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Prostate Pathology of Genetically Engineered Mice: Definitions and Classification. The Consensus Report from the Bar Harbor Meeting of the Mouse Models of Human Cancer Consortium Prostate Pathology Committee

¹ Department of Pathology and Vanderbilt Prostate Cancer Center, and ² Department of Urologic Surgery and Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center, Nashville, Tennessee; ³ Department of Pathology, University of California at Los Angeles, Los Angeles, California; ⁴ National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina; ⁵ Department of Pathology, Baylor College of Medicine, Houston, Texas; ⁶ Departments of Pathology and Urology, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts; ⁷ Departments of Pathology and Urology, Washington University of St. Louis, St. Louis, Missouri; ⁸ The Jackson Laboratory, Bar Harbor, Maine; ⁹ Veterinary and Tumor Pathology Section, Office of Laboratory Animal Resources, National Cancer Institute, Frederick, Maryland; and ¹⁰ Center for Comparative Medicine, University of California, Davis, Davis, California

ABSTRACT

The Pathological Classification of Prostate Lesions in Genetically Engineered Mice (GEM) is the result of a directive from the National Cancer Institute Mouse Models of Human Cancer Consortium Prostate Steering Committee to provide a hierarchical taxonomy of disorders of the mouse prostate to facilitate classification of existing and newly created mouse models and the translation to human prostate pathology. The proposed Bar Harbor Classification system is the culmination of three meetings and workshops attended by various members of the Prostate Pathology Committee of the Mouse Models of Human Cancer Consortium. A 2-day Pathology Workshop was held at The Jackson Laboratory in Bar Harbor, Maine, in October 2001, in which study sets of 93 slides from 22 GEM models were provided to individual panel members. The comparison of mouse and human prostate anatomy and disease demonstrates significant differences and considerable similarities that bear on the interpretation of the origin and natural history of their diseases. The recommended classification of mouse prostate pathology is hierarchical, and includes developmental, inflammatory, benign proliferative, and neoplastic disorders. Among the neoplastic disorders, preinvasive, microinvasive, and poorly differentiated neoplasms received the most attention. Specific criteria were recommended and will be discussed. Transitions between neoplastic states were of particular concern. Preinvasive neoplasias of the mouse prostate were recognized as focal, atypical, and progressive lesions. These lesions were designated as mouse prostatic intraepithelial neoplasia (mPIN). Some atypical lesions were identified in mouse models without evidence of progression to malignancy. The panel recommended that mPIN lesions not be given histological grades, but that mPIN be further classified as to the absence or presence of documented associated progression to invasive carcinoma. Criteria for recognizing microinvasion, for classification of invasive gland-forming adenocarcinomas, and for characterizing poorly differentiated tumors, including neuroendocrine carcinomas, were developed and are discussed. The uniform application of defined terminology is essential for correlating results between different laboratories and models. It is recommended that investigators use the Bar Harbor Classification system when characterizing new GEM models or when conducting experimental interventions that may alter the phenotype or natural history of lesion progression in existing models.

Introduction and Objectives

The increased generation of potential models of prostate neoplasia in genetically engineered mice (GEM) and their use in investigations of possible cancer therapies in prostate carcinoma (Pca) mandate the development of a standardized pathology classification scheme. Because mice and other rodents do not spontaneously develop Pca, histological criteria have been developed based on the disorders observed in newly created GEM models and by efforts to translate these lesions to the familiar histopathology of human Pca and its precursor lesions. Because the goal of the Mouse Models of Human Cancer Consortium (MMHCC) is to model human neoplasia, use of criteria and terminology applied to human prostate pathology is logical. However, as detailed herein, there are anatomical and natural history issues that impact on the ability to make straightforward analogies between GEM models of Pca and the human disease being modeled. Furthermore, in addition to pathological criteria, other criteria that can be incorporated into characterization and validation of GEM models include genetic and other molecular alterations and the natural history of the prostate lesions, and the similarity of these aspects to human Pca.

GEM models will be useful for delineating novel causative molecular alterations in the development and/or progression of Pca and useful in testing interventions that will translate to treatments in human Pca patients if such models are similar, at least in some regards, to this heterogeneous human neoplasia at initiating or secondary molecular alterations. Because histopathologic features are a phenotypic consequence of these underlying molecular alterations, pathology assessment will be useful for characterizing new models and for detecting potentially meaningful changes as a consequence of genetic crosses or therapeutic interventions.

Protocols for proper tissue submission are necessary for characterizing the pathology of the prostate and other organs in new GEM models. Tissue-based analysis of biological parameters including proliferation, apoptosis, and microvessel density can contribute to model characterization and provide mechanistic insight into effects of genetic manipulations and therapeutic interventions.

Hence, the specific objectives of the MMHCC Prostate Pathology Committee to facilitate characterization and application of GEM models of prostatic disease were as follows: (a) development of a classification scheme for disorders of the prostate and related organs in GEM; (b) provision of histopathologic definitions for these disorders; (c) collection and annotation of images illustrating these disorders; and (d) collection, organization, and distribution of pathology protocols useful in characterization of prostate disorders in GEM.

Because GEM are being used to model human neoplasms for investigational purposes, the pathological classifications of the disorders in various organ sites in GEM are intended to facilitate translation of GEM research to critical issues in understanding the causes and discovering more effective treatments for human malignancies. For GEM models to have application to specific or broad subsets of human Pca patients, it is fundamental to understand GEM prostate pathology in the context of human prostate pathology. This is a driving principle in the development of the classification scheme presented herein. Knowledge of the basic anatomical and histological similarities and differences between the mouse and human prostate is necessary. As such, this report includes considerations of prostate anatomy, and clinical and pathological features of the full spectrum of both benign and malignant human prostate disorders, similarities to which have been or can potentially be encountered in GEM models. Each section for the classification scheme is in general divided into definition of that disorder, criteria for recognition of the disorder in the GEM prostate, clinical considerations and morphological features in human prostate pathology, and the pathological and biological features of that entity in GEM models.

The images illustrating the various lesions in the mouse pathology classification were taken from the slides of the models provided to the MMHCC Prostate Pathology Committee (Table 1) or additional materials made available to the authors. Where possible, multiple models are illustrated for a specific lesion, to emphasize the common features of the disorder and to allow visualization of the process against different backgrounds, and so forth. The inclusion of a model or the reference to a model regarding a specific lesion is not intended as a potential endorsement or criticism of that specific model. Similarly, the illustration of a specific lesion in a single slide is not intended to be taken as a generalization regarding the natural history of that model. Characterization of GEM models is an integrated endeavor incorporating pathology, natural history, and molecular characterizations. This classification scheme and accompanying images illustrate how pathology characterization can be applied to existing models, and, hence, provides guidelines for characterization of future models as well.

General Considerations

Several general principles regarding characterization of GEM models for Pca were elaborated at the Bar Harbor Pathology Workshop (Table 2) .

The low incidence of pathology in the prostate of wild-type mice suggests that any of the lesions described in GEM, including inflammatory and other non-neoplastic disorders, could be a consequence of the genetic manipulation involved. These lesions could be due to systemic effects (e.g., immunological or endocrinologic effects) or be a direct consequence of a genetic alteration in the prostate. Some differences in the frequency of spontaneous lesions could also exist between different genetic strains of mice. These factors should be borne in mind when evaluating mild phenotypes occurring in a small percentage of examined animals.

Importance of Adequate Controls and of Blinded Histopathologic Assessment
The characterization of new GEM models should include comparison with appropriate controls, particularly aged-matched mice of identical genetic background. The importance of aged-matched control mice is especially true for aged GEM mice and those with only mild phenotypes. Blinded histopathologic analysis is highly desirable in all of the studies that use histopathology as an end point. Blinded histopathologic analysis for data collection can be performed in a blinded fashion after initial review of possible pathology in GEM mice. Characterization of GEM models for Pca should, when at all possible, include the analysis by an experienced pathologist, and preferably one with experience in both human and mouse prostate pathology. Members of the MMHCC Prostate Pathology Committee are available for review of pathology material generated by investigators engaged in GEM research, both within and outside the MMHCC. Centralized review of particularly promising models, including in future Pathology Workshops, is desirable.

