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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

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Abstract and Figures

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.
Histology of the mouse prostate. A, intermediate power photomicrograph of adult wildtype 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, wildtype mouse prostate shows the well-defined basal cell layer that is often circumferential and extends into the normal, short luminal in-foldings (arrowheads).
… 
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).
… 
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.
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[CANCER RESEARCH 64, 2270–2305, March 15, 2004]
Meeting Report
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
Scott B. Shappell,
1,2
George V. Thomas,
3
Richard L. Roberts,
1
Ron Herbert,
4
Michael M. Ittmann,
5
Mark A. Rubin,
6
Peter A. Humphrey,
7
John P. Sundberg,
8
Nora Rozengurt,
3
Roberto Barrios,
5
Jerrold M. Ward,
9
and
Robert D. Cardiff
10
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 En-
gineered 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 Com-
mittee 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 clas-
sification of mouse prostate pathology is hierarchical, and includes devel-
opmental, 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 progres-
sion to invasive carcinoma. Criteria for recognizing microinvasion, for
classification of invasive gland-forming adenocarcinomas, and for char-
acterizing poorly differentiated tumors, including neuroendocrine carci-
nomas, 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. Be-
cause 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 log-
ical. 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 cri-
teria 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 molec-
ular 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 sec-
ondary 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 character-
izing the pathology of the prostate and other organs in new GEM
models. Tissue-based analysis of biological parameters including pro-
liferation, apoptosis, and microvessel density can contribute to model
characterization and provide mechanistic insight into effects of ge-
netic manipulations and therapeutic interventions.
Hence, the specific objectives of the MMHCC Prostate Pathol-
ogy 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
Received 4/9/03; accepted 1/19/04.
Grant support: National Cancer Institute Mouse Models of Human Cancer Consor-
tium Grant, U01 CA-98013, and a Department of Defense Prostate Cancer Center Grant.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
Note: S. Shappell is currently at the Oppenheimer Urologic Reference Laboratory in
Nashville, TN.
Requests for reprints: Scott B. Shappell, Oppenheimer Urologic Reference Labora-
tory (OUR Lab), 1854 Airlane Drive, Suite 17A, Nashville, TN 37210. Phone: (615)
874-0410; Fax: (615) 232-8009; E-mail: scottshappell@ourlab.net.
2270
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of pathology protocols useful in characterization of prostate dis-
orders 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 ma-
lignancies. For GEM models to have application to specific or
broad subsets of human Pca patients, it is fundamental to under-
stand 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 path-
ological 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 ma-
terials 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 characteriza-
tions. 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.
A brief protocols section is included, primarily to address issues of
tissue submission and immunohistochemical assays to support model
classification or utilization.
General Considerations
Several general principles regarding characterization of GEM mod-
els for Pca were elaborated at the Bar Harbor Pathology Workshop
(Table 2).
Table 1 Genetically engineered mouse (GEM) models of prostatic neoplasia reviewed by the Bar Harbor Pathology Workshop
Category Model Transgene or
knockout Background Promoter or selectivity of
knockout Reference
Cell cycle C3(1)-SV40 SV40 early region C3(1) (82)
TRAMP SV40 early region C57B1/6 FVB Short PB (83)
TRAMP SV40 early region C57B1/6 Short PB
11
LPB-Tag 12T10 SV40 large T
antigen CD1 Long PB (37, 38)
LPB-Tag 12T5 SV40 large T
antigen CD1 Long PB (37)
LPB-Tag 12T7s SV40 large T
antigen CD1 Long PB (37)
LPB-Tag 12T7f SV40 large T
antigen CD1 Long PB (37)
CR2-SV40 SV40 early region FVB CR2 (33)
p27 /p27 knockout C57 Genomic knockout,
heterozygous
12
Growth factors/signal
pathways MT-TGF
Rat TGF
B6D2F1 MT
13
PB-ras H-ras PB
14
PB-FGF8b FGF, isoform b B6D2F1 ARR
2
PB (102)
Receptors MT-DNIIR TGF
RII dominant-
negative B6D2F1 MT (103)
15
Tumor suppressors PTEN /PTEN knockout Balb/c 129 Genomic knockout,
heterozygous
16
PTEN /PTEN knockout SVJ129 Genomic knockout,
heterozygous (35)
12
Men1
TSM/1
Men1 knockout NIH Black Swiss
129/SvEvTacFBR Genomic knockout,
heterozygous (104)
Homeobox genes Nkx 3.1 /Nkx 3.1 knockout 129/SvImj C57B1/6J Genomic knockout,
homozygous (54)
Bigenics LPB-Tag 12T-
7f MT-DNIIR See above (105)
MT-TGF
MT-
DNIIR See above
17
Nkx /⫺⫻Pten /See above (35)
Pten /⫺⫻p27 /See above
12
Nkx /⫺⫻Pten /
⫺⫻p27 /
See above
12
Pb-ras /⫹⫻mxil
/
See above (106)
14
11
R. Herbert, unpublished observations.
12
C. Abate-Shen, unpublished observations.
13
R. J. Coffey, unpublished observations.
14
N. Schreiber-Agus, unpublished observations.
15
H. Moses, unpublished observations.
16
H. Wu, unpublished observations.
17
S. Cutler and R. J. Coffey, unpublished observations.
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Low Frequency of Spontaneous Genitourinary Pathological
Lesions in Mice
The low incidence of spontaneous pathological lesions in the wild-
type mouse prostate was emphasized. This is true not only for neo-
plastic proliferations, but also for non-neoplastic disorders, and in-
cludes aged mice and mice of different genetic backgrounds. In a
recent survey of 612 control B6C3F1 mice for 2-year toxicology and
carcinogenicity studies conducted in the National Toxicology Pro-
gram, not a single example of a spontaneous carcinoma was observed
in the prostate of these wild-type mice, with fairly uniform sampling
of anterior prostates (APs), dorsolateral prostates (DLPs), and ventral
prostates (VPs; Ref. 1). Epithelial hyperplasia was rare, noted in 0.7%
of VPs, and 0.9% of APs and DLPs (1). Adenocarcinoma of the
seminal vesicles was noted in 2 mice (0.3%). Other pathological
diagnoses were also uncommon in 2-year-old control B6C3F1 mice.
Whereas lymphocytic infiltration in the prostate was fairly common
(30% of DLPs and VPs, and 20% of APs), more pronounced
degrees of inflammation, including neutrophilic infiltrates within
prostate acini that may constitute a designation of prostatitis, were
uncommon (1, 3, and 5% for AP, VP, and DLP, respectively; Ref.
1). Atrophy was noted in only the AP of 2 of 612 mice and possible
mucinous metaplasia in only 1 DLP (1).
The low incidence of pathology in the prostate of wild-type mice
suggests that any of the lesions described in GEM, including inflam-
matory 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 compar-
ison with appropriate controls, particularly aged-matched mice of
identical genetic background. The importance of aged-matched con-
trol 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 investi-
gators engaged in GEM research, both within and outside the
MMHCC. Centralized review of particularly promising models, in-
cluding 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 back-
grounds 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 character-
istics of a model. Individual GEM models may show different his-
topathologic 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, progres-
sion 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 mod-
els that are already known in some human Pcas will identify models
that may be useful for testing targeted therapeutic strategies. There-
fore, 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 pros-
tate help support the application of GEM models for the study of
molecular alterations that accompany the development and progres-
sion 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.
Table 2 General principles in pathologic characterization of genetically engineered
mouse (GEM) models of prostate disease
Specify breeding strategy and genetic background, including if previously described
model being employed with different breeding protocol.
Phenotypes should be compared to age-matched controls of similar genetic
background. This is particularly crucial for milder or more subtle phenotypes.
Histopathologic assessment for model characterization and documentation of lesion
progression should be by blinded analysis.
The time course of lesion development and the frequency of lesions at individual time
points should be specified in the description and characterization of a new GEM
model.
All prostatic lobes should be included in the analysis.
The histopathology of other male accessory glands should be included in description/
characterization of possible GEM models of prostate disease.
Pathology material should be made available to members of the Mouse Models of
Human Cancer Consortium Prostate Pathology Committee if a new GEM model is
established with a goal of future research application or sharing with the broader
research community.
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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 lobeshas
been supplanted by the term zonesbased on the concept of specific
zones within the human prostate. These zones are anatomically recog-
nizable, have characteristic histological features, and, importantly, have
specific predisposition to benign or malignant neoplastic diseases. As
described by McNeal (46), 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 ejacula-
tory ducts enter the urethra, near the prostatic utricle at the verumonta-
num, 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, CE). 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.
The TZ is located interiorly between the urethra and the surrounding
PZ and CZ. In the young postpubertal adult, architectural and histological
differences in the glands of the TZ and the PZ are not well defined.
Therefore, the morphological distinction between TZ and PZ is made
primarily by the after the factinvolvement of the TZ by BPH (Fig. 1,
A, B, H, and J). The TZ is the exclusive site of BPH in the human (Fig.
1, Aand H; Fig. 4). BPH histological alterations in the TZ are increas-
ingly common with age in the human prostate, and are present in as many
as 8090% of radical prostatectomy (RP) specimens (removed for Pca;
Ref. 6). In contrast, only 20% of clinically significant Pcas originate in
the TZ (5, 8, 9). TZ tumors often have characteristic, if not specific,
histology (Refs. 10, 11; Fig. 1, Hand I), and may arise as a result of
genetic alterations and precursor lesions differing from those Pcas occur-
ring in the PZ (12). In contrast to its common occurrence in the PZ,
prostatic intraepithelial neoplasia (PIN), including high-grade PIN
(HGPIN), is rarely seen in the TZ. Rather, a lesion referred to as atypical
adenomatous hyperplasia (or adenosis) is thought to be a precursor lesion
for usual TZ tumors (12). TZ tumors are often composed extensively of
low grade Gleason pattern 2 carcinoma (Fig. 1, Hand I). Hence, many TZ
tumors detected clinically are Gleason score 4 or 5, although TZ tumors
not uncommonly contain higher-grade foci. TZ tumors detected clinically
appear to have a better prognosis than clinically detected PZ tumors
(13, 14).
The PZ contains 75% of the glandular tissue in the normal human
prostate and is the most frequent site of Pca origin (810). The PZ is
located particularly on the posterior and lateral aspects of the prostate
(Fig. 1, A, B, H, and J). This location explains why most palpable tumors
(i.e., clinical stage T2 versus T1c) are located in the PZ (14, 15) and why
transrectal biopsies are typically targeted to the PZ rather than the TZ.
The PZ origin of most Pcas also dictates important anatomical relation-
ships for prostate capsule penetration, or extracapsular extension (ECE),
by Pca. In the human prostate, the glands are surrounded by a prominent
stroma of contractile spindle cells and collagen (6, 16). This fibromus-
cular stroma, which is much more abundant in the human compared with
the rodent, extends beyond the outer perimeter of the glands and forms a
more or less distinct capsule,separating the prostate from periprostatic
fat. The capsule is best defined histologically in the posterior and lateral
portions of the human prostate (Fig. 1, Hand J; Refs. 6, 16). Standard
pathological staging of RP specimens addresses the absence or presence
of ECE (i.e., stage pT2 or pT3 tumors, respectively), a major prognosti-
cally significant cutoff for increased risk of progression after surgical
treatment (8, 9, 17, 18). Nerve bundles, which facilitate ECE, are located
particularly in the posterolateral aspect of the gland, with the largest nerve
plexus at the base and a smaller one at the apex (5). The typical site of
ECE is, thus, at the posterolateral aspect of the human prostate gland,
particularly at the base, which is also a common route for invasion of
seminal vesicles, which are at the superior posterior aspect of the prostate
(5, 8, 9).
