: Prostate basal cell lesions can have architectural and cytologic atypia that mimic prostate adenocarcinoma. Immunohistochemical stains for basal cell markers are most helpful in the differential diagnosis. All of the published studies show basal cell lesions are positive for basal cell keratins, whereas adenocarcinoma is negative for both. We reported two cases of prostate basal cell lesions with negative basal cell keratin expression by immunohistochemistry. STUDY DESIGN: We reported the histologic and immunohistochemical profiles of two cases of basal cell lesions of the prostate. RESULTS: Histologically, both cases were highly suspicious for prostate adenocarcinoma with infiltrative growth pattern and significant nuclear atypia. The atypical glands in both cases were negative for basal cell keratins. However, both lesions were positive for another basal cell marker, p63, confirming that they were basal cells in origin, rather than prostate adenocarcinoma. CONCLUSION: Prostate basal cell lesions can occasionally be negative for basal cell keratins by immunohistochemistry and therefore may be misdiagnosed as prostate adenocarcinoma. We recommend using both p63 and basal cell keratins simultaneously in the workup of atypical prostate lesions to avoid such a misdiagnosis.     

P63 is expressed in basal and myoepithelial cells of human normal and tumor salivary gland tissues.

p63 is essential for epithelial cell survival and may function as an oncogene. We examined by immunohistochemistry p63 expression in human normal and tumor salivary gland tissues. In normal salivary glands, p63 was expressed in the nuclei of myoepithelial and basal duct cells. Among 68 representative salivary gland tumors, 63 displayed p63 reactivity. In all tumor types differentiated towards luminal and myoepithelial lineages (pleomorphic adenomas, basal cell adenomas, adenoid cystic carcinomas, and epithelial-myoepithelial carcinomas), p63 was expressed in myoepithelial cells, whereas luminal cells were always negative. Similarly, in mucoepidermoid carcinomas, basal, intermediate, and squamous cells expressed p63, in contrast to luminal mucous cells. p63 reactivity was also restricted to basal cells in Warthin tumors and oncocytomas. Myoepitheliomas and myoepithelial carcinomas all expressed p63. The only five negative tumors were three of four acinar cell carcinomas and two of three adenocarcinomas. In conclusion, p63 is expressed in the nuclei of normal human salivary gland myoepithelial and basal duct cells. p63 expression is retained in the modified myoepithelial and basal cells of human salivary gland tumors, which suggests a role for p63 in oncogenesis of these complex tumors.     

p63 in prostate biology and pathology

The identification of stem cells and differentiation programs regulating the development and maintenance of the normal prostate epithelium is essential for the identification of the cell type(s) and molecular alterations involved in the development and propagation of prostate cancer (CaP). The p53-homologue p63 is highly expressed in normal prostate basal cells and is a clinically useful biomarker for the diagnosis of CaP. Importantly, p63 has been shown to play a critical role in prostate development. Recent experimental evidence also suggests that this gene is essential for normal stem cell function in the prostate as well as other epithelial organs. Future studies aimed at better defining the role of p63 in the renewal of the adult prostate epithelium are likely to shed new light on the mechanisms involved in prostate carcinogenesis.     

Critical role of p63 in the development of a normal esophageal and tracheobronchial epithelium

The trachea and esophagus originate from the foregut endoderm during early embryonic development. Their epithelia undergo a series of changes involving the differentiation of stem cells into unique cell types and ultimately forming the mature epithelia. In this study, we monitored the expression of p63 in the esophagus and the trachea during development and examined in detail morphogenesis in p63^–/– mice. At embryonic day 15.5 (E15.5), the esophageal and tracheobronchial epithelia contain two to three layers of cells; however, only the progenitor cells express p63. These progenitor cells differentiate first into ciliated cells (p63^–/-tubulin IV⁺) and after birth into mature basal cells (p63⁺/K14⁺/K5⁺/BS-I-B4⁺). In the adult pseudostratified, columnar tracheal epithelium, K14⁺/K5⁺/BS-I-B4⁺ basal cells stain most intensely for p63, whereas ciliated and mucosecretory cells are negative. In stratified squamous esophageal epithelium and during squamous metaplasia in the trachea, cells in the basal layer stain strongest for p63, whereas p63 staining declines progressively in transient amplifying and squamous differentiated cells. Generally, p63 expression is restricted to human squamous cell carcinomas, and adenocarcinomas and Barrett's metaplasia do not stain for p63. Examination of morphogenesis in newborn p63^–/– mice showed an abnormal persistence of ciliated cells in the esophagus. Significantly, in both tissues, lack of p63 expression results in the development of a highly ordered, columnar ciliated epithelium deficient in basal cells. These observations indicate that p63 plays a critical role in the development of normal esophageal and tracheobronchial epithelia and appears to control the commitment of early stem cells into basal cell progeny and the maintenance of basal cells. retinoic acid; stem cell; carcinoma; basal cell; differentiation