Importance of Genetic Background
The genetic background could have modifying influences on lesion development and/or progression in GEM models of prostate disorders. Some examples of the possible influence of different genetic backgrounds on neoplastic progression have already been observed (2 , 3) . Therefore, great care must be taken in describing the genetic background and breeding strategies involved in the creation of new GEM models and in the production of mice obtained from other laboratories.

The Role of Natural History in Model Characterization
The importance of the natural history of neoplastic progression was stressed as fundamental to the characterization of any given model. The time course of lesion development and progression, and any defined accompanying molecular alterations are important characteristics of a model. Individual GEM models may show different histopathologic features at different ages, and a careful description of the frequency of specific lesions at specific time points is vital. These may be the attributes used to validate a model for relevance to a particular aspect of the biology of human Pca.

Certain clinically relevant general features of the natural history of human Pca are well known. These features, such as development of invasive gland-forming cancer from in situ precursor lesions, progression to locally advanced disease, metastases to lymph nodes and bone, and progression to hormone refractory disease, may be desirable in a mouse model. It is unlikely that any given model will faithfully mimic even these general attributes. Accurate descriptions of the temporal progression of histopathologic alterations and of the molecular changes detected with progression are important parameters of model characterization. Identification of molecular alterations in GEM models that are already known in some human Pcas will identify models that may be useful for testing targeted therapeutic strategies. Therefore, the histopathology, natural history, and accompanying molecular and genetic alterations will be part of the information available on National Cancer Institute websites for GEM models of Pca.

Anatomical Considerations

Anatomy of the Human Prostate and the Zonal Origin of Pca
Some anatomical similarities between the mouse and human prostate help support the application of GEM models for the study of molecular alterations that accompany the development and progression of Pca. Both species have male accessory organs that develop from the Wolffian ducts and the urogenital sinuses. Both species have androgen-sensitive organs and form lobular glands that have a similar triad of distinctly differentiated epithelial cells and similar functions. However, there are also some crucial differences between the prostatic glands in the two species. These include differences in the gross and microanatomy that have implications for pathological interpretation in mouse models and for the use of the mouse for modeling some clinicopathologic characteristics of human Pca.

Although distinct lobes can be recognized in the developing human prostate, the adult human prostate is not divided into discreet lobar structures. The past use of terms, such as lateral and posterior "lobes" has been supplanted by the term "zones" based on the concept of specific zones within the human prostate. These zones are anatomically recognizable, have characteristic histological features, and, importantly, have specific predisposition to benign or malignant neoplastic diseases. As described by McNeal (4, 5, 6) , the human prostate is composed of the anterior fibromuscular stroma, the periurethral transition zone (TZ), the peripheral zone (PZ), and the central zone (CZ; Fig. 1 ). The TZ is particularly associated with benign prostatic hyperplasia (BPH) and the PZ with Pca. The CZ surrounds the ejaculatory ducts (Fig. 1C) and comprises an increasing portion of the prostate from where the ejaculatory ducts enter the urethra, near the prostatic utricle at the verumontanum, to the base. The CZ glands have characteristic morphology, with large complex glands showing a more irregular luminal border, with epithelial tufting, papillary formations, and frequent Roman arches or even cribriforming (Fig. 1, C–E) . The histological characteristics of the CZ and its spatial relationship to the ejaculatory ducts have lead to a suggested origin from the Wolffian duct (7) . However, there is currently insufficient data to support a Wolffian duct rather than urogenital sinus origin of the CZ. The CZ is rarely the site of origin for Pca, although it can be secondarily involved by extension from a PZ tumor.

View larger version (105K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Gross and microscopic anatomy, and zone of origin of prostate adenocarcinoma in the human prostate. A, gross photograph showing a cross-section of a prostatectomy specimen in which the transition zone (TZ) is markedly expanded by fleshy nodules of benign prostatic hyperplasia (BPH; black arrowheads). The TZ is demarcated from the posteriorly and laterally located peripheral zone (PZ) by fibrous tissue (black arrows) compressed by the expanding TZ. Medially, the urethra (white arrows) is slit-like due to compression by the BPH-expanded TZ. Lateral aspects of the PZ are indicated (white arrowheads). B, gross photograph showing a cross-section of a prostatectomy specimen in which the TZ and PZ appear somewhat spongy due, in part, to dilated, atrophic glands. Note the homogenous, tan-gray tumor nodule in right posterolateral PZ (arrowhead). This is a common area of involvement for usual PZ tumors. A tumor in this posterior location would likely be palpable as a discrete, firm nodule on digital rectal examination. Prostate carcinoma is not typically discernible grossly, especially with smaller tumors detected by PSA screening (urethra, arrow). C–E, low, intermediate, and high magnification photomicrographs of normal central zone (CZ) glands in a prostatectomy specimen. C, the CZ surrounds the ejaculatory ducts (arrowhead), which penetrate the prostate parenchyma and empty into the urethra at the verumontanum, a raised posterior ridge at approximately the junction of the mid and apical third of the prostate. The CZ is located in the posterior medial aspects of the prostate and occupies more tissue toward base. D and E, CZ glands are larger in diameter than usual PZ glands and have more irregular luminal contours due to papillary infoldings. Roman arches (arrowheads), imparting a cribriform architecture (arrowheads), are common in CZ glands. However, normal CZ glands lack cytologic atypia, a feature that helps to distinguish them from prostatic intraepithelial neoplasia on transrectal biopsy. F, high magnification of normal benign PZ glands in radical prostatectomy specimen. Compared with usual acinar prostate carcinoma, benign glands are larger and have a tufted or undulating luminal border. Benign glands have two distinct cell layers, the basal cells and the differentiated luminal secretory cells. Basal cells are not always discernible or distinguishable from adjacent underlying stromal cells by light microscopy. Secretory cells may be variably stratified or pseudostratified but lack features of cytologic atypia that are characteristic of prostatic intraepithelial neoplasia. Secretory cells in benign glands typically exhibit a clear to granular, faintly eosinophilic cytoplasm, which is variably disrupted at the luminal border due to ongoing apocrine-type secretion. G, intermediate magnification of HMWCK immunostaining (CK 903) of benign prostate glands in radical prostatectomy specimen. The basal cell layer is circumferentially intact in multiple, adjacent gland profiles. Basal cell hyperplasia is evident focally (arrowhead). H and I, typical human TZ tumor. H, whole mount section, in which TZ and PZ are easily identified due to the expansion of the TZ by BPH nodules (arrowheads) composed of hyperplastic glandular and stromal elements. Tumor (*), a Gleason score 2 + 3 = 5 carcinoma, is outlined by ink dots and is clearly located within the TZ and extending into the anterior aspect of the prostate (black arrow). Urethra and periurethral region where prostatic ducts enter, shown by white arrow at level of verumontanum. I, high-power photomicrograph, showing a common TZ tumor morphology, corresponding to Gleason pattern 2. Tumor is composed of intermediate to large glands, with ample, fairly clear cytoplasm. Nuclei are basally located and some are pyknotic. Scattered large more vesicular nuclei with prominent nucleoli were also present confirming the carcinoma diagnosis. Occasional intraluminal dense pink secretions (more typical of carcinoma than benign glands) are noted (arrowheads). J and K, typical human PZ tumor. J, whole mount section showing outlined PZ tumor (*), a Gleason score 3 + 4 = 7 carcinoma, in right posterolateral aspect of the gland. Expansion of the TZ by nodules of glandular and stromal hyperplasia (BPH changes, arrowheads) is evident. Note the extension of the PZ laterally (arrows). K, intermediate power photomicrograph of tumor in J, showing stromal invasion by discrete, well-formed glands (arrows) in a Gleason pattern 3 component and the transition to a higher grade Gleason pattern 4 focus (*), where more solid-like growth of fused glands is evident. Note occasional crystalloids and dense pink secretions within lumens of carcinoma glands (arrowheads).