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 predom-
inantly 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 pros-
tate glands do not possess such a basal cell layer, with the atypical cells
presumably representing aberrantly differentiated or neoplastic counter-
parts of secretory cells. Pragmatically, the absence of an immunopheno-
typically 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 hyper-
plasia (adenosis; Refs. 8, 9, 18, 2426).
Anatomy and Histology of the Mouse Prostate
In contrast to the human, the rodent prostate is divided into anatomi-
cally 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, AF).
The abundant intervening dense fibromuscular stroma surrounding the
glands and their immediate stroma of adjacent lobulesfound 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
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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). CE, 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. Dand 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). Hand 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 35 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). Jand K, typical human PZ tumor. J, whole mount section showing outlined PZ tumor (), a Gleason
score 3 47 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).
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anatomical organization of the prostate between the human and mouse.
These different anatomical features also create potential differences re-
garding the biology of neoplastic extension outside of the prostate in the
human (e.g., amount of stroma, location of nerves, presence of a cap-
sulebetween 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.
The anatomically distinct lobes of the mouse prostate have distinctive
histology and biochemistry. The mouse DP is lined by simple columnar
and occasionally slightly stratified and tufting epithelium (Fig. 2, Band
C). The moderate degree of infolding is intermediate between the AP and
the flatter luminal borders of the LP and VP. The secretory cells of the DP
have lightly eosinophilic granular cytoplasm, and the central to basally
located small uniform nuclei contain inconspicuous or small nucleoli.
Gland lumens contain homogenous eosinophilic secretions (Fig. 2C). The
LP has flatter luminal edges, with only sparse infoldings, with the
abundant luminal space containing more particulate eosinophilic secre-
tions (Fig. 2B). The epithelium is cuboidal to low columnar, with more
clear to lightly granular cytoplasm and small uniform basally located
nuclei. The mouse DLP has sometimes been stated to be the most
homologous to the human PZ (16, 2830). The embryologic develop-
ment of the mouse prostate has been examined in detail and reviewed
previously (2729). The specific developing lobes identifiable in the
embryo remain recognizable in the postnatal and adult mouse prostate, as
the lobes described above. However, these developing lobes are recog-
nizable as such in the human only in the embryo, but not in the adult (28).
It was, therefore, the consensus opinion of the Bar Harbor Pathology
Panel that there is no existing supporting evidence for a direct relationship
between the specific mouse prostate lobes and the specific zones in the
human prostate. It is possible in the human prostate that the zones
described above are contributed to by more than one embryologically
recognizable lobe.Until the relationships of mouse prostate lobes and
human prostate zones can be more precisely defined, no data currently
exists that would defend an a priori conclusion that one lobe of the mouse
prostate is more relevant to human Pca than another lobe.
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 ultra-
structural 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
Fig. 2. Histology of the mouse prostate. A, in-
termediate power photomicrograph of adult wild-
type mouse anterior prostate. The anterior prostate
shows the most complex luminal architecture com-
pared 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 lat-
eral 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 (arrow-
heads). Low- (D) and high- (E) magnification pho-
tomicrographs of mouse ventral prostate. Only a
thin rim of fibromuscular stroma surrounds indi-
vidual glands (arrowheads), with loose connective
tissue extending between individual gland profiles.
This is in contrast to the greater amount of con-
tractile 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 (arrow-
heads).
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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 HM-
WCK 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) dem-
onstrates 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 anat-
omy 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 secre-
tions 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 cheeseappearance, 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 overexpress-
ing 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 periure-
thral glands are also androgen regulated derivatives of the urogenital
sinus. The BUGs in the male mouse are analogous to Cowpers 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. Cowpers glands can
occasionally be seen in apical portions of RP specimens and are rarely
sampled accidentallyin transrectal biopsies, where they may cause
diagnostic confusion. In the human, Cowpers 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 patholog-
ical 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
biphasicappearance, 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 abun-
dant 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 blocsections (e.g., see
Fig. 12 in the Protocolssection) 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 secre-
tory 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.
The Bar Harbor Pathology Workshop: Materials
and Methods
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 facil-
itate classification of existing and newly created mouse models and their
translation to human prostate pathology. The classification system de-
scribed 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 pre-
sented, 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 anno-
tated 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 1821, 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 outsideprostate 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
18
R. D. Cardiff, unpublished observations.
19
P. A. Humphrey, unpublished observations.
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models of GEM were made available through generous donation by inves-
tigators 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 sec-
tioned, 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. pre-
pared 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. 111 were prepared from images of the slide
sets of the Bar Harbor meeting obtained with a Nikon Professional Digital SLR
D1 camera, resolution 2012 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 magni-
fications of 40, 100, and 400, respectively.
Pathological Classification of Disorders of the Prostate in
GEM (The Bar Harbor Classification: Table 3)
Disorders of Development
Agenesis/Aplasia and Hypoplasia (Fig. 3)
Definitions
Agenesis [1]. Agenesis (aplasia) is a disorder of development in
which there is the absence of an organ due to the lack of formation and
development of its primordium in the embryo.
Hypoplasia [2]. Hypoplasia is a disorder of development that dif-
fers from aplasia in that the organ is not completely absent, but there
is inadequate development or underdevelopment of the organ.
Criteria/Explanation. Agenesis is characterized by the lack of any
identifiable prostate lobes on gross examination at necropsy. It should
be confirmed by the absence of histologically recognizable prostate in
appropriate microscopic sections. In hypoplasia, the prostate should
be small for age and may or may not have associated histological
abnormalities (Fig. 3A).
Discussion. Absence of the prostate should be distinguished
from a small prostate due to developmental reasons, that is hyp-
oplasia, and both disorders should be distinguished from secondary
regression, as in atrophy. Developmental abnormalities of the
prostate may be associated with more widespread abnormalities of
the genitourinary tract and/or other organ systems. In humans, the
prostate is not appropriately developed in patients with disorders of
androgen synthesis or signaling. The prostate is small in patients
who have autosomal recessive deficiency of type II 5
-reductase
(42), due to absent or inadequate formation within the prostate of
dihydrotestosterone from circulating testosterone. Dihydrotestos-
terone is necessary for normal prostate development as well as the
development of BPH. 5
-Reductase occurs in two different
isozyme forms, type 1 and type 2. Although individual studies
differ slightly regarding tissue specific expression, the prostate
predominantly expresses type 2, whereas type 1 is expressed
primarily in liver and skin (43).
Examples of abnormal prostate development have been reported
in GEM, including as part of a constellation of more widespread
abnormalities. Regarding androgen metabolism defects, 5
-reduc-
tase type 2 knockout mice have abnormal development of the
prostate, similar to human patients with autosomal recessive 5
-
reductase type 2 deficiency, whereas 5
-reductase type 1 knockout
mice have an apparently normal prostate phenotype (43). Exami-
nation of the 10% of p57
Kip2
-deficient mice that survived beyond
Table 3 Hierarchical classification of disorders of the mouse prostate and other male
accessory glands: The Bar Harbor Classification
a
Prostate
A. Disorders of development:
1. Agenesis/aplasia [1]
2. Hypoplasia [2]
3. Metaplasia [3]
4. Atrophy [4]
B. Inflammatory disorders
1. Prostatitis
1.1 Active/chronic active [5]
1.2.1 Granulomatous prostatitis [6]
1.2.2 Granuloma [7]
2. Abscess [8]
3. Descriptive modifiers
1. Coagulative necrosis [9]
2. Apoptosis [10]
3. Fibrosis [11]
C. Non-neoplastic proliferations of the prostate: hyperplasia
1. Epithelial hyperplasia [12]
1.1 Focal
1.1.1 With atypia
1.1.2 Without atypia
1.2 Diffuse
1.2.1 With atypia
1.2.2 Without atypia
2. Stromal hyperplasia [13]
2.1 Focal
2.1.1 With atypia
2.1.2 Without atypia
2.2 Diffuse
2.2.1 With atypia
2.2.2 Without atypia
3. Combined epithelial and stromal hyperplasia (extentfocal or diffuse and
presence or absence of atypia should be specified) [14]
D. Neoplastic proliferations of the prostate [15]
1. Benign
1.1 Adenoma [16]
1.2 Papillary adenoma or papilloma [17]
2. Prostatic intraepithelial neoplasia (PIN)/neoplastic proliferation of potential
premalignant potential
2.1 With documented progression to invasive carcinoma [18]
2.2 Without documented progression to invasive carcinoma [19]
3. Carcinoma (invasive)
3.1 Microinvasive carcinoma [20]
3.2 Invasive carcinoma [21]
3.2.1 Adenocarcinoma [22]
3.2.1.1 Well differentiated
3.2.1.2 Moderately differentiated
3.2.1.3 Poorly differentiated
3.2.2 Neuroendocrine carcinoma [23]
3.2.2.1 Small cell carcinoma
3.2.3 Squamous cell carcinoma [24]
3.2.4 Spindle cell/sarcomatoid carcinoma [26]
3.2.5 Undifferentiated carcinoma [27]
3.2.6 Mixed carcinoma (specify components); adenosquamous carcinoma [25]
E. Neoplastic proliferation of the stroma
1. Benign neoplasms [28]
2. Malignant neoplasms: sarcoma
2.1 Leiomyosarcoma
2.2 Rhabdomyosarcoma
2.3 Chondrosarcoma
2.4 Osteosarcoma
2.5 Sarcoma, NOS [29]
F. Neoplastic proliferations of stroma and epithelium
1. Carcinosarcoma [30]
Periurethral and bulbourethral glands
A. Disorders of development
B. Atypical hyperplasia
C. Carcinoma
1. Adenocarcinoma
2. Neuroendocrine carcinoma
3. Undifferentiated carcinoma
a
Numbers in brackets refer to definitions in text.
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weaning showed immaturityof the prostate (most likely com-
patible with hypoplasia), as well as of the seminal vesicles and
testes (44).
p63 knockout mice also have abnormal prostate development. p63
is selectively expressed in the basal cell layer of a variety of epithelial
tissues, including human and mouse prostate (45). In contrast, in
human Pca, p63 immunostaining is absent (46), similar to the loss
of a basal cell layer in malignant prostate glands as determined by
immunostaining for HMWCK. p63 knockout (p63 /) mice die at
birth and show severe defects in the development of multiple epithe-
lial organs (47). Histological analysis of the periurethral region in day
20
W. Tu, R. Coffey, R. J. Matusik, unpublished observations.
21
N. Schreiber-Agus, R. D. Cardiff, unpublished observations.
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.
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1 p63 /male mice demonstrated that the prostate does not develop
in these animals (agenesis; Ref. 46).
Homeobox (HOX) genes are important in prostate development and
cell determination within the developing male genitourinary tract (29,
48), and HOX genes have been implicated in the development and
progression of human Pca (49, 50). Hoxd-13 is expressed in mesenchyme
and epithelium of the lower genitourinary tract in the perinatal period,
including focally in the budding nascent ducts of the developing mouse
prostate (51). Transgenic Hoxd-13-deficient mice have multiple abnor-
malities in development of the male accessory glands, including agenesis
of the BUGs, diminished seminal vesicle luminal folding, and decreased
size and ductal branching in the DP and VP (hypoplasia; Ref. 51).
Hoxa-13 is also widely expressed in the developing lower genitourinary
tract. In animals with a spontaneously occurring heterozygous mutation
involving one Hoxa-13 allele, there is decreased size and branching of the
DLP and VP (hypoplasia), as well as abnormal seminal vesicle morphol-
ogy (52). GEM mutants for both Hoxb-13 and Hoxd-13 show hypoplasia
of the VP duct tips, and in Hoxb-13 mutants, the VP epithelium is
composed of simple cuboidal rather than tall columnar cells, and expres-
sion of VP-specific secretory proteins is lost (53). In mice homozygous
for a null mutation in the androgen-regulated murine Nkx3.1 homeobox
domain, Bhatia-Gaur et al. (54) reported abnormalities in prostate ductal
morphogenesis and reduced development of secretory differentiation in
both the prostate and BUGs, compatible with hypoplasia. PIN-like le-
sions are also described in this mouse (54).
Metaplasia (Fig. 3)
Definition. Metaplasia [3]. Metaplasia is the replacement of one
adult cell type (epithelial or mesenchymal) by another adult cell type.
Criteria/Explanation. Prostatic epithelial metaplasia is recog-
nized histologically by the replacement of normal prostatic epithelium
(basal and luminal secretory cells) by non-neoplastic transitional
(urothelial), squamous, or mucinous epithelium, with cytologic fea-
tures similar to other tissues in which these epithelia are found. The
histological type of metaplasia observed in GEM should be specified.
Although regarded as non-neoplastic, if such metaplasia develops
nuclear atypia (potentially constituting dysplasia), it too should be
noted and described.
Discussion. In the human prostate, well-recognized forms of meta-
plasia include transitional (urothelial) metaplasia, mucinous metapla-
sia, and squamous metaplasia. In the human prostate, in addition to the
prostatic urethra, urothelial or transitional epithelium typically ex-
tends a variable length along primary periurethral ducts (Fig. 3B).
Urothelial metaplasia is often seen admixed with more typical secre-
tory cells for variable lengths along secondary periurethral ducts and
is not uncommonly seen in prostatectomy specimens or biopsies to
involve even more distal PZ glands (6). Transitional metaplasia is
commonly observed after antiandrogen therapy (6, 8, 9). Transitional
or urothelial metaplasia is recognized by its resemblance to normal
bladder urothelium (Fig. 3C), and its cells commonly exhibit focal
nuclear grooves characteristic of urothelial cells. Also, similar to
normal urothelium, it stains strongly and uniformly with antibodies to
HMWCK, such that this property cannot be used to distinguish basal
cell hyperplasia and transitional metaplasia. Indeed the two may be
seen together, for example, in response to antiandrogen therapy. The
prostatic urethra not uncommonly shows squamous metaplasia, pos-
sibly in response to chronic irritation, and squamous metaplasia can be
seen focally in more peripheral regions of the human prostate and
adjacent to infarcts involving BPH nodules in the TZ (6). Squamous
metaplasia can occur more extensively in the prostate in response to
antiandrogen treatment or with estrogen treatment for Pca (6), and is
in fact a well-documented response to estrogens in a variety of
species, including in rodents. Mucinous metaplasia, in which prostate
secretory cells show cytoplasmic mucin, often with basilar displace-
ment of the nucleus similar to intestinal goblet cells, is usually a very
focal process (6).
Metaplasia is rarely noted in wild-type mouse prostates. In a survey
of 2-year-old B6C3F1 mice, a form of possible mucinous metaplasia
(with less pronounced goblet cells compared with GEM examples
described herein) was noted in only 1 of 612 mice (1). Metaplasia in
the prostate of GEM may arise in response to similar initiating stimuli
as described above for human prostate or may more uniquely arise as
a consequence of the specific genetic manipulation. Foci compatible
with transitional metaplasia have been observed in the partially
atrophic or regressing PIN-like lesions in faster growing lines of
LPB-large T-antigen (Tag) mice after castration (Refs. 29, 55; Fig.
3D).
22
Intestinal or adenomatous metaplasia was noted in GEM cre-
ated with H-ras expressed in the prostate with a probasin promoter
(Pb-ras /), with or without crossing to mxil /mice (Fig. 3E).
23
Atrophy (Fig. 3)
Definition. Atrophy [4]. Atrophy is an adaptive or secondary re-
sponse in a previously normally developed organ that is characterized
by shrinkage due to loss of cellular substance, leading to diminished
tissue or organ size if sufficient numbers of cells are involved.
Criteria/Explanation. Grossly, the presence of atrophy may be
reflected by smaller prostatic lobes (compared with age-matched
wild-type mice if due to genetic manipulation or compared with
control mice if due to treatment effect). Histologically, atrophy is
manifested by shrunken or dilated glands lined by epithelium with less
secretory cytoplasm.
Discussion. In GEM, a smaller prostate due to atrophy needs to be
distinguished from hypoplasia. In addition to the documentation of
normal prostate development (e.g., at earlier time points or before an
experimental manipulation) the atrophic prostate should contain an
essentially normal number of ductular and glandular structures,
whereas these should be perceptively reduced in hypoplasia.
Atrophy is a common spontaneoushistological alteration in the
human prostate, in which shrunken or dilated glands are lined by a
flattened epithelial lining. In the TZ (e.g., in BPH nodules) and occasion-
ally in the PZ, it can take the form of cystically dilated glands imparting
a swiss cheese appearance. In the PZ, atrophy is more often seen as
angulated dark-appearing glands in which a lobular configuration is
maintained, and the flat luminal lining gives a high nuclear:cytoplasmic
ratio and, hence, a hyperchromatic appearance (Fig. 3F). A possibly
related entity is postatrophic hyperplasia, recognized histologically as a
somewhat lobular configuration of small acini surrounding a larger, more
angulated gland with flatter lining cells (56). The cells in the small gland
profiles have more cytoplasm than usual atrophy and can have prominent
nucleoli, especially when associated with admixed inflammation, leading
to diagnostic difficulties regarding distinction from Pca (57). A possible
relationship to inflammation has been suggested for usual forms of
atrophy, and possible preneoplastic potential has been suggested for
inflammation-associated atrophy (so-called proliferative inflammatory
atrophy) and postatrophic hyperplasia (18, 58, 59). Cell proliferation rates
are actually higher in these usualforms of atrophy and postatrophic
hyperplasia compared with normal benign glands (60). In contrast, atro-
phy seen with hormone deprivation in humans may be more of an
activeinvolution, possibly involving apoptosis.
In contrast to its common occurrence in prostates of middle aged
and older humans, spontaneous atrophy in the wild-type mouse pros-
22
S. B. Shappell, unpublished observations.
23
R. D. Cardiff, N. Schreiber-Agus, unpublished observations.
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tate is apparently extremely uncommon. In the survey of control
2-year-old B6C3F1 mice, atrophy was noted only in the AP of 2 of
612 mice and 1% of seminal vesicles (1). Atrophy, with dilated
gland profiles and flattened epithelium, typically associated with
prominent inflammation, has been observed in castrated GEM com-
pared with the PIN lesions in intact similarly aged mice from these
models (Refs. 29, 55, 61; Fig. 3I). Of note, HGPIN in the human also
appears to be an androgen-responsive lesion (62).
Inflammatory Disorders
Prostatitis, Active or Chronic Active (Fig. 3)
Definition. Active Prostatitis [5]. Active prostatitis (also referred
to as chronic active prostatitis or chronic prostatitis) is an inflamma-
tory process characterized by infiltration of inflammatory cells into
prostatic stroma and glandular elements.
Criteria/Explanation. Prostatitis is recognized histologically by
the presence of neutrophils and/or mononuclear inflammatory cells
within the prostatic glandular epithelium and/or gland lumens, not
simply by the presence of increased inflammatory cells (including
lymphocytes) within prostatic stroma. Reactive epithelial proliferation
secondary to inflammation, which can have mild cytologic atypia,
should be described and the lesion classified as inflammation (pros-
tatitis). Active prostatitis can be secondary to infectious etiologies or
occur without documentable infection.
Discussion. In the human prostate, the presence of neutrophils
within prostatic parenchyma is not necessarily indicative of an acute
process, and typically reflects an ongoing (active) more chronic
process. This is the rationale for the application of the terms active or
chronic active for this entity in both the human and mouse prostate.
When the term prostatitis is used without additional qualification, it is
regarded as synonymous with active or chronic active prostatitis.
Clinicopathologic entities involving prostate inflammation or re-
lated disorders in the human include acute prostatitis, chronic bacterial
prostatitis, chronic abacterial prostatitis, and prostadynia (63). These
disorders are typically diagnosed by clinical features and laboratory
procedures, although the histology when seen has characteristic fea-
tures (6, 8, 63). Chronic prostatitis is common and is usually caused
by organisms typically associated with urinary tract infections. His-
tological features of chronic active prostatitis are not uncommonly
seen in transrectal biopsy (performed for abnormal digital rectal
examination or elevated serum PSA), and may or may not correlate
with symptoms.
Importantly, chronic prostatitis is not diagnosed in human or mouse
by just the presence of possibly increased lymphocytes within the
prostate stroma. In the human prostate, lymphocytes, including small
lymphoid aggregates, are common in the prostate stroma. Similarly, in
mouse prostates, scattered lymphocytes in the stroma and small
lymphoid aggregates in the fibromuscular stroma or surrounding loose
connective tissue can be observed. In the pathology survey of 2-year-
old control B6C3F1 mice, such lymphocytic infiltration in the pros-
tatic stroma was noted in 30% of DLPs and VPs, and 20% of APs
(1). Although this feature can be assessed in GEM (as it could be
variable depending on genetic manipulation), it should not be classi-
fied as chronic prostatitis or active prostatitis. In the mouse prostate,
chronic active prostatitis is characterized by neutrophils and mono-
cytes (not readily distinguishable from lymphocytes in routine histol-
ogy sections) within the prostatic epithelium and gland lumens (Fig.
3G). This type of active inflammation, including with neutrophils and
cellular debris within gland lumens, was noted in only approximately
35% of DLPs and VPs, and 1% of APs in 2-year-old control
wild-type B6C3F1 mice (1). As in the human prostate, inflammation
in the mouse prostate can be accompanied by reactive epithelial
proliferation and nuclear atypia, including the presence of nucleoli,
although such alterations are expected to be relatively mild (1). Such
lesions in the GEM prostate should be classified as inflammatory,
with specification of reactive atypia, rather than proliferative, as in
hyperplasia or PIN. Although the distinction could at times be diffi-
cult, attention should be given to areas relatively free or devoid of
inflammation, where recognition of epithelial hyperplasia can be
made with more confidence.
In addition to its importance in producing symptoms, recent atten-
tion has begun to focus on inflammation-associated atrophy as a
predisposing condition or precursor for Pca in the human (18, 58, 59,
64). Although little attention has focused thus far on inflammatory
disorders in the GEM prostate, similar inflammatory infiltrates can
occur in the GEM prostate (Fig. 3I). Regarding possible etiologies of
active inflammation in the GEM prostate, urinary stasis complicating
obstruction due to large size of the prostate as a consequence of the
genetic manipulation can likely contribute to and be complicated by
infection. Consideration should also be given to modulation of the
immune or inflammatory response as a consequence of nonprostate
selective genetic manipulations.
Granulomatous Prostatitis
Definition. Granulomatous Prostatitis [6]. Granulomatous prosta-
titis is a type of chronic prostatic inflammation, characterized micro-
scopically by a dominant component of granulomas or cell types
characteristic of granulomas, that is macrophages (histiocytes or tissue
macrophages).
Granuloma [7]. A granuloma is a focal lesion composed of cir-
cumscribed accumulations of histiocytes/macrophages, often with
multinucleated giant cells.
Criteria/Explanation. Granulomatous prostatitis is recognized
when the inflammatory process within the prostatic parenchyma has
an extensive component of granulomatous inflammation as defined
above. In the prostate, such granulomas may be centered around
disrupted glandular elements or within the stroma and may be accom-
panied by central necrosis or be non-necrotizing. With less histolog-
ically defined granulomas, granulomatous prostatitis is recognized by
the presence of a prominent infiltrate of histiocytes/macrophages,
typically with abundant eosinophilic cytoplasm (epithelioid histio-
cytes). These cells are admixed with other inflammatory cells, in-
cluding within the epithelial compartment, such that focal areas may
resemble chronic active prostatitis as defined above. Granulomatous
prostatitis may be due to infectious or noninfectious etiologies.