View larger version (172K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Epithelial hyperplasia in the human and mouse prostate. A–C, low, intermediate, and high magnification photomicrographs demonstrating histological features of benign prostatic hyperplasia (BPH) in a section from a radical prostatectomy specimen performed for Pca. Similar changes can be found in specimens from simple prostatectomy and transurethral resections of the prostate performed for symptomatic BPH. BPH consists of nodules of hyperplastic glandular and stromal elements that occur in the transition zone of the human prostate. A, low power shows the circumscribed, nodular growth pattern. B and C, glands may be increased in number and may have increased epithelial tufting (arrowheads), but are otherwise fairly normal in appearance. BPH is not associated with cytologic atypia (i.e., nuclear and nucleolar enlargement) that is seen in prostatic intraepithelial neoplasia (PIN) in the peripheral zone. Stromal hypercellularity without atypia is commonly noted in foci of BPH. D, basal cell hyperplasia at edge of BPH nodule in human prostate. High power photomicrograph showing some gland profile with a well-defined basal cell layer (arrowheads), other gland profiles with stratified or multiple layers of small basal cells (arrows), and some composed of solid balls or nests of basal cells (*). Typical stromal hypercellularity seen with basal cell hyperplasia in the transition zone is appreciated at top and far bottom left. E, clear cell cribriform hyperplasia in transition zone of human prostatectomy specimen. This entity is occasionally observed as an incidental finding in association with BPH nodules, but can also be seen in the central zone. In contrast to cribriform carcinoma or cribriform high-grade PIN, there is no significant cytologic atypia in clear cell cribriform hyperplasia. Basal cells are often quite conspicuous in these foci, either as a well-defined, circumferential layer (arrowheads) or as small focal tufts of basal cell hyperplasia. Mild stromal hypercellularity can be appreciated focally (*). F and G, epithelial hyperplasia in mouse prostate. Low- and high-power photomicrographs of a cribriform proliferation within a pre-existing gland lumen is noted (*), with essentially normal surrounding stroma (arrowhead). Anterior prostate from 22-month Nkx 3.1 -/- mouse. In the focus shown, there is no appreciable cytologic atypia. Atypia, if present in hyperplasia, should be noted and described. Some of these mice show foci of epithelial atypia that progresses in extent and severity, compatible with mouse PIN. As epithelial proliferation and nuclear atypia are the morphological hallmarks of PIN, criteria of focality and progression need to be addressed for the appropriate distinction of hyperplasia with atypia and PIN (see text for details).

The PZ is also the predominant, essentially exclusive, site of PIN in the human prostate (8 , 9 , 12 , 18) . Epithelial hyperplasia analogous to that seen in TZ BPH does not occur in the PZ. Instead epithelial proliferation occurs within the confines of pre-existing normal gland profiles, and is designated as low- or high-grade PIN based predominantly on nuclear features as described below (12 , 19) .

Histology and Phenotype of Human Prostate Glands
In the human prostate, benign glands are composed of a basal epithelial cell layer and differentiated secretory luminal cells (i.e., two cell types), with some immunophenotypically defined transitional or intermediate forms and a small subpopulation of cells showing neuroendocrine (NE) differentiation (6 , 20 , 21) . Benign glands are larger than typical cancer glands, and have an undulating or slightly tufting luminal contour due in part to stratification or pseudostratification of secretory cells and the mechanisms of cellular secretion (Refs. 6 , 22 ; Fig. 1F ). Basal cells in benign human prostate glands are the dividing or progenitor cell (a subset of which may be the true prostatic "stem cells"), giving rise to the differentiated secretory cells lining the gland lumens and, most likely, to NE cells as well (21 , 23) . Basal cells tend to be oriented parallel to the basement membrane, are not always conspicuous by light microscopy, and can be difficult to distinguish from underlying spindle stromal cells (6) . They are routinely recognized by immunostaining with antibodies to high molecular weight cytokeratin (HMWCK; Fig. 1G ). Malignant prostate glands do not possess such a basal cell layer, with the atypical cells presumably representing aberrantly differentiated or neoplastic counterparts of secretory cells. Pragmatically, the absence of an immunophenotypically defined basal cell layer is a useful adjunct for recognizing malignant glands and distinguishing them (particularly in biopsies) from small gland profiles of benign glands or certain well-described mimics of Pca, such as atrophy, partial atrophy, and atypical adenomatous hyperplasia (adenosis; Refs. 8 , 9 , 18 , 24, 25, 26 ).

Anatomy and Histology of the Mouse Prostate
In contrast to the human, the rodent prostate is divided into anatomically distinct lobes. The mouse prostate can be separated into the AP or coagulating gland, the VP, and dorsal and lateral lobes, often grouped together as the DLP (Figs. 2 and 12 ; Ref. 27 ). The lobes are generally invested by a thin mesothelial-lined capsule that separates the various lobes from each other. This capsule may not always be appreciated grossly, but can often be seen in microscopic sections. The individual mouse prostate lobes are composed of a series of branching ducts or tubules that end blindly (Fig. 2) . The glandular prostate is separated from the mesothelial-lined capsule by various amounts of loose fibroadipose connective tissue that contains the major vascular channels, nerves, and ganglia. The individual ductules making up each lobe of the mouse prostate are surrounded by a thin fibromuscular tunica that is composed of only a few layers of bland spindle cells that are smooth muscle actin immunopositive and interspersed in eosinophilic collagen (Fig. 2, A–F) . The abundant intervening dense fibromuscular stroma surrounding the glands and their immediate stroma of adjacent "lobules" found in the human prostate is not present in the mouse (compare to Fig. 1, A, B, H, and J ; Ref. 16 ). Hence, there are clear, fundamental differences in the anatomical organization of the prostate between the human and mouse. These different anatomical features also create potential differences regarding the biology of neoplastic extension outside of the prostate in the human (e.g., amount of stroma, location of nerves, presence of a "capsule" between prostate and periprostatic fat, seminal vesicle proximity to sites commonly involved by ECE in the human). Therefore, it was the opinion of the Bar Harbor Pathology Panel that mouse models may not be adequate or suited to address these particular clinicopathologic or staging issues related to ECE in human Pca.

View larger version (131K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Histology of the mouse prostate. A, intermediate power photomicrograph of adult wild-type mouse anterior prostate. The anterior prostate shows the most complex luminal architecture compared with the other lobes of the mouse prostate, including frequent mucosal folds protruding into the gland lumens (arrowheads). See text for a more detailed histological description. B, low-power photomicrograph showing adjacent dorsal (D) and lateral (L) prostate lobes of an adult, wild-type mouse. A thin rim of fibromuscular stroma and then more peripherally located loose connective tissue surrounds individual glands in both the lateral and dorsal lobes. Note the differences in the epithelial cell thickness and luminal diameters in the lateral and dorsal prostate, and the increased amount of eosinophilic secretory product in the glands in the dorsal prostate compared with the lateral prostate (see text for details). C, high-power photomicrograph of adult, wild-type mouse dorsal prostate. Note the thin rim of fibromuscular stroma surrounding individual gland profiles (arrowheads). Low- (D) and high- (E) magnification photomicrographs of mouse ventral prostate. Only a thin rim of fibromuscular stroma surrounds individual glands (arrowheads), with loose connective tissue extending between individual gland profiles. This is in contrast to the greater amount of contractile fibromuscular stroma surrounding all of the gland lobules in the human prostate. See text for descriptions of nuclei, cytoplasm, and nature of secretions. F, a high molecular weight cytokeratin (CK5) immunostained section from adult, wild-type mouse prostate shows the well-defined basal cell layer that is often circumferential and extends into the normal, short luminal in-foldings (arrowheads).