Discussion. So-called nonspecificgranulomatous prostatitis is
the most common form of noninfectiousgranulomatous prosta-
titis in humans, being present in up to 1% of transrectal biopsies
(6). It is likely due to inflammatory reaction to prostatic secretions,
potentially complicating intraprostatic duct obstruction and acinar
rupture. Histologically, it is characterized by a mixed inflamma-
tory infiltrate, without well-formed granulomas or giant cells, but
with sheet-like growth of histiocytes (macrophages; Fig. 3H).
Admixed eosinophils are an important diagnostic feature (6). The
most common form of infectious granulomatous prostatitis in
humans is an iatrogenic condition, caused by the instillation of
Mycobacterium Bacillus Calmette-Gue´rin for the treatment of uri-
nary bladder carcinoma in situ or superficially invasive bladder
cancer. Necrotizing or non-necrotizing granulomatous inflamma-
tion can be seen in the human prostate as a part of systemic
infections with fungal organisms or tuberculosis (6). Fungal or
other infectious etiologies could in theory lead to granulomatous
inflammation in the prostate of GEM models, especially if the
genetic manipulation resulted in immunocompromise.
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Abscess
Definition. Abscess [8]. An abscess is a focal area of acute inflam-
mation and tissue destruction (necrosis). It differs from active pros-
tatitis by the presence of neutrophils as the vastly predominant in-
flammatory cell type, the markedly greater extent/density of the
inflammatory infiltrate, and the presence of associated tissue destruc-
tion. It is essentially always caused by infection, particularly bacterial
and fungal.
Criteria/Explanation. Abscess is recognized by sheet-like infil-
tration of neutrophils, with a cavity formed in areas of frank tissue
destruction. It may be surrounded by a wall of granulation tissue, with
prominent small capillary formation, and may resolve by leaving a
cavity surrounded by fibrosis.
Discussion. With the availability of antibiotic treatment, abscess is
rare in the human prostate (6). It can be seen as a complication of
acute prostatitis, potentially associated with urinary obstruction and
infection with coliforms, or due to hematogenous seeding from an-
other source, usually associated with staphylococcal infection. It is
more likely to occur in the setting of immunocompromise. If identi-
fied in the prostate of GEM, local or systemic infection should be
suspected. Frequent occurrence could also accompany immunosup-
pression due to the specific genetic manipulation involved.
Modifiers of Inflammation
Coagulative Necrosis
Definition. Coagulative Necrosis [9]. Coagulative necrosis is a
type of cell death due to hypoxia or toxins identified by loss of nuclear
detail with retention of cell outlines.
Criteria/Explanation. In the Bar Harbor Classification, necrosis is
essentially synonymous with coagulative necrosis. Coagulative necro-
sis is a form of tissue necrosis in which the basic outline of the cells
and tissues is maintained. Coagulative necrosis may occur in a manner
potentially liberating cellular contents, and typically involving multi-
ple cells and potentially large areas of contiguous cells within a tissue,
and associated with an inflammatory response at the periphery. It is
quite characteristic of hypoxic cell death (infarction). Necrosis (co-
agulative necrosis) may be manifested by soft, friable tissue grossly.
It is confirmed microscopically by confluent areas with loss of nuclear
staining, with cell and tissue outlines still evident as eosinophilic in
H&E-stained sections.
Discussion. Necrosis in the Bar Harbor Classification is distin-
guished from cell death due to inflammatory injury in prostatitis and
possibly associated with infection, which is properly classified as a
form of prostatitis or abscess as described above. In any organ,
coagulative necrosis is often seen as a consequence of hypoxic cell
death, as in infarction. It is seen in the human prostate, for example,
when BPH nodules undergo infarction, where coagulative necrosis
may be surrounded by atypical reactive epithelium and squamous
metaplasia (6). It has been seen either focally or more extensively in
the prostate and seminal vesicles in occasional older transgenic ani-
mals with markedly enlarged prostates and subsequently engorged
seminal vesicles,
13
which is likely due to an ischemic process.
Apoptosis
Definition. Apoptosis [10]. Apoptosis or programmed cell death is
a form of cell death triggered by various signals, which initiate a
cascade of intracellular events identified morphologically by a frag-
mentation of the nuclear chromatin and hyalinization of the cyto-
plasm.
Criteria/Explanation. Generally apoptotic cells can be recognized
by rounded red cytoplasm and pyknotic, fragmented nuclear chroma-
tin. In addition to its usual microscopic appearance, it can be identi-
fied and even quantitated using ancillary techniques, such as terminal
deoxynucleotidyl transferase-mediated nick end labeling assays and
immunohistochemistry for enzymes activated as part of the cell death
program, described elsewhere.
Discussion. Atrophy of the prostate after castration is in part the
result of apoptosis of the epithelium. In several GEM models of
prostatic neoplasia, increased epithelial proliferation has also been
noted to be accompanied by increased apoptosis.
Fibrosis
Definition. Fibrosis [11]. Fibrosis is a deposition of extracellular
collagen by activated fibroblasts.
Criteria/Explanation. The histological features of fibrosis may
evolve with time. Early on, activated fibroblasts are surrounded by
abundant eosinophilic staining collagenous material, possibly with
edematous stroma, and adjacent chronic inflammation and granulation
tissue, if occurring as a sequelae of inflammatory injury. With time,
these areas of scarring evolve to hypocellular regions with dense
eosinophilic collagen.
Discussion. In human and veterinary pathology, fibrosis is a well-
recognized potential consequence of inflammation (i.e., scarring). In
addition to a sequelae of inflammatory injury, transgenic manipula-
tions involving growth factors or their receptors can lead more di-
rectly to stromal cell or fibroblast activation, with increased deposi-
tion of extracellular matrix. Such stromal alterations have been
observed in the prostates of GEM, and should be considered when the
fibrosisor stromal hyalinization is not accompanied or preceded by
prominent inflammation.
Non-Neoplastic Proliferations of the Prostate: Hyperplasia
Epithelial Hyperplasia (Fig. 4)
Definition. Epithelial Hyperplasia [12]. Epithelial hyperplasia is a
non-neoplastic increase in epithelial (glandular) tissue compared with
age-matched wild-type control mice.
Criteria/Explanation. Epithelial hyperplasia is recognized as ei-
ther an increase in glandular spaces or as an increase in epithelial cells
within normal-appearing gland profiles, the latter primarily reflected
by stratification of epithelial cells. Pronounced forms can achieve
tufting, micropapillary, and even cribriform architecture. In the Bar
Harbor Classification, epithelial hyperplasia is additionally classified
as focal or diffuse. The modifier term focal refers to the involvement
of one or a few gland spaces. Diffuse refers to a more extensive and
uniform process. In practice, this can be distinguished as involvement
of 50% of gland profiles in adequately sampled prostate lobes.
Epithelial hyperplasia can be accompanied by cytologic (nuclear)
atypia, which should be noted. Criteria for cytologic atypia include
nuclear enlargement, pleomorphism, chromatin abnormalities, and
increased prominence of nucleoli, as described. Epithelial hyperplasia
is distinguished from mouse PIN (mPIN) by criteria of focality and
progression for the latter, as detailed below. If accompanied by
stromal proliferation, the lesion should be classified as mixed epithe-
lial and stromal proliferation, as described below. Hyperplasia is the
appropriate designation for epithelial proliferations in the mouse
prostate that are not reactive to inflammation and that do not satisfy
the definition of mPIN. Appropriate modifiers regarding extent and
the presence of atypia should be included.
Discussion. A variety of characteristic epithelial hyperplasias oc-
cur in the human prostate, most notably adenomatous or glandular
hyperplasia in the TZ as part of the glandular and stromal hyperplasia
typical in BPH (Fig. 4, AC; Ref. 6). Basal cell hyperplasia can be
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seen in association with BPH (Fig. 4D), as well as in a morpholically
distinct form in the PZ, which, when accompanied by prominent
nucleoli, can mimic PIN (6, 65). Another described morphological
entity is clear cell cribriform hyperplasia (Fig. 4E), which is also most
commonly observed in the TZ with BPH (6). Importantly, the creation
of new glandular spaces is not a diagnostic criterion specific for
adenocarcinoma per se in the human prostate, nor should this be
regarded as an absolutely specific feature for adenocarcinoma in
prostate of GEM. Glandular proliferation is not accompanied by
appreciable cytologic atypia in human BPH. Hypercellular BPH nod-
ules can be observed, and atypical adenomatous hyperplasia, or ad-
enosis, is characterized by a proliferation of admixed larger and small
gland profiles, with similar nuclear and cytoplasmic features in each,
and retention of at least a fragmented basal cell layer (6). Other lesions
with increased glandular or epithelial tissue include sclerosing adeno-
sis, also typically seen in the TZ, which can have nuclear atypia and
mimic higher-grade Pcas (6). Hence, many benign lesions in the
human prostate demonstrate increased glandular spaces in addition to
or instead of increased epithelial cell stratification within pre-existing
gland or duct spaces.
Epithelial proliferation classified as hyperplasia is not routinely
recognized per se in the human prostate PZ. Lesions referred to as
postatrophic hyperplasia are described above with atrophy. Epithelial
proliferation in normal PZ gland spaces is usually accompanied by at
least mild nuclear enlargement and atypia, and classified as low-grade
PIN (9, 65). However, the anatomical restrictions of hyperplasia as a
typical TZ histological alteration and PIN as a generally PZ-restricted
entity in human prostate cannot be translated to specific lobes or
regions within lobes of the mouse prostate. Hence, hyperplasia is the
appropriate designation for epithelial proliferations in the mouse
prostate not satisfying the definition of mPIN.
Hyperplasia defined as an increase in glandular tissue compared
with age-matched wild-type control mice could certainly be a devel-
opmentalconsequence of transgene expression during prostate de-
velopment. Attention should be given to the extent (diffuse versus
focal) or possible uniform involvement of such epithelial prolifera-
tions (even if atypia is present) in effort to distinguish a generalized
phenomenon versus a possible manifestation of the development of an
epithelial neoplasm in GEM. Hyperplasia may also be seen as an
increase in epithelial cells within otherwise normal-appearing gland
spaces. Prostate epithelial hyperplasia has been observed in GEM
(Fig. 4, Fand G). The presence or absence of atypia should be noted.
Fig. 4. Epithelial hyperplasia in the human and mouse prostate. AC, 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. Band 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 (). Fand 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).
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As epithelial proliferation with nuclear atypia is the histological
hallmark of PIN, atypia should be diagnosed with caution and pref-
erably in a blinded fashion so that confidence can be had in the
presence of this potentially subjective feature. As described below,
other criteria are necessary for a classification of PIN in the mouse
prostate, such as focality and progression.
Epithelial proliferation can result in an increase in basal cells or
secretory cells or both, but as proliferative capacity is likely limited to
basal cells, an increase in luminal cells likely represents accompanying
cellular differentiation. When possible, the cell types involved (i.e.,
increased compared with wild-type controls) should be specified. Ad-
junctive immunohistochemical stains can be helpful, as described in the
protocol section. Regarding classification of focal versus diffuse, al-
though some gland lumens will be cut more longitudinally and others
more transversely in any given section, and many of these are different
profiles of the same connecting ducts or glands, the semiquantitative
estimation of the percentage of involvement or rigid counting of gland
spaces involved may be a useful objective parameter. Also, as normal
proliferation and apoptosis in the rodent prostate may occur differentially
along proximal and distal portions of ducts and glands, attention should
be given if possible to differential involvement of proximal and distal
portions of the duct/gland profiles within well-oriented sections (see
Protocolsection regarding sectioning). This is desirable, as genetic
manipulations could accentuate, modulate, or negate these normal cell
turnover mechanisms. As a supplement to blinded histopathologic assess-
ment, more objective parameters of epithelial proliferation and turnover
can be used, such as immunostaining for proliferation markers and tissue
stains for apoptosis (see Protocolsection). Such parameters can be
assessed in an objective quantitative fashion (either by blinded counting
or image analysis approaches), as long as sampling is random and equal
and/or attention is given to the proximal versus distal portion of examined
prostate lobes.
Stromal and Combined Epithelial and Stromal Hyperplasia
(Fig. 5)
Definitions Stromal Hyperplasia [13]. Stromal hyperplasia is a
non-neoplastic increase in the cellularity of the stromal component of
the prostate compared with age-matched controls.
Combined Epithelial and Stromal Hyperplasia [14]. Combined
epithelial and stromal hyperplasia is the simultaneous non-neoplastic
increase of both epithelium (as described above) and stroma.
Criteria/Explanations. Stromal hyperplasia is recognized by an
increase in the density of stromal cells compared with age-matched
control mice. Cells are oval to spindle with general similarity to
normal prostatic stroma, and their increase may be accompanied by an
increase in the extracellular matrix, which should be noted. Stromal
hyperplasia may be focal or diffuse. Focal refers to one or multiple
areas comprising less than half of the sampled tissue. Diffuse stromal
hypercellularity involves 50% of the sampled stromal compartment.
Stromal hyperplasia may be accompanied by cytologic atypia, includ-
ing nuclear enlargement, hyperchromasia, pleomorphism, and prom-
inent nucleoli, which should be noted. Particularly if accompanied by
nuclear atypia, increased stromal cells must be distinguished from
undifferentiated or sarcomatoid carcinoma. The resemblance to nor-
mal prostatic stroma (which may be more evident at earlier time
points), spatial relationship to normal or proliferative noncarcinoma-
tous epithelial elements, and immunohistochemistry (e.g., negative for
pan-keratin and possibly positive for smooth muscle actin) may be
helpful. Stromal hyperplasia should also be distinguished from stro-
mal neoplasms based on criteria described below. Combined epithelial
and stromal hyperplasia should be distinguished from adenoma and
papilloma based on criteria described below.
Discussion. Stromal hypercellularity in the human prostate is an
extremely common, almost characteristic, accompaniment to the glandu-
lar hyperplasia that occurs in the TZ in BPH (6). Stromal hypercellularity
is noted to accompany other typically TZ lesions, such as basal cell
hyperplasia and sclerosing adenosis (6), whereas it is not a usual accom-
panying feature (i.e., conspicuous by light microscopy) to the malignant
glandular elements in typical PZ Pcas in the human (8, 9).
Prominent stromal hypercellularity has been observed along with
epithelial cell proliferation in multiple GEM models (Fig. 5, AC).
Whether a direct consequence of transgene expression or a possible
paracrine effect from transformed epithelial cells is not defined. When
uniform or diffuse, it is certainly unlikely to be a desmoplastic
response to what is hopefully a focal invasive event in carcinoma (see
below). Often the stromal hypercellularity in GEM prostate has been
noted to be progressive and to have a general resemblance, especially
in the early stages, to normal prostate stroma. In some models (Fig. 5,
Aand C), there is also noted a more condensed hypercellular stroma
in closer proximity to the proliferating atypical epithelial compart-
ment. Atypia and mitotic activity in this stroma can be conspicuous.
In addition to possible classification as combined epithelial and stro-
mal hyperplasia, in some models, the epithelial lesion accompanying
the stromal alterations is properly regarded as mPIN (as defined
below), and there has been development of subsequent invasive car-
cinoma. Such lesions can be designated as mPIN with hypercellular
stromaor mPIN with stromal hyperplasia.Atypia in the stromal
component should be addressed as well. In contrast to these hyper-
plastic processes, focally prominent proliferations of epithelium and
associated stroma raise consideration of discrete neoplasms, poten-
tially classifiable as adenoma or papilloma.
Neoplastic Proliferations of the Prostate
Benign Epithelial Neoplasms: Adenoma, Papillary Adenoma, or
Papilloma (Fig. 5)
Definitions. Neoplasm [15]. A neoplasm is an autonomous new
growth. The designations of adenoma and papilloma are intended for
true neoplastic epithelial proliferations that lack the hallmarks of
malignancy, such as destructive invasion and metastatic potential.
Adenoma [16]. An adenoma is a benign neoplasm of gland-form-
ing epithelium.
Papillary Adenoma [17]. A papillary adenoma or papilloma is a
benign neoplasm of gland-forming epithelium with a well-defined
fibrovascular stroma, wherein the vascular core and surrounding
stroma is covered by neoplastic epithelium in a manner imparting a
papillary or branching pattern.
Criteria/Explanation. Although in theory such lesions are clonal
neoplasms, certainly this is not routinely demonstrated for the vast
majority of lesions classified as such. Instead, these benign tumors are
recognized by a combination of pathological features, including dis-
crete growth of epithelial elements (with variable stromal component)
that can be expansile or nodular or protrude into a lumen, but without
destructive invasion. The stromal component may include a prominent
vasculature, either throughout or at the base of the lesion. In papil-
loma, there is a well-defined fibrovascular stroma or stalkthat is
covered by an epithelial lining in a manner that assumes a character-
istic papillary growth pattern (Fig. 5, FH). Papillary adenoma and
papilloma are, hence, synonymous. Cytologic atypia can be present in
adenoma or papilloma, and if so, it should be described. Furthermore,
if such atypia progresses (i.e., is increasingly prominent in lesions
from older GEM), this should be noted and described. Such lesions
may potentially parallel the development of high-grade dysplasia in
some human adenomas in organs other than prostate. Similarly, if
focally architecturally distinct epithelial regions are identified, such as
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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 Aand Bas 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 Aand Brepresent 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-likeadded 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-likecan 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. FH, 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 ARR
2
Pb-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.
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showing greater crowding, or accentuated atypia or apparent invasion
into the stromal compartment of the lesion itself, this should be noted
and described. Such lesions potentially parallel the development of
early invasive carcinoma in some adenoma-carcinoma sequences in
organs other than prostate.
Discussion. There are essentially no counterparts in human prostate
pathology, in terms of benign neoplasms of variably differentiated secre-
tory epithelial cells, compared with the common occurrence of adenomas
in such tissues as colon. There are rare basal cell lesions described as
basal cell adenomas (8, 9), but in general, this category in GEM was
created based on already observed or possible lesions more comparable
with those in other tissue sites or in veterinary pathology.
When occurring in the background of extensive or even diffuse
epithelial and stromal hyperplasia, focal intraluminal polyploid pro-
trusions may represent foci of essentially intraluminal herniations,
with growth into an area of less resistancethan surrounding stroma
or adjacent tissue (Fig. 5, Dand E). Some such foci in a few SV40 or
Tag-based models develop a very characteristic edematous stroma as
well, and lesions either representing combined epithelial and stromal
hyperplasia or true neoplasms have been described by the term phyl-
lodes-likeon the basis of their histological resemblance to phyllodes
tumors in the human. These tumors are particularly characteristic
(although not necessarily common) in the human breast. Histologi-
cally similar lesions have rarely been described in the human prostate
(8, 9). The hyperplastic versus neoplastic nature of the observed
lesions in the prostate of GEM, which are seen especially with
advancing age in certain models, is not established, and no consensus
was reached on this matter. We do not recommend the term phyllodes
tumor as a precise designation for these foci because of a lack of
demonstrated homology to these uncommon neoplasms in the human
(e.g., in terms of potential clinical behavior or natural history and the
lack of more widespread background epithelial and stromal abnor-
malities in the human). However, the descriptive adjective phyl-
lodes-likemay be useful in describing the characteristic histology of
such combined epithelial-stromal lesions.
A lesion homologous to papilloma is not recognized in the human
prostate per se, but lesions satisfying typical histological criteria for
papilloma in human and veterinary pathology have been observed in
the prostate and seminal vesicles of GEM. When seen in a background
of more widespread hyperplasia or PIN, such lesions again need to be
distinguished from hyperplasia and are recognized by their focally
distinct growth pattern. Destructive invasion is lacking. Given the
possible background of widespread hyperplasia occurring throughout
the GEM prostate, molecular data to support that such foci are truly
distinct clonal neoplasms and not part of a more widespread process
would be welcomed. For now, such lesions can be classified as
adenomas or papillomas, or as combined epithelial and stromal pro-
liferations, but they should be described in detail (including the
presence or absence of atypia), as should the histology of the rest of
the prostate in which they are arising.
Neoplastic Proliferation of Premalignant Potential: mPIN (Figs. 6
and 7)
Definition. mPIN [18]. mPIN is the neoplastic proliferation of
epithelial cells within preexisting or normal basement membrane
confined gland spaces. In the Bar Harbor classification, mPIN is
further classified as mPIN: (a) with documented progression to inva-
sive carcinoma [18]; or (b) without documented progression to inva-
sive carcinoma [19].
Criteria/Explanation. mPIN is recognized histologically as pro-
liferation of atypical epithelial cells within pre-existing glands. These
basement membrane-bound glands may be architecturally similar to
the wild-type prostate or increased uniformly as a consequence of
transgene expression, but are still contrasted with those of invasive
carcinoma (discussed below). Proliferation is most commonly recog-
nized by stratification of epithelial cells. The epithelial cells demon-
strate nuclear atypia. With progressive neoplastic epithelial growth,
the focus can acquire a tufting, micropapillary, or cribriform growth
pattern. In rare instances, the pattern may be flat, in which prolifer-
ation is not conspicuous, but the nuclear atypia and its progression are
sufficient for the diagnosis of mPIN. Nuclear atypia can be in the form
of nuclear enlargement, nuclear membrane irregularity, hyperchroma-
sia, chromatin clumping, prominent nucleoli, or a combination of
these features. As the category of epithelial hyperplasia can have
atypia as described above, two other features that must be present for
a designation of mPIN are focality and progression. The lesion should
begin focally, as a manifestation of neoplasia, rather than being
present uniformly throughout the prostate, as a perhaps more direct
consequence of transgene expression or other genetic manipulation.
Progression refers to either increased extent of involvement or in-
creased nuclear atypia or both, with both being particularly support-
ive. Rigid criteria for the degree of these changes that constitute
progressionwere not established (see discussion below for guide-
lines). However, they should be well documented, described, and
hopefully illustrated over an appropriate time frame (depending on the
rapidity of neoplasia development) in any given new model. Examples
are shown in Figs. 6 and 7. Although initial evaluation of the spectrum
of possible lesions in a new model can be unblinded, subsequent
evaluation of lesions in mice of different ages using objective schemes
to describe architectural and cytologic abnormalities that may allow
for documentation of temporal progression should be blinded.
Within the Bar Harbor classification, the natural history of an mPIN
lesion is an important component of its classification. Progression to
invasion is recognized based on the criteria for microinvasive or more
extensively invasive carcinoma as detailed below. It was the consensus
opinion of the Pathology Panel that mPIN should not at present be graded
histologically, because insufficient data exists to support general mor-
phological criteria for a high-grade designation that might predict pro-
gression to or association with invasive carcinoma in any given model.
This issue is considered in more detail in the discussion below.
Discussion. mPIN presumably shares some of the molecular alter-
ations characteristic of carcinoma; however, the neoplastic cells have
not invaded through the basement membrane into surrounding stroma.
Regarded as a potential precursor for invasive carcinoma, by itself,
PIN lacks metastatic potential. Because of the biological significance
of PIN and the frequency with which GEM models develop lesions
potentially classifiable as mPIN, there first follows a consideration of
the biology, pathology, and clinical aspects of human PIN.
Human PIN. Human PIN is the neoplastic proliferation of epi-
thelial cells within preexisting gland spaces, which occurs predomi-
nantly (almost exclusively) in the PZ (Fig. 6A; Refs. 8, 9, 66, 67). The
proliferating, stratified cells are neoplastic and less-differentiated
counterparts of secretory or luminal cells and do not immunostain
with antibodies to high molecular weight CK. In fact, with increasing
grades of PIN the basal cell layer is progressively lost or fragmented
(19).