View larger version (91K): [in this window] [in a new window] [Download PPT slide]   Fig. 12. Technique for histological examination of the prostate with en bloc submission. A, exposed intact prostate, bladder, and seminal vesicles [genitourinary (GU) bloc] after linear ventral abdominal incision. The white curvilinear seminal vesicles are readily apparent. B, schematic diagram of removed GU bloc (anterolateral view), after transection of the urethra (UR). VP, ventral prostate; LP, lateral prostate; DP, dorsal prostate; SV, seminal vesicles; CG, coagulating gland or anterior prostate; DD, ductus deferens; UB, urinary bladder. Horizontal black line indicates the level of transverse sectioning through the urethra to include both dorsolateral prostate and ventral prostate, at or near level of SV junction. C, removed GU bloc from animal in A, corresponding to that illustrated schematically in B. The amputated segment of distal urethra is longitudinally oriented at bottom. D, same bloc after transverse section through urethra as indicated in B, generating the lower and middle portions of tissue shown. An additional transverse section through seminal vesicles and anterior prostate has been made (top). The free portions of the seminal vesicles can be embedded on end for sectioning. The two portions after the transverse cut through the urethra should be embedded with their cut surfaces downward (sectioning into the cut surfaces). E, microscopic section illustrating the resulting tissue section, allowing typically adequate visualization of DP, LP, and ventral prostate, as well as other tissues that may have pathology (e.g., ampullary glands, shown, and periurethral glands, not well visualized in section, but typically demonstrable in deeper sections). 1, urethra in cross-section; 2, paired ductus deferens in cross-section; 3, paired ampullary glands in cross-section; 4, ventral prostate; 5, lateral prostate; 6, dorsal prostate.

The mouse AP is closely apposed to the seminal vesicles, along its entire curving length (1 , 16 , 27 , 31) . Histologically, it normally demonstrates a more papillary and cribriform growth pattern than the other lobes, with cuboidal to columnar epithelial cells containing typically central nuclei with inconspicuous to small nucleoli, and eosinophilic granular cytoplasm. The gland lumens contain abundant slightly eosinophilic secretions (Fig. 2A) . Despite the complex growth pattern of the epithelium in the AP and its close spatial relationship to the Wolffian duct-derived seminal vesicles, the mouse AP is clearly derived from the urogenital sinus (27) . The mouse VP has flatter luminal edges and only focal epithelial tufting or in-folding (Fig. 2, D and E) . The abundant luminal spaces typically contain homogenous pale serous secretions. The nuclei are small, uniform, typically basally located, and have inconspicuous to small nucleoli (Fig. 2E) .

The glands of each of the mouse prostate lobes appear to have normal cell populations homologous to the human prostate, including luminal secretory cells, a basal cell layer, and a minor population of NE cells. In the mouse, as in normal human prostate glands, a basal cell layer is not conspicuous by routine light microscopy, and ultrastructural studies had reported previously the lack of a continuous basal cell layer in normal mouse prostate glands (32) . Antibodies to HMWCK (66 kDa and 57 kDa), which identify the basal cell layer in benign human glands, had been reported to not identify a similar phenotypic basal cell layer in normal mouse glands (33) . However, a more recent study using a rabbit polyclonal antibody to mouse cyto-keratin (CK) 5 showed staining of a basal cell layer in histologically normal prostate glands (Ref. 34 ; Fig. 2F ). Similar results have been achieved with antibodies to CK14 (35) ,¹⁸ and even with a murine antibody against human HMWCK that is commonly used in human prostate pathology (36) . Whether decreased immunostaining for HMWCK will be observed in PIN lesions in GEM models (34) and/or absence of immunostaining for HMWCK will have diagnostic utility in recognizing invasive adenocarcinoma in GEM models as in human Pca remains to be thoroughly addressed. Limited markers exist for secretory cell differentiation in the mouse prostate, such as antibodies to DLP protein (37) . Immunostaining for chromogranin (CG) demonstrates a very minor population of immunophenotypically defined NE cells in the normal mouse prostate (33 , 38) . Such cells appear to represent <0.3% of the normal mouse prostate cell population.¹⁹

Ampullary Glands
The ampullary glands in the mouse are androgen-dependent glandular outpouchings of the proximal ductus deferens, with one gland on each side. The single proximal ducts enter into the ductus deferens proximal to the seminal vesicles. The ampullary glands are of Wolffian duct origin, in contrast to the urogenital sinus origin of the prostate, and add secretions to the semen that contribute to fertility (39) . They have no known human counterpart. However, familiarity with their gross and microscopic anatomy is important for the proper interpretation of lesions that may be noted in the male accessory glands of GEM (40) . Although they may be separately dissected to facilitate their identification, they can also be identified by their characteristic location and their characteristic secretions when seen in sections of male reproductive organs submitted en bloc as described in the protocols section below (e.g., see Fig. 12 ). The epithelium is simple columnar and in comparison to the prostate ducts, is surrounded by a denser fibromuscular stroma. The secretions have a characteristic "swiss cheese" appearance, with holes noted in the dense eosinophilic secretions. Enlargement with epithelial hyperplasia was noted in the ampullary glands (and other Wolffian duct-derived tissues, such as seminal vesicle), but not the prostate lobes, in mice overexpressing int2/Fgf-3 under the control of the mouse mammary tumor virus long terminal repeat (40) .

Bulbourethral Glands
As with the prostate, the bulbourethral glands (BUGs) and periurethral glands are also androgen regulated derivatives of the urogenital sinus. The BUGs in the male mouse are analogous to Cowper’s glands in human males, which are located subjacent to the urethra in the suburothelial connective tissue at the membranous portion of the urethra, immediately distal to the prostatic apex. Cowper’s glands can occasionally be seen in apical portions of RP specimens and are rarely sampled "accidentally" in transrectal biopsies, where they may cause diagnostic confusion. In the human, Cowper’s glands are rarely the site of origin of carcinoma. As some strategies for targeting transgenes to the mouse prostate have also resulted in transgene expression and pathology in these other male accessory glands, familiarity with their location and histological features is important for adequate pathological characterization of GEM models of Pca.

In the mouse, the BUGs are located more distally along the urethra, separated more distinctly from the prostate than in the human, and lie under the bulbocavernous muscle (28) . In contrast to the prostate, which does not show histologically distinct acini and excretory ducts, the periurethral glands and BUGs have a "biphasic" appearance, with lobules of secretory acini and central excretory ducts that are lined by cuboidal epithelium and empty into the urethra. The secretory acini of the BUGs are arranged in lobules, the cells of which have basally located nuclei and abundant pale mucinous cytoplasm (41) .

Periurethral Glands
In the human, the periurethral glands (glands of Littre) are located along the penile or spongy urethra. Again, these glands are rarely the site of cancer origin. Of note, in keeping with their developmental relationship to the prostate, these tissues can express prostate-specific antigen (PSA). In the mouse, the periurethral glands are located in the suburothelial tissue distal to the portions of the urethra that have the openings of the prostate ducts (more proximal than the BUGs). They are not routinely dissected from the prostate and other male accessory tissues grossly, but are commonly seen in "en bloc" sections (e.g., see Fig. 12 in the "Protocols" section) or in sections of remaining tissue (including urethra, and proximal SV and prostate ducts) submitted after dissection and separate submission of individual prostate lobes. The periurethral glands are composed of lobules of acini and short excretory ducts that open into the urethra (e.g., see Fig. 11 ). In wild-type mice, the acinar epithelium is cuboidal with oval nuclei and a denser more eosinophilic granular cytoplasm compared with secretory cells of the BUGs (41) . A variable outpouching, called the urethral diverticulum, is occasionally found in some mouse strains. It is lined by a urethral mucosa with associated periurethral glands. Unaware prosectors may confuse this diverticulum with the BUG, because it is frequently at the same anatomical level as the BUG, or with an abnormal urethra.