Grading Human PIN. Human PIN is now classified as low or high
grade, replacing a previous three-tiered system whereby PIN I is
low-grade PIN, and PIN II and PIN III are high-grade PIN (HGPIN;
Refs. 8, 9, 65, 66, 68, 69). With increasing severity (grade), there is
greater nuclear enlargement and increasingly prominent nucleoli (8, 9,
66). Macronucleoli (23
m) are typical and diagnostic of HGPIN
(Fig. 6B). HGPIN architecturally can show multiple growth patterns,
including tufting and micropapillary (most common; Fig. 6B), flat or
cribriform (70).
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Biological and Clinical Significance of HGPIN. In the human,
HGPIN is considered the likely precursor lesion for the majority of
invasive Pcas in the PZ (Fig. 6A; Refs. 12, 18). Multiple lines of
evidence support the relationship between HGPIN and PZ-located Pca
(8, 12, 19, 67). These include the earlier age occurrence of HGPIN
compared with invasive Pca in autopsy studies. There is an increased
incidence of HGPIN (but not low-grade PIN) in autopsy or RP
prostates with cancer compared with those without. There is fre-
quently close spatial association of HGPIN and invasive acinar-
forming Pca (Fig. 6A). Numerous studies examining various molec-
ular markers, including expression of oncogenes, growth factors, and
their receptors, have demonstrated alterations in HGPIN that are
intermediate between benign prostate and Pca or that are similar in
HGPIN and Pca (18, 19). Similar to the frequent multifocality of
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 36) 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.
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Fig. 7. Prostatic intraepithelial neoplasia (PIN) in non-SV40/Tag mouse models. Aand 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
Cand D, mPIN in dorsolateral prostate sections of PTEN /⫺⫻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 /⫺⫻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 /⫺⫻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 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 ARR
2
Pb-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 /⫺⫻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 glandswithin 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.
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invasive Pca in the human prostate, HGPIN is frequently multifocal,
and genetic evidence supports that HGPIN and Pca within a prostate
are commonly multiclonal (19, 71).
Significance Itof Cribriform Lesions with Intact Basal Cell Lay-
ers in Human Pca. Although in the human, HGPIN as a potential
precursor lesion for invasive Pca may demonstrate a cribriform
growth pattern within pre-existing ducts and glands (70), several
recent studies have shown that cribriform HGPINhas a worse
prognosis than cribriform invasive carcinoma in RPs or imparts an
independent increased risk for progression when identified in RPs (72,
73). Such data have raised speculation that these lesions, which are
associated with higher grade and larger volume Pcas, actually repre-
sent the spread of invasive carcinoma within ducts, so-called intra-
ductal carcinoma,a postinvasive rather than preinvasive lesion (72
74), which is supported by recent molecular evidence (75).
The possible biological implications of these lesions in the human
prostate should not prematurely be extended to mouse models based
solely on architectural and histological similarities of noninvasive
cribriform proliferations. Atypical cribriform lesions within ducts
have been observed in numerous GEM models of Pca. In the mouse,
however, these can be seen without associated invasion anywhere in
the prostate (e.g., in contrast to the large volume of typically Glea-
son score 7 invasive carcinoma in human RPs) or with only small foci
of invasion. Hence, these lesions likely represent PIN in the mouse
(i.e., as a true potential precursor lesion), as they have typically been
interpreted.
Invasion in Association with HGPIN. The earliest invasive forms
of Pca associated with HGPIN in the human prostate (conceptually
analogous to microinvasion in the mouse prostate classification) are
not clearly defined. Not well recognized in the human is the penetra-
tion through the HGPIN gland basement membrane of individual
tumor cells or small groups of cells with possible cytologic alterations
or stromal response. Possible early invasive lesions composed of
microscopic foci of larger HGPIN-like glands with closely arranged
sproutingsmall acini have been described in RPs (76), but the
application of such concepts to diagnosing early invasive carcinoma
on biopsy is not established (77). Issues relevant to microinvasion in
GEM models are described below in the section of microinvasive
carcinoma.
mPIN. General Considerations Regarding Possible Grading of
mPIN. In some previously published descriptions of GEM models,
grading PIN as low grade or high grade has been accomplished based
predominantly on progressive nuclear atypia. However, it should be
borne in mind that human HGPIN represents a broader concept than
just its morphological features; that is, by virtue of its being seen in
association with invasive carcinoma and by having documented mo-
lecular alterations similar to Pca, it is considered to have true potential
for progression to invasion (12, 18, 19). Obviously, documentation of
progression to invasion and characterization of progressive molecular
alterations in PIN and invasive Pca have not been accomplished in all
of the mouse models. Because of the association of HGPIN with
invasive Pca in the human, consideration was given to classifying as
high grade only those mPIN lesions that occur in models that have
documented progression to invasive carcinoma (and in which the
invasion appears to be related to the PIN lesions). However, it was the
opinion of the Pathology Panel that this definition may discourage
development and characterization of models that could be useful for
deciphering early changes leading to PIN in Pca development and for
testing possible chemoprevention agents. Furthermore, because insuf-
ficient data exists on morphological features of PIN that correlate with
progression (regardless of the specific mouse model), it was the
consensus opinion of the Pathology Panel that mPIN should not at
present be graded based on histological criteria as low grade or high
grade. Any attempts to do such within the context of a formal
classification scheme applicable to all of the GEM models would be
premature.
should be additionally noted that based on the developed criteria for
mPIN, in which focality and documentation of progression (of extent
and atypia, not necessarily to invasive carcinoma) are required, many
published models stating that they have PIN or PIN-like lesions would
not actually satisfy the diagnostic criteria for mPIN. Investigators
should place more emphasis on satisfying such criteria for a proper
designation of mPIN in a GEM model. The number of foci or
percentage of gland lumens involved, and descriptions of progressive
architectural abnormalities and nuclear atypia with increasing age
should allow for adequate classification of a GEM model as having
mPIN. A histopathologic classification scheme for PIN lesions in one
non-SV40 model, incorporating lesions also seen in other non-SV40
models, was described recently by a group of investigators including
a member of the MMHCC Prostate Pathology Committee (78). PIN
was divided into four categories based on progressive architectural
and cytologic abnormalities (78). Although in this published study, it
was not clear if all of the histological assessments were blinded, the
application of this scheme allowed for the documentation of lesion
progression in Nkx /⫺⫻PTEN /mice (35, 78). Lesions similar
to these PIN categories have been noted in several other non-SV40
based models listed in Table 1 (78). Detailed time course analyses
(with sufficient numbers of animals at a range of time points) have not
been reported in other models using this classification scheme to
determine whether similar progression can be documented. However,
it is likely that such a scheme can be applied in a blinded manner to
objectively document progression of extent and severity of epithelial
proliferations to allow for recognition of models satisfying mPIN
criteria in the Bar Harbor Classification scheme (78). The reproduc-
ibility and interobserver agreement of this or any other mPIN classi-
fication scheme have not been documented thoroughly. This scheme
(78) and lesions illustrated herein provide the investigator some idea
of the phenotypes of mPIN lesions that may develop in non-SV40
based models. For now, although other descriptions may be adequate
for documentation of progression, utilization of this particular scheme
for documentation of progression for recognizing mPIN is the sole
recommended application of this described scheme, and it is not
recommended as a histological grading scheme for dividing mPIN
into low or high grades. Histological classification schemes capable of
documenting progression of mPIN lesions within an individual model,
such as that described by Park et al. (78), may also be useful for
blinded histological assessment of possible therapeutic benefit of
chemoprevention strategies designed to inhibit or reduce progression.
The Bar Harbor Classification scheme does allow for grading/
classification of mPIN, but stipulates that natural history be consid-
ered when classifying mPIN (i.e., whether mPIN is or is not associated
with progression to invasive carcinoma). Although there are obvious
histological differences in PIN lesions in different GEM models, it
was the consensus opinion of the Pathology Panel upon uniform
review of 20 models that these histological differences do not yet
translate to differences in potential for development of invasive car-
cinoma that are an important component of the concept of a high-
grade PIN lesion. For example, PIN has progressed to invasive car-
cinoma in multiple SV40 early region or large T antigen-based
models. If based on these observations, the profound nuclear atypia in
the PIN lesions of these mice were to serve as the histological
24
C. Abate-Shen, R. D. Cardiff, unpublished observations.
25
S. Cutler, R. J. Coffey, R.L. Roberts, S. B. Shappell, unpublished observations.
26
H. A. Moses, Jr., R.J. Matusik, R. L. Roberts, S.B. Shappell, unpublished obser-
vations.
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hallmarks of HGPIN in GEM, few if any non-SV40-based models
would have lesions classified as HGPIN. The morphological appear-
ance of the PIN lesions in SV40 and non-SV40 models is quite
distinct (see below). At the time of the Bar Harbor Pathology Work-
shop, none of the non-SV40 based models available for review (Table
1) demonstrated unequivocal invasion with any significant frequency.
However, it appears that some recently created non-SV40-based mod-
els show progression to invasive and metastatic carcinoma (7981).
Future consensus conferences in which such models are reviewed may
allow for the development of a grading scheme to be applied to
non-SV40-based models, which may allow for predicting which new
models may show progression to invasive carcinoma. Any such future
histological mPIN grading scheme would likely be different from
such a scheme for SV40-based models.
Classification of mPIN in the Bar Harbor Classification. Al-
though mPIN should not be graded morphologically, this category is
divided into those models that have documented progression to inva-
sion or the capacity to invade and those that do not (Table 3). The
natural history of a model is, thus, an important component of how an
mPIN lesion should be classified. Criteria for invasion are detailed
below. Because human HGPIN is associated with invasive carcinoma
and when seen in isolation, may potentially progress to invasive
carcinoma, the classification of mPIN with documented progression to
invasive carcinoma is in some ways biologically analogous to human
HGPIN. mPIN without documented invasion can be considered ini-
tially as a tentative category when a PIN lesion is observed in a new
mouse model. If invasion is subsequently documented, the classifica-
tion is appropriately modified to reflect this. The frequency with
which such invasion should occur to warrant such designation was not
established. However, it is intended that this should not be an exceed-
ingly rare, isolated, or equivocal event. Progression should be repro-
ducible. For example, development of unequivocal invasive carci-
noma in 510% of animals in a particular age range of a model
would be far more convincing than a single microscopic focus of
possible invasion in a single mouse.
The potentialfor invasion or the capacity to progress to inva-
sionare important concepts in precursor lesions. As such, it was
additionally decided that demonstration of progression to invasive
carcinoma of a PIN lesion in a transplant model would warrant
classification as PIN, with documented progression.The criteria for
recognizing subsequent invasion in a transplant model were not ex-
tensively discussed or clearly delineated, but in general, invasive
carcinoma should be recognized on the basis of the sound criteria for
invasion in the intact prostate as described below. Furthermore, stan-
dardized protocols for such acceptable transplantation models were
not developed or approved, and may be a focus for future workshops.
For now, complete details should be provided by investigators using
this experimental approach, including the age of the source animal,
the size, lobe, and histological nature of the transplanted material, and
the time course for subsequent histological investigation of the trans-
planted tissue. In contrast, crossing of one GEM line with another
(Table 1) with resulting progression of PIN lesions to invasion (which
has been specifically reported in some instances; Refs. 30, 79) does
not warrant classification of the PIN lesions in the parent lines as PIN
with invasion. Such bigenic lines are considered distinct models,
and should be characterized fully and classified separately.
Descriptions and Biology of mPIN. A spectrum of lesions has thus
far been observed in GEM models that satisfy criteria for mPIN.
Although sharing some general characteristics with PIN lesions in the
human prostate, the morphology of PIN in some GEM models is
distinct from that of the human prostate PZ in many aspects, including
with regard to nuclear features. These differences are particularly
pronounced for SV40 early region and Tag transgenic mouse lesions
(Fig. 6). Tufting, micropapillary, and cribriform PIN lesions have
been observed in SV40 or large T antigen-based GEM models. How-
ever, compared with human HGPIN, in SV40-based mPIN lesions, the
atypical nuclei appear to be more elongated, are more hyperchromatic,
and have a greater mitotic and apoptotic rate (Fig. 3, CH; Ref. 16).
In at least one SV40 model, the high proliferative and apoptotic rates
have been confirmed by PCNA immunostains and tissue assays for
apoptotic bodies. These indices increased with time in PIN lesions,
paralleling other nuclear abnormalities in PIN, and were indeed much
higher than those reported in human prostate (32). Some PIN lesions
in SV40-based models have even demonstrated necrosis (e.g., CR2-
SV40),
12
which is not seen in human HGPIN, but such lesions were
still compatible with in situ lesions and not intraductal carcinoma as
described for human Pca (73). All of the reported SV40 early region
and Tag antigen-based models have progressed from PIN lesions to
invasive carcinoma (33, 37, 38, 8284).
Tufting, micropapillary, and cribriform patterns of epithelial pro-
liferation have also been noted in mPIN lesions in non-SV40-based
models, with cribriforming and even duct distension particularly
likely to be seen in older ages (35, 78). Compared with SV40-based
models, nuclear atypia appears to be of a different quality in PIN
lesions of these models, including growth factor and tumor suppressor
gene manipulated GEM models (Table 1). Although nuclear enlarge-
ment occurs, there is less hyperchromasia and less nuclear membrane
abnormalities. Nucleoli, occasionally multiple, are prominent (Fig. 7).
The nuclear atypia in these models is thus more reminiscent of that
seen in human HGPIN. Although progression to invasion has been
reported by some investigators (30, 34), it was not consistently present
in any of the models reviewed by the Pathology Panel. Since the time
of the Bar Harbor meeting, at least three non-SV40-based models
have been described that reportedly progress to unequivocal invasive
carcinoma (79, 80, 81). All of these models have had participation by
members of the MMHCC Prostate Pathology Committee in their
pathology characterization. As mentioned above, future review may
allow for development of histological mPIN grading schemes predic-
tive of progression in non-SV40-based models.
Modifiers of PIN Lesions in Individual Models. For GEM lesions
properly classified as mPIN, the pattern of growth should be described
(e.g., flat, tufting, micropapillary, cribriform, and combinations). The
extent of involvement should be described, with documented temporal
progression as detailed above (e.g., as focal or diffuse or as the
number of ducts/glands involved compared with the total assessed, or
expressed as percentage). Nuclear atypia and its progression should be
described. In all of the studies, but especially in models with perhaps
more subtle alterations (e.g., Fig. 7, Eand F), blinded pathological
analysis compared with aged-matched controls should be performed.
Supportive objective studies using tissue markers can be used, such as
proliferation and apoptosis assessment, which are known to be altered
in human PIN (19), and thus far in mPIN lesions that have been
examined (30, 32, 54).
Malignant Proliferations of the Epithelium
Microinvasive Carcinoma (Fig. 8)
Definition. Microinvasive Carcinoma [20]. Microinvasive carci-
noma is the earliest recognizable form of invasive carcinoma, with
penetration of malignant cells through the basement membrane of
PIN-involved glands into the surrounding stroma. It is distinguished
from invasive carcinoma by the greater extent of the invasive focus of
carcinoma in the latter.
Criteria/Explanation. Microinvasive carcinoma in the prostate of
GEM is recognized by the extension of individual tumor cells or small
nests or acini of cytologically atypical cells into the thin rim of stroma
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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 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. GI, invasive adenocarcinoma. Anterior prostate from 38-week-old (C57Bl/6TRAMP/⫹⫻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.
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underlying PIN-containing ducts or glands. Criteria for recognition of
microinvasion in the GEM prostate include adequate spatial separa-
tion from larger PIN glands of small atypical acini, nests, or individual
atypical cells; unequivocal penetration through the basement mem-
brane of PIN glands based on special stains, immunohistochemistry,
or electron microscopy; and a stromal response (e.g., desmoplasia).
Discussion. Microinvasive Carcinoma in Human Pca. A histo-
logically recognizable form of microinvasive carcinoma as defined
herein is not well characterized for human Pca. Clinically, when
HGPIN and adjacent small acini of atypical cells are present on
transrectal biopsy, it is important to distinguish a diagnosis of
HGPIN suspicious for invasionor HGPIN with adjacent small
atypical glands(which may warrant a repeat biopsy) versus HGPIN
with invasion(which is sufficient for definitive therapy, such as
radical prostatectomy; Refs. 65, 77, 85). However, even in the latter
situation, this refers to small acini of malignant cells nearby larger
HGPIN glands, but which are morphologically incompatible with just
tangential sectioning of outpouchings of HGPIN glands. This is dif-
ferent from a recognizable penetration through the basement mem-
brane of HGPIN containing glands of more closely adjacent individ-
ual tumor cells or small numbers of tumor cells as described for
microinvasive carcinoma in GEM. Hence, despite the biologically
important stage of initial invasion in Pca progression, the concept of
microinvasive carcinoma has little clinical meaning in the human.
Furthermore, because of sampling issues, a single small (i.e., 1 mm)
focus of adenocarcinoma (with or without associated HGPIN) on
biopsy cannot predict a small, clinically insignificant tumor that can
be managed conservatively (8688).
Microinvasive Carcinoma in GEM: General Considerations. De-
spite the above issues in human Pca, there are several reasons why
microinvasive carcinoma is considered to be a definable and useful
category in the prostate pathology of GEM. Biologically, the ability to
identify early invasive carcinoma may allow for the characterization
of the genetic alterations necessary for this crucial stage in the
progression of Pca. Secondly, because many models are being gen-
erated that appear to be predominantly characterized by PIN, having
histological criteria (or criteria based on ancillary techniques such as
immunohistochemistry) to make a definitive diagnosis of microinva-
sion will allow for the identification of which models truly progress.
Thirdly, unlike the difficulty in recognizing such a stage in human Pca
progression, it appears that such a stage of early progression can be
reliably identified in the mouse. Early invasive carcinomas in certain
GEM models do appear to take the form of individual cells or small
numbers of cells invading through the basement membrane into
surrounding stroma. The ability to sample evolving mouse lesions
with time in contrast to tissue sampling issues in human patients is one
advantage for defining these lesions. Recognition of microinvasion in
the GEM prostate may be facilitated by the more uniform or smoother
gland contours, with a generally flat or smooth epithelial-stromal
interface. However, given the extremely thin rim of fibromuscular
stroma in the mouse prostate, there cannot be a physically large
amount of separation between PIN glands and invasive foci in a
normal, non-desmoplastically thickened stroma. Hence, recognizing
microinvasive foci arising from PIN is still a potentially challenging
issue in mouse prostate pathology (16). The ability to define micro-
invasion in the mouse prostate is supported by several convincing
reported examples, particularly in models based on the SV40 early
region or large T antigen (Refs. 32, 33, 38; Fig. 8), but also potentially
in growth factor and cell cycle regulator manipulated models (30).
Blinded histopathologic analyses in GEM models support that this
stage of microinvasive Pca, relative to HGPIN, increases with time
(33, 38). Furthermore, histopathologic analyses in bigenic models to
begin addressing key factors in tumor progression have shown that
this is a definable end point, with documentable inhibition of progres-
sion to microinvasion from HGPIN lesions (89).
Microinvasive Carcinoma in GEM: Definitive Classification of Mi-
croinvasive Carcinoma and Possible Use of Adjunctive Techniques.
Obviously, greater confidence in the classification of microinvasion is
obtained for lesions in GEM models that clearly progress to unequiv-
ocally more extensive invasion (Fig. 8C). Possible criteria for more
pronounced degrees of invasion include obvious destructive or exten-
sive involvement of stroma, extension into periprostatic fat or loose
connective tissue surrounding the contractile stroma, perineural inva-
sion, lymphovascular invasion, and metastases (16).
For demonstrating unequivocal penetration of carcinoma into sur-
rounding stroma in models with PIN and questionable invasion,
adjunctive methodologies (special stains, immunostains, electron mi-
croscopy) may be warranted, especially in models not showing pro-
gression to more definitive and unequivocal invasive carcinoma over
the time periods being investigated (78). However, these adjunctive
techniques have not been rigorously applied and validated for this
specific issue of invasive versus in situ lesions. Application of such
techniques to tissue sections in models clearly progressing to micro-
invasive and more extensive invasive carcinoma may allow for the
establishment of novel criteria to apply to models with more ques-
tionable early invasion from PIN.
Microinvasive Carcinoma in GEM: Stromal Alterations with Early
Invasion. It is important to note that routinely detectable stromal
alterations are not a typical feature in invasive human Pca. Although
phenotypic alterations may be demonstrable using special techniques,
prostate stroma does not show histologically defined inflammation,
edema, or desmoplasia in response to usual invasive acinar carcinoma.
Stromal hypercellularity is not seen in association with invasive Pca
as a common or characteristic feature, and in fact, can sometimes
argue against carcinoma in suspicious foci, being more typical in
certain benign mimics of carcinoma in biopsy or transurethral resec-
tions of the prostate specimens (such as basal cell hyperplasia and
sclerosing adenosis; Refs. 8, 9). This does not mean that stromal
hypercellularity or desmoplasia cannot accompany invasive carci-
noma in GEM models and that it will not be a useful feature in
recognizing invasion in some models. However, it is crucial that such
stromal alterations be truly focal and found in association with the
possible invasive focus in question. It should not be observed more
uniformly in association with proliferating atypical epithelium as a
possible paracrine effect or as a possible more direct consequence of
transgene expression in stromal cells. Attention should be paid to the
focality of desmoplasia. Different pathologists may have different
sensitivities or skills for recognizing such alterations, and it can be
complicated by a more general background of stromal hypercellularity
in some models (16, 29, 37, 83). No consensus opinion was reached
regarding the validity of proposed examples of such desmoplasia in
association with invasive carcinoma in the study sets for the Bar
Harbor Pathology Workshop. However, this feature has been reported
in association with invasion in some models (83), may be dependent
in part on the genetic background of the mouse, and has been observed
by some of the involved pathologists.
27
Desmoplasia is certainly not
a uniformly appreciable feature of early invasive carcinoma in general
in GEM prostates, as it has not been observed or described in some
published models having convincing early invasion (33, 38).
27
M. M. Ittmann, unpublished observations.
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Invasive Carcinoma (Figs. 8 and 9)
Definition. Invasive Carcinoma [21]. Invasive carcinoma is a ma-
lignant epithelial neoplasm that exhibits destructive growth in prostate
parenchyma.
Criteria/Explanation. An invasive malignant epithelial neoplasm
has a growth pattern that in contrast to PIN is incompatible with
architecturally normal glands, and in contrast to a benign tumor,
exhibits destructive local invasion or is capable of metastasizing.
Invasive carcinoma in the mouse prostate is distinguished from mi-
croinvasive carcinoma by an increased size or extent of the invasive
focus (see criteria, below). Invasive carcinoma may show glandular
differentiation classifiable as adenocarcinoma, show cytologic fea-
tures of other types of carcinoma, such as NE or squamous cell
carcinoma, or show no morphological features of specific differenti-
ation, as in undifferentiated carcinoma.