View larger version (170K): [in this window] [in a new window] [Download PPT slide]   Fig. 11. Neoplastic involvement of periurethral and bulbourethral glands in genetically engineered mice. A and B, atypical hyperplasia in periurethral glands in a 22-week-old C(3)1-SV40 mouse. At low-power magnification (A), hyperchromasia in multiple foci of periurethral glands is evident (arrowheads), without apparent architectural distortion. Urethral lumen is to the top/top-right. A single prostate gland profile is seen at bottom, below the muscle between it and the periurethral glands. At higher magnification (B), multiple acinar and/or duct profiles show atypical cells (arrowheads), with nuclear enlargement, hyperchromasia, and chromatin clumping. Stromal invasion is not present. C and D, extensive involvement of periurethral region and prostate by a poorly differentiated carcinoma [with possible neuroendocrine (NE) differentiation] in a 38-week-old C(3)1-SV40 mouse. Low power (C) shows a large tumor focus involving periurethral region (urethral lumen, black *, top), as well as extensive destructive involvement of prostate (white *, bottom left). Distinguishing the actual site of origin could be difficult. Note more normal-appearing prostate gland lumens at right. The single small nodule of tumor in this region (arrow) would be most compatible with secondary involvement at this site (i.e., extension or spread from tumor of periurethral region or from other part of prostate) as no prostatic intraepithelial neoplasia is present in adjacent portions of prostate. Prostatic intraepithelial neoplasia is commonly noted with invasive tumor originating in the prostate. D, high-power magnification of the tumor in C, showing occasional gland or rosette formation (arrowhead) Most tumor cells show scant cytoplasm, with a high nuclear:cytoplasmic ratio. Many of the nuclei are hyperchromatic, with occasional nuclear molding, features typical of NE differentiation. The differential diagnosis includes poorly differentiated adenocarcinoma versus NE carcinoma. Tumors with morphology typical of NE carcinomas (as illustrated for the prostate in Fig. 9 and for other accessory glands below) should be designated as such. Immunohistochemistry or electron microscopy can be used to confirm the NE nature suggested by the characteristic histological appearance. Adenocarcinomas in human, and potentially in mice, may show features of NE differentiation upon ultrastructural or immunohistochemical analysis. In lesions with definitive and predominant glandular differentiation, this can be designated as carcinoma, or adenocarcinoma, with NE differentiation, rather than as NE carcinoma. E, involvement of bulbourethral gland by a NE carcinoma in a 44-week LPB-Tag 12T-10 mouse. Focal tumor cell apoptosis is present in center (arrowheads). Urethral lumen is to top left (*). F, involvement of periurethral glands by NE carcinoma in a 17-week-old LPB-Tag 12T-7s mouse. Urethral lumen (*) and urethral mucosa are to top left. Nuclear features and rosetting (arrowhead) typical of NE differentiation identifiable by light microscopy alone are present. Note similar morphological appearance of tumors in E and F to NE carcinomas arising in the prostate as shown in Fig. 9 . G, extensive involvement of periurethral region (center) and proximal portions of prostate (far right and far left) by poorly differentiated neoplasm compatible with NE carcinoma (including poorly differentiated or small cell carcinoma) in 24-week-old TRAMP mouse. Tumor bulges into urethral lumen (*). Focal residual normal periurethral glands are noted at left and bottom left relative to urethral lumen (arrowheads). Whether such tumors in this region are ever found without morphologically identical large tumor apparently arising in the prostate has not been described in this model. The androgen regulation of the periurethral and bulbourethral glands mandates careful examination of these tissues in transgenic mouse models made with androgen-regulated promoters. H, focal atypical hyperplasia apparently involving duct of periurethral gland (arrowheads) in a CR2-SV40 mouse. These tall atypical epithelial cells appear to conform to the normal duct lining. There is nuclear enlargement, hyperchromasia, and chromating clumping, and ample eosinophilic cytoplasm. Residual periurethral gland acini are seen (arrows). As this promoter targets a neuroendocrine epithelial cell population in the prostate in an apparently androgen-insensitive manner, it is tempting to speculate whether a minor NE cell population in these other accessory glands is the target for this lesion. In contrast to at least the C3(1)-SV40 and occasional LPB-Tag tumors shown herein, however, these lesions have not been noted to progress to frank carcinoma.

The Pathological Classification of Prostate Lesions in GEM is the result of a directive from the MMHCC Prostate Steering Committee to provide a specific hierarchical taxonomy of disorders of the mouse prostate to facilitate classification of existing and newly created mouse models and their translation to human prostate pathology. The classification system described herein is the culmination of three meetings and workshops attended by various members of the Prostate Pathology Committee of the MMHCC. In April 2001, an initial 2-day meeting was held at Vanderbilt University Medical Center (organized by S. B. S. and attended by S. B. S., R. L. R., R. H., N. R., R. B., J. M. W., and R. D. C.), in which models were presented, slides were reviewed, and approaches to classification of mouse prostate disorders were discussed. These processes were continued and a hierarchical taxonomy of mouse prostate diseases was drafted with annotated images at the 2-day Pathology Committee meetings (attended by S. B. S., G. V. T., R. L. R., N. R., J. M. W., and R. D. C.) accompanying the MMHCC Steering Committee meeting in San Francisco in July 2001. Finally, a formal 2-day Pathology Workshop was held at The Jackson Laboratory in Bar Harbor, Maine, in October 2001, preceding the National Cancer Institute MMHCC-sponsored Conference on Modeling Human Pca in Mice at The Jackson Laboratory on October 18–21, 2001. This session was organized by S. B. S. and attended by members of the MMHCC Prostate Pathology Committee (S. B. S., G. V. T., R. L. R., R. H., J. M. W., and R. D. C.), a representative of The Jackson Laboratory (J. P. S.), and three invited "outside" prostate pathology experts (M. M. I., M. A. R., and P. A. H.), who were chosen by the Prostate Pathology Committee Chairman on the basis of their well-recognized expertise in human prostate pathology, their research interests in Pca, and their experience with characterization and utilization of GEM models of Pca. The combined efforts leading to the Bar Harbor Classification represent a balanced effort of investigational human (M.D. and/or M.D./Ph.D.) pathologists (S. B. S., R. L. R., G. V. T., M. M. I., M. A. R., P. A. H., R. B., and R. D. C.) and veterinary (D.V.M. and/or D.V.M./Ph.D.) pathologists (R. H., J. P. S., N. R., and J. M. W.), typically with specific research interests in Pca and in studies using GEM.

For the Bar Harbor meeting, paraffin blocks and/or glass slides from 24 models of GEM were made available through generous donation by investigators from across the country. A complete set of these slides was brought to the Bar Harbor meeting, and slides and images derived from them were shared with all of the pathologists. Provided paraffin blocks were sectioned, and H&E stained before hand and supplemented with sets of H&E-stained slides from individual investigators to allow for the creation of personal study sets of individual slides from 22 of these models (Table 1) . Some models included different ages of mice and/or different tissues, such that study sets of 93 slides from these 22 models were provided to the 10 panelists at the Bar Harbor meeting. All of these slides were reviewed in detail at the meeting and the slide sets were retained by the individual pathologists for later review.

S. B. S. organized the meetings with input from R. D. C. S. B. S. prepared the entire text of this article, incorporating ideas and comments made at the time of the meeting and subsequently by the Panel members. The classification scheme was generated by combined opinions of all of the participants, and all of the listed authors were valuable contributors to the material of this report.

The photomicrographs in Figs. 1 2 3 4 5 6 7 8 9 10 11 were prepared from images of the slide sets of the Bar Harbor meeting obtained with a Nikon Professional Digital SLR D1 camera, resolution 2012 x 1324 pixels and 12 bits per color, attached to an Olympus BX50 5-headed microscope with U-PLAN objectives. Images were captured using Nikon Capture software and processed in PhotoShop, with final figures generated as Jpegs at 200 d.p.i. In figure legends, images referred to as low, intermediate, and high magnification typically represent original magnifications of x40, x100, and x400, respectively.

View larger version (177K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Hypoplasia, metaplasia, atrophy, and inflammation in human and mouse prostate. A, abnormal or delayed development, consistent with hypoplasia, in section of anterior prostate from genetically engineered mouse (GEM). The prostate was small and ill-defined grossly. Although the illustrated lesion is from a suboptimal histologic section, reduced gland profiles with associated prominent stroma (arrowheads) can be appreciated, surrounded by conspicuous nerve ganglia (*). (Low-power photomicrograph of anterior prostate from 19-week-old homozygous SMAD3 knockout mouse, Smad3 -/-.20 B, human prostate from prostatectomy specimen, low-power photomicrograph showing normal primary periurethral ducts, which are typically at least partially lined by urothelium, extending out from urethra (with partially denuded lining) in lower left (*). C, High-power photomicrograph of transrectal biopsy specimen from human prostate showing prominent transitional metaplasia. Patient in blinded chemoprevention trial, and may or may not have received long-term treatment with 5-reductase inhibitor. Note, normal urothelium as well as transitional metaplasia will immunostain with antibodies to high molecular weight cytokeratin, similar to basal cells and basal cell hyperplasia. Transitional metaplasia, which can be seen with basal cell hyperplasia, is recognized by histological resemblance to normal urothelium, including such features as frequent nuclear grooves. D, transitional metaplasia in GEM prostate. High-power photomicrograph showing portions of three gland profiles with epithelial stratification, flattening of cells toward the surface, dense eosinophilic cytoplasm, and nuclear features also compatible with transitional metaplasia (arrowheads). Intraepithelial (and stromal) inflammatory cells (*) and some epithelial reactive changes are present as well. Section from DLP of an LPB-Tag 12T7s mouse castrated at 22 weeks and sacrificed at 29 weeks. E, mucinous metaplasia in prostate epithelium of GEM, characterized by large cytoplasmic vacuoles compressing nuclei to basal aspect in cells highly reminiscent of intestinal goblet cells (e.g., arrowheads top right). High-power photomicrograph of prostate from 24-month-old Pb-Ras mouse.21 F, atrophy (spontaneous, not treatment-related) in peripheral zone of human prostatectomy specimen. Low power showing dilated glandular profiles with flat lumens on left (arrowheads) and very typical shrunken lobules on right (arrows). These appear hyperchromatic on scanning magnification because of the high nuclear:cytoplasmic ratio due to scant cytoplasm. Recognition of lobular architecture is a useful feature. Especially when associated with inflammation, atypia with mildly enlarged nucleoli can be present. An at least partial basal cell layer would be present on high molecular weight cytokeratin immunostaining. G, active prostatitis histologically (i.e., not necessarily associated with clinical symptomatology) in section of a human prostatectomy specimen. High power shows multiple gland profiles involved, with intraluminal and intraepithelial inflammatory cells, including neutrophils (arrowheads), as well as inflammation in surrounding stroma. Glands are partially atrophic, with dilated, angular profiles, and shrunken eosinophilic cytoplasm. Reactive atypia can be present, and mitotic figures can even occasionally be found. H, nonspecific granulomatous prostatitis in human prostatectomy specimen. High-power photomicrograph shows sheet-like growth of histiocytes and admixed inflammatory cells (*), without well-formed granulomas or giant cells (see text for details). I, atrophy and inflammation, including "active prostatitis," in section of castrated GEM (LPB-Tag 12T7s castrated at 22 weeks, sacrificed at 29 weeks). Three dilated gland profiles with somewhat flattened epithelium (arrowheads) and inflammatory cells within epithelium, stroma, and periprostatic connective tissue are present (arrows). Residual hypercellular stroma, not well demonstrated, is seen focally adjacent to dilated gland in bottom left (*). The extent of intraepithelial and stromal inflammation in this example is more pronounced than that typically seen in human specimens from patients treated with antiandrogens before radical prostatectomy for prostatic carcinoma.

View larger version (164K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Combined epithelial and stromal proliferations in genetically engineered mice (GEM): prostatic intraepithelial neoplasia (PIN) versus hyperplasia versus benign neoplasms. A, combined epithelial and stromal proliferation in a GEM prostate. Low-power photomicrograph of dorsolateral prostate (DLP) of a LPB-Tag 12T5 mouse at 19 weeks, shows marked lobular expansion by a fairly symmetric and uniform proliferation of atypical epithelial cells (arrowheads) with hypercellular stroma (arrows). In stromal hyperplasia, the stromal elements may be fairly normal in appearance but show increased cellularity or they may show cytologic atypia, which should be described. Occasionally, hyperplastic stromal elements are more condensed and consist of crowded spindle cells with scant cytoplasm more immediately adjacent to atypical epithelium. In the LPB-Tag 12T5 mouse and related fast-growing LPB-Tag lines this epithelial lesion begins focally and quickly progresses in extent to an essentially diffuse lesion, and occasionally progresses to invasion. Thus, it has been regarded as PIN with a morphology distinct from PIN occurring within pre-existing gland spaces (see text). In SV40 or large T-antigen GEM models with such exuberant and diffuse atypical glandular and stromal hyperplasia, the distinction between true invasion versus herniation of glandular and stromal proliferations into periprostatic loose connective tissue or fat can sometimes be difficult (see text for details). B, photomicrograph showing a lobular expansion of glands in a 16-week-old TRAMP mouse prostate by atypical prostatic epithelium and a mildly hypercellular stroma (arrowheads). This is a somewhat uniform and symmetric epithelial lesion in which the small, peripheral acini appear to connect to the larger, more central lumen (*), with similar cytologic atypia. The stromal hyperplasia shown here is not in response to invasion and can be seen diffusely surrounding all three of the illustrated gland profiles. The distinction between lesions like those shown in A and B as PIN versus adenocarcinoma can be difficult. Histological features that help to distinguish adenocarcinoma, such as architecturally distinct foci and desmoplasia, are described in the text. The consensus of the Pathology Committee was that lesions like those shown in A and B represent in situ lesions. Compare these in situ lesions to the well-differentiated adenocarcinoma shown in Fig. 8E. C , hypercellularity of stroma (arrowheads) admixed with proliferating atypical glands (arrows) in 24-week-old TRAMP mouse prostate. These markedly hypercellular stromal elements consist of spindle cells with scant cytoplasm (arrowheads). Foci with these characteristics are also common in AP and DLP of fast-growing LPB-Tag lines (see text), and are different in appearance from the more smooth muscle-appearing hypercellular stroma also noted. These foci are usually seen in immediate apposition to atypical epithelium. The reactive versus neoplastic nature of this type of stromal proliferation or possible epithelial-mesenchymal transformation have not been thoroughly addressed. Cytologic atypia and mitotic activity can be noted. Possible prostatic stromal origin for poorly differentiated spindle cell lesions in metastatic foci should be considered in GEM models with such characteristics. Ancillary techniques described in the "Protocols" section can be useful for distinguishing metastatic carcinoma versus sarcoma. D, markedly atypical epithelial and admixed stromal proliferation in DLP of 25-week LPB-Tag 12T7s mouse. The overall histological appearance of the lesion shown here is very similar to the background glandular and stromal proliferations seen in these mice, and may constitute a simple physical herniation or protrusion of glands and stroma into duct lumens (*). Whether these lesions thus represent a focal exaggeration of the atypical epithelial hyperplasia or mouse PIN and stromal hyperplasia versus distinct neoplasms is not established. These foci are common with increasing age in the fast growing LPB-Tag lines and can show associated stromal edema, with an appearance reminiscent of phyllodes tumors in human breast, as have been described in TRAMP mice. E, low-power photomicrograph of multiple gland or duct lumens with intraluminal epithelial and stromal proliferations (*) in prostate of a TRAMP mouse. Lesions with these characteristics have had the descriptor "phyllodes-like" added to them, because of their histological resemblance to this human tumor, most often found in the breast. Lesions with these histological features are very rarely encountered in the human prostate. In the mouse lesions, the surface of the intraluminal component is typically covered by epithelium, and the polyploid portion contains an admixture of small glands and stroma. The small gland profiles in the polyploid portion often appear to connect to the surface epithelium, and the stroma is variably hypercellular, hyalinized, or edematous. Histologically, such foci have many features compatible with the designation of papilloma. In SV40 or Tag-based models, they are always seen in a background of more general atypical epithelial and stromal proliferation. A consensus was not reached on the nature of these lesions based solely on their histological features. Whether they constitute hyperplasia, as a focal exaggeration of a more general process, or distinct clonal neoplasms arising against a hyperplastic background remains to be established. If encountered, either of these classifications is appropriate; however, their histological features should be described along with the appearance of the rest of the prostate. The term "phyllodes-like" can be added as a purely histological descriptive adjective, but this term does not imply any biological relationship of these lesions to phyllodes tumors found in human tissues. F–H, low-, intermediate-, and high-power photomicrographs of a discrete papillary lesion compatible with papillary hyperplasia or a papillary adenoma (papilloma) in a GEM prostate. F, this lesion protrudes into and partially fills the lumen (arrowheads) of the lateral prostate from a 73-week-old ARR2Pb-FGF8b mouse. The lesion shows an expansile rather than destructive growth pattern. G, a well-vascularized stroma (arrows) is associated with the epithelial proliferation. Papillary structures (arrowheads) are present although the papillae are less evident in foci where the epithelium is more crowded. H, focal cytologic atypia with nuclear enlargement and macronucleoli (arrowheads) are also evident in these regions. In addition to discreet papillary lesions like the one illustrated here, lesions consistent with mouse PIN are also seen in these mice.

View larger version (139K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Prostatic intraepithelial neoplasia (PIN) lesions in human prostate and SV40 and large T-antigen (Tag)-based mouse models. A, invasive acinar-forming Gleason pattern 3 (Gleason score 3 + 3 = 6) adenocarcinoma (arrowheads) in association with high-grade (HG) PIN (arrows) in human radical prostatectomy specimen. Compare the smaller glands of the invasive carcinoma to the larger (normal)-sized HGPIN containing gland. The HGPIN gland demonstrates nuclear stratification, enlargement, and atypia, with hyperchromasia apparent at this lower magnification. Note the substantial amount of intervening stroma (*) between the unequivocally invasive glands and the adjacent HGPIN gland. B, human HGPIN, with tufting intraluminal proliferation of markedly atypical epithelial cells (arrowheads), with nuclear enlargement and prominently enlarged nucleoli (arrows). Macronucleoli are characteristic of human HGPIN. The cytologic atypia is similar to that typically appreciable in invasive adenocarcinoma. C, mouse prostatic intraepithelial neoplasia (mPIN) showing focal involvement of multiple gland profiles (arrowheads) by stratified cells with nuclear enlargement and atypia, resulting in a hyperchromatic appearance evident at this intermediate magnification. Section of C3(1)-SV40 mouse prostate at 9 months. Foci of residual more normal-appearing epithelium are clearly present (arrows). Progression in extent and the degree of cytologic atypia is compatible with mPIN. In this model, as in other reviewed SV40 and Tag-based models, there is documented progression to invasive carcinoma, often in association with such mPIN lesions. mPIN with documented progression to invasive carcinoma is a specific subcategory designated in the Bar Harbor Classification. D, PIN in GEM prostate, with extensive involvement of most illustrated gland profiles (arrowheads). Tufting and focally cribriform atypical epithelial proliferations are noted, with general maintenance of normal duct/gland architecture. Nuclear hyperchromasia is appreciable even in this low-power photomicrograph. Section of prostate from 8-week-old TRAMP mouse. mPIN in this model has documented progression to association with invasive tumor. E, high-power photomicrograph demonstrating architectural and cytologic features of mPIN in 3-month-old C3(1)-SV40 mouse. Nuclear stratification, enlargement/elongation, and hyperchromasia are pronounced (arrowheads). Irregular nuclear membranes, occasional prominent nucleoli, and mitoses are also appreciable in such lesions. F, mPIN in TRAMP mouse prostate, showing epithelial tufting and marked nuclear hyperchromasia, completely involving three shown gland profiles (*), with a portion of one adjacent gland profile showing somewhat more normal cells (arrowhead). G, PIN in ventral prostate of 24-week LPB-Tag 12T-10 mouse showing tufting and micropapillary proliferation of atypical epithelial cells (arrowheads), within an otherwise architecturally normal pre-existing gland, without associated hypercellular stroma. Nuclear enlargement/elongation and particularly hyperchromasia are evident (arrows). Progression to invasive carcinoma is documented in this mouse. H, PIN in CR2-SV40 mouse, showing focal stratification of atypical epithelial cells with enlarged, hyperchromatic nuclei (arrowheads). More normal appearing gland profiles are seen at top right and bottom left (*). These lesions progress in extent, compatible with mPIN, and are associated with documented invasive carcinoma. Such early PIN foci show colocalization of Tag antigen and neuroendocrine markers in this model.

View larger version (135K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Prostatic intraepithelial neoplasia (PIN) in non-SV40/Tag mouse models. A and B, mPIN in anterior prostate of 11-month-old PTEN +/- mouse. A, intermediate magnification showing prominent cribriform epithelial proliferation within preexisting gland profiles (arrowheads), with more normal-appearing prostate gland profiles at bottom (arrows) and portion of seminal vesicle at right (*). B, higher magnification showing two adjacent involved gland profiles (arrowheads) with tufting and cribriform growth and possibly mildly reactive surrounding stroma (*). Epithelial nuclear atypia includes enlarged nuclei with vesicular chromatin and prominent nucleoli (arrows), similar to that typical in human high-grade PIN. The degree of atypia and progression in extent are compatible with mouse PIN (mPIN). Spontaneous progression to invasive carcinoma is not a characteristic outcome in this genomic knockout model, with only rare possible invasion noted in older (>1 year) mice.18 C and D, mPIN in dorsolateral prostate sections of PTEN +/- x p27 +/- mice. C, intermediate magnification showing focal prominent cribriform proliferation (arrowheads) within a gland lumen in an 8-month-old mouse. D, higher magnification showing cytologic atypia in cribriform mPIN in 6-month-old PTEN +/- x p27 +/- mouse, with enlarged nuclei and scattered prominent macronucleoli (arrows). Note the essentially normal surrounding thin fibromuscular stroma. Progression to frank invasion has not been observed in these mice,24 although it was reported in up to 25% of PTEN +/- x p27 -/- mice by other investigators (30) . E, intermediate power photomicrograph of cribriform epithelial proliferation in multiple glands (arrowheads) in anterior prostate of 16-week-old metallothionein (MT)-transforming growth factor mouse. Atypia and progression in this mouse are compatible with mPIN.25 F, high-power photomicrograph showing mild degrees of focal epithelial stratification (arrowhead) and nuclear atypia, characterized by mild nuclear enlargement and occasional prominent nucleoli (arrows). Ventral prostate section from 16-week-old MT-DNIIR mouse. Atypia and progression are compatible with mPIN.26 Characterization of models with potentially subtle phenotypes including relatively mild epithelial proliferation and atypia is supported by the inclusion of adequate age-matched controls and blinded histopathologic analysis. Additional possible supportive objective analyses (e.g., for documenting progression) include quantitative assessment of indices of proliferation and apoptosis. See text for details. G, mPIN, showing complex cribriform and microacinar epithelial proliferation, with nuclear atypia, extensively involving a gland lumen (arrowheads). A relatively uninvolved gland profile is shown at bottom right (arrows). High-power photomicrograph of lateral prostate from 24-month-old Pb-ras x mxil +/+ mouse.21 H, mPIN, showing cribriform epithelial proliferation, with nuclear atypia, including enlarged nuclei and focally prominent nucleoli. Section of lateral prostate from 82-week-old ARR2Pb-FGF8b mouse. I, mPIN, showing cribriform epithelial proliferation involving multiple pre-existing gland lumens (arrowheads) of the anterior prostate of an 8-month-old Nkx +/- x PTEN +/- mouse (low power). J, high magnification of same section as in I, showing complex cribriform growth pattern, including supporting delicate microvessels (arrowheads), and nuclear atypia, with enlarged nuclei and prominent nucleoli (arrow). Immunostaining for endoglin (CD105) demonstrated increased vessels in these cribriform lesions, which filled some duct lumens. This raises consideration about newly formed associated vessels and stroma and the existence of "back to back glands" within duct lumens, although the overall duct and lobular architecture was not altered (35) . This lesion is described in the classification scheme of Park et al. (78) , serving to document lesion progression sufficient for classification as mPIN as described in the text. Whether such lesions will eventually be associated with progression to unequivocal destructive invasion into the surrounding fibromuscular stroma in non-SV40 models remains to be more fully characterized, as described in the text.

View larger version (162K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Microinvasive carcinoma and invasive adenocarcinoma in prostates of genetically engineered mice. A, microinvasion occurring in association with prostatic intraepithelial neoplasia (PIN) lesion in LP of 7-month-old C3(1)-SV40 mouse. High power magnification showing extension of single cells and cords and small nests of cells (arrowheads) into thickened stroma underlying cribriform mouse PIN. B, microinvasive carcinoma in association with PIN in lateral prostate of 40-week-old LPB-Tag 12T-10 mouse. Small nests of atypical cells with hyperchromatic nuclei, generally scant cytoplasm, and without evident glandular formation are invading into the stroma (arrowheads) surrounding a PIN-containing gland. C, progression to more extensive invasion in mouse prostate cancer model. Section of prostate from CR2-SV40 mouse showing almost circumferential invasion into the thickened stroma (arrowheads) surrounding a residual mouse PIN-containing gland (between *). Invasion is in the form of individual cells and cords and nest of cells, with apparent focal rosetting (arrows). A PIN-containing gland is also seen adjacent to this focus (middle), with more normal-appearing gland at bottom right corner. D, a microinvasive focus of well differentiated adenocarcinoma (demarcated by arrowheads). Section of prostate from 16-week LPB-Tag 12T-7f x MT-DNIIR bigenic mouse. Such a lesion can stand out at low magnification as a more crowded small acinar focus compared with the more diffuse and symmetric lobular expansion by atypical epithelial proliferation (arrows) and hypercellular stroma. On higher magnification as shown, definitive alterations in nuclear and cytoplasmic features are evident, with larger more vesicular nuclei and more densely eosinophilic cytoplasm, compared with adjacent PIN. Similar cytologic alterations are well known with early invasive carcinomas (compared with associated in situ lesions) in a variety of carcinomas in the human, such as cervical and urothelial. This is in contrast to the fairly similar nuclear features of high-grade PIN and associated invasive acinar Gleason pattern 3 carcinoma in human prostate cancer. E, focus of well-differentiated adenocarcinoma in mouse prostate. Section of 24-week-old TRAMP mouse. On the left, there is extension of a focally distinct group of smaller and well-formed acini into surrounding stroma and connective tissue (arrowheads). Compared with widespread and essentially diffuse background of PIN containing glands or the diffuse symmetric lobular expansion with admixed large and connecting small gland profiles that is seen in some of the SV40 or large T antigen-based models with androgen-dependent promoters, the low power focality and architecturally distinct nature of the glands in question is a useful feature for distinction from possible complex in situ or atypical hyperplastic lesions. Although not characteristic of invasive adenocarcinoma in the human prostate, in optimal histological sections, a desmoplastic response in the surrounding fibromuscular stroma or surrounding looser connective tissue can also facilitate recognition of such foci in the mouse prostate. The invasive focus in this example is uniformly and completely composed of discernible gland formations, indicating the designation of well differentiated, as explained in the text. F, invasive adenocarcinoma in association with mouse PIN in LPB-Tag 12T-10 mouse prostate. Nests of tumor cells extending into the stroma show definitive gland formation (arrowheads), with clear lumens or light eosinophilic secretions, rather than features of neuroendocrine rosetting. G–I, invasive adenocarcinoma. Anterior prostate from 38-week-old (C57Bl/6TRAMP/+ x FVB)F1 TRAMP mouse, provided by National Institute of Environmental Health Sciences.11 G, intermediate power showing unequivocal invasive small acinar adenocarcinoma (arrowheads), extensively extending into stroma and periprosatic loose connective tissue, with two remaining PIN-involved glands seen at top and bottom right. H, higher magnification, showing discreet (arrowheads) and occasionally fused small glands, with nuclear enlargement and nucleoli. I, intermediate power showing very pronounced extension of malignant glands into surrounding periprostatic loose connective tissue (*), with possible desmoplastic response (arrowheads). Definitive well-formed glands are present. Because of occasional admixed more solid nests and fused glands, the lesion could be appropriately classified as a moderately differentiated adenocarcinoma in the Bar Harbor Classification scheme. This was considered by the Pathology Panel to be the best histological example of unequivocal invasive adenocarcinoma, with a uniform consensus designation as such. Such a focus was seen in only this particular mouse in material supplied for review.

View larger version (178K): [in this window] [in a new window] [Download PPT slide]   Fig. 9. Invasive neuroendocrine (NE) carcinomas and carcinomas with morphological features suggestive of NE differentiation in prostate and metastases in genetically engineered mice models. A–C, invasive NE carcinoma in CR2-SV40 mouse. A, intermediate power showing invasive carcinoma (*) in association with prostatic intraepithelial neoplasia (PIN) in multiple gland profiles (arrowheads). B and C, low- and high-power photomicrographs of extensively invasive carcinoma. The invasive foci show a generally solid or sheet-like proliferation, but with evident rosettes throughout (arrowheads in C). On high magnification, cells with typical nuclear features of NE carcinoma focally have a moderate amount of eosinophilic cytoplasm, particularly evident in areas of rosette formation (arrowheads in C). Note that focal glandular differentiation can be seen in human NE carcinomas. The tumors in these mice are mucin-negative. In addition to the morphology illustrated here, foci in which tumor cells have less cytoplasm and more oval or spindle hyperchromatic nuclei can be seen, similar to human small cell carcinoma. The PIN, invasive, and metastatic lesions in this model show cytologic, immunophenotypic, and ultrastructural features indicative of NE differentiation. D–I, invasive and metastatic NE carcinoma in the LPB-Tag 12T-10 model. D, intermediate power showing extensive invasion by NE carcinoma, including extensive areas of "less differentiated" small cell carcinoma (*), in ventral prostate of a 40-week-old mouse. An entrapped PIN gland is seen (white arrowhead), as are two other PIN containing profiles at top and bottom left (black arrowheads). Some foci in the invasive tumor show "punched out" cribriform like areas with eosinophilia due to cell cytoplasm in areas of rosette formation. Areas in this field with more solid growth and extreme cellularity, with closely spaced oval or spindle cells show focal crush "artifact" or Azzopardi effect, which is also characteristic of human small cell carcinoma. E, intermediate magnification, showing extensive involvement of periprostatic tissue by NE tumor (*), encroaching on adjacent PIN-gland and its surrounding stroma (arrowhead). Extensive rosette formation is evident. F, strong focal chromogranin i