The distinction of invasive carcinoma from microinvasive carcinoma
(defined and described above) is essentially one of degree. Recom-
mended criteria include one or more of the following, extension into a
widened (potentially desmoplastic) stroma (e.g., Fig. 8C; compared with
a more normal width stroma in microinvasive carcinoma; e.g., Fig. 8, A
and B); extension into the loose connective tissue and periprostatic fat
surrounding the contractile fibromuscular stroma (e.g., Fig. 8, GI; Fig. 9;
Ref. 38); and a focus size of 1 mm (33). Other possible morphological
indicators of invasive carcinoma include perineural invasion, lymphovas-
cular invasion, and metastases. Criteria for classification of invasive
carcinomas into specific subtypes based on patterns of differentiation
(Table 3) are described below.
Discussion. In contrast to PIN, because of the presence of lym-
phatics and vessels in prostatic stroma and surrounding tissue, inva-
sive carcinomas in human and mouse have metastatic potential, as
well as the increased capacity to cause morbidity due to local/regional
effects. In addition to local invasive growth within prostatic paren-
chyma (including the contractile rim of fibromuscular stroma and the
surrounding looser connective tissue in the GEM prostate), other
properties of invasive carcinoma in human Pca that may be recog-
nized in the GEM prostate include perineural invasion, lymphovas-
cular invasion, and metastases. These properties are discussed in more
detail, followed by definitions and characteristics of the subclassifi-
cations of invasive carcinomas in GEM prostates.
Perineural Invasion in Pca. Human Pca has a well-recognized char-
acteristic tendency to invade perineural spaces. In human prostate
pathology, properly defined perineural invasion is pathognomonic of
carcinoma on biopsy (8, 9). It is observed in 20% of transrectal
biopsies with Pca, in which case it may correlate with ECE in
subsequent RP (90, 91) and/or help in the decision to sacrifice the
neurovascular bundle ipsilateral to the side with perineural invasion,
potentially reducing the risk of positive surgical margins at foci of
ECE in RPs (92). Perineural invasion is present in the vast majority
(7590%) of totally submitted RP specimens. The prognostic signif-
icance is the subject of ongoing investigation (93).
In the mouse prostate, nerve bundles are not histologically conspic-
uous within the thin rim of fibromuscular stroma surrounding indi-
vidual acini, but rather are seen in the surrounding loose connective
tissue. Prostatic perineural invasion by carcinoma has been observed
in multiple GEM models (29, 33). However, these particular models
develop obvious destructive prostatic stromal and periprostatic inva-
sion by carcinoma, so that it is not yet known if perineural invasion
will be observed in mouse models as the only definitive parameter of
invasion in occasional cases.
Lymphovascular Invasion in Pca. Lymphovascular invasion can be
identified in a significant minority of human RP specimens, including
those without identifiable pelvic lymph node metastases (94, 95). The
frequency of lymphovascular invasion is greater in stage pT3 (versus
pT2) tumors, and in such patients, tumor lymphatic invasion may have
independent prognostic significance for biochemical progression (el-
evated serum PSA) after RP (94, 95).
Lymphovascular invasion has been reported with carcinoma in the
prostate of GEM (33, 37), presumably in lymphatic spaces in the loose
connective tissue between acini rather than within the thin rim of
fibromuscular stroma surrounding acini. It is also presumed that
lymphovascular invasion in mouse prostate tumor models will un-
commonly be identified in the absence of obvious (stromal) invasive
carcinoma, but there is little specific information regarding this thus
far. Furthermore, as in routine human surgical pathology, caution
must be exercised regarding possible artifactual histologically identi-
fied lymphovascular invasion. The presence of large amounts of
potentially fragile or friable in situ atypical proliferations can lead to
implantation of tumor within vessel spaces either during grossly
cutting poorly fixed specimens or during histological sectioning.
Attention should be paid to the usual morphological features of true
lymphovascular invasion as described in human Pca (e.g., shape of
focus, adhesion to vessel wall, and subtle cytologic changes; Ref. 94)
as well as presence or absence of associated unequivocal invasion, its
extent, and its spatial relationship to the focus of possible lympho-
vascular invasion.
Metastases in Pca. The presence of metastatic Pca is essentially
diagnostic of invasive carcinoma, as neoplasia confined to in situ
basement membrane-contained lesions should not have access to
lymphatic or vascular spaces necessary for regional or systemic
spread. However, as in human Pca, tumor metastasis would not be
expected to be a very sensitive indicator of invasive disease in GEM.
For example, all of the patients undergoing RP have documented
invasive disease; however, in most current practice settings, lymph
node metastases are present in only 25% of RP and bilateral pelvic
lymph node dissection specimens (14, 17, 96).
All of the GEM models of Pca reported thus far as developing
metastatic tumor have had unequivocal invasion at similar or earlier
time points. However, metastases are occasionally noted in individual
animals without documented invasive histologically similar primary
tumors in the prostate. This could represent a sampling issue, as there
is some indication that metastases from mouse Pcas can occur in
association with very small foci of invasive tumor (38). However, if
this situation is encountered, several other explanations should be
considered: (a) the metastasis could come from another completely
unrelated site, which should always be excluded in transgenic mice
made with nonselective promoters or in genomic knockout models;
(b) the primary could be in other male accessory glands, such as the
periurethral or BUGs, which develop invasive tumors histologically
similar to prostate tumors in some models made with reportedly
prostate-selective promoters; and (c) a poorly differentiated spindle
cell tumor in a distant site could actually represent a metastatic
sarcoma, which should be a consideration in models that are devel-
oping stromal hyperplasia and possibly neoplasia in association with
neoplastic epithelial proliferation. In this last case, ancillary studies,
such as CK immunostains and electron microscopy, can be useful for
distinguishing carcinoma from sarcoma. Sarcomas typically metasta-
size by hematogenous routes, very characteristically to liver and lung.
However, several GEM models developing NE carcinomas have
shown liver and lung metastases, and this pattern of visceral metas-
tasis is also characteristic of Pca with NE differentiation in the human
(97). Lymph node metastases are quite characteristic of carcinoma, in
general, and pelvic lymph nodes are a typical site for metastasis of Pca
in the human. Para-aortic/abdominal lymph node metastases have
been noted in multiple GEM models of prostatic neoplasia.
It was the consensus opinion of the Pathology Panel that immuno-
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Fig. 9. Invasive neuroendocrine (NE) carcinomas and carcinomas with morphological features suggestive of NE differentiation in prostate and metastases in genetically
engineered mice models. AC, 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). Band 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. DI, 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 differentiatedsmall 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 outcribriform 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 artifactor 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 immunostaining (apical cytoplasmic granular, arrowheads)inthein situ component (PIN) of 12-month-old LPB-Tag
12T-10 mouse, which also had extensive invasive NE carcinoma. G, focal strong cytoplasmic granular chromogranin immunostaining () in liver metastasis. H, Metastatic NE
carcinoma () in the liver of a 40-week-old 12T-10 mouse. Liver metastases were common with increasing age in this mouse, and often showed prominent rosetting. NE
differentiation was demonstrated in such metastases by chromogranin immunostaining, as in G, and by electron microscopy. I, two pulmonary micrometastases () in 44-week
LPB-Tag 12T-10 mouse. Lung metastases are typically smaller than liver lesions, are often in alveolar septa or peribronchial arterial spaces, and are more typically composed
of oval or spindle cells with scant cytoplasm and without prominent rosette formation. JL, invasive carcinoma with morphological features suggestive of NE differentiation
in TRAMP mouse. Jand K, intermediate- and high-power photomicrographs of prostate from 24-week-old TRAMP mouse showing extensive destructive invasion (in J)
between residual PIN glands (arrowheads in J). The tumor shows relatively solid growth, but with evident cribriform or gland-like spaces, recognizable at lower magnifications,
and confirmed at high magnification (arrowheads in K). This lesion, reported previously as moderately differentiated adenocarcinoma, shows most nuclei contain granular
chromatin. Admixed spindle or oval cells with more hyperchromatic and/or pyknotic nuclei and abundant rosette or rosette-like spaces are noted. The consensus opinion of
the Pathology Panel was that this morphology was highly suggestive of a carcinoma with NE differentiation, based on experience with human tumors as well as the
morphological similarity to well-documented NE carcinomas in other genetically engineered mice (e.g., compare with BE). Similar morphology has been noted in metastatic
foci. L, less-differentiated focus in prostate from another 24-week-old TRAMP mouse. Tumor reported previously as poorly differentiated carcinoma was felt to show
morphological features of NE differentiation, quite similar to human small cell carcinoma. Tumor is composed of closely spaced oval and spindle cells with scant cytoplasm
and hyperchromatic nuclei. Metastatic foci, including pulmonary metastases, can show similar morphology. Similar cytologic features have been noted in the invasive and
metastatic tumors in other mouse models (i.e., CR2-SV40 and LPB-Tag 12T-10) in which the tumor has been confirmed to show immunophenotypic and ultrastructural NE
differentiation. The foci in TRAMP as shown in Land regarded as small cell carcinoma by the Pathology Panel have been subsequently shown to be synaptophysin
immunopositive, whereas such staining was not noted in foci as shown in Jand K.
28
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stains for a particular transgene (e.g., large T antigen in the case of
SV40 or Tag-based models) cannot be used as a priori evidence of
origin from the prostate for a potential metastatic focus. This is poor
pathology practice, and numerous examples are recognized of trans-
gene expression in other sites besides the prostate with anticipated
prostate selective promoters. However, in well-characterized models
in which extensive necropsy studies (covering age ranges in question)
have shown absence of transgene expression in other organs (the list
can never be too complete), transgene expression documented by in
situ hybridization or immunostains can be an important adjunct to
more traditional means of establishing the site of a primary tumor for
metastases in question. Currently, well-documented markers for pros-
tate origin independent of a specific transgene (analogous to human
PSA) are not defined for mouse. More data need to be acquired for
newer techniques, such as mass spectrometric analysis of protein
expression patterns (38), for identifying or confirming a prostate
origin for a metastatic focus.
Classification of Invasive Carcinoma in GEM. When an invasive
carcinoma is identified, it should be histologically classified as to a
specific type of carcinoma if possible, such as adenocarcinoma or NE
carcinoma, or otherwise classified as an undifferentiated carcinoma
(Table 3). The use of adjunctive techniques such as immunohisto-
chemistry or electron microscopy is also described below for some
specific applications.
Adenocarcinoma (Fig. 8)
Definition. Adenocarcinoma. Adenocarcinoma is an invasive ma-
lignant neoplasm of epithelium that demonstrates unequivocal glan-
dular formation.
Criteria/Explanation. Although these tumors may show immuno-
phenotypic or ultrastructural features indicative of NE differentiation,
these are not extensive or uniform, and adenocarcinomas lack the
characteristic light microscopic features of NE differentiation detailed
for NE carcinoma below. In the Bar Harbor Classification, adenocar-
cinomas of the prostate of GEM should be additionally designated as
well, moderately, or poorly differentiated, according to the extent or
percentage of glandular formation.
Well-differentiated adenocarcinomas refer to those in which all or
the majority of the invasive focus is composed of discreet, well-
formed glands (Fig. 8, Eand F). Moderately differentiated adenocar-
cinomas refer to tumors in which extensive gland formation is still
evident, but there are admixed foci showing gland fusion or solid
areas (Fig. 8, GI). Poorly differentiated adenocarcinomas are those in
which invasive foci are composed predominantly of nests or solid
sheets, but in which glandular formation is focally present.
Discussion. It was the opinion of the Pathology Panel that specific
percentages for the amount of an invasive tumor showing gland
formation would not be specified for the purpose of the classification
of differentiation of adenocarcinoma in GEM. If progressive dedif-
ferentiation is observed in a given model, with less gland formation
observed in invasive foci with age, the approximate percentage of
gland formation in individual tumors can be stated as a function of
time. It should be noted that the terms designating differentiation in
the GEM classification of prostate adenocarcinoma do not translate to
similar terms in human prostate pathology. Although differentiation in
human Pca is more often stated by Gleason score or pattern, there is
a general translation to these more descriptive terms, such that well-
differentiated tumors correspond to typically TZ located Gleason
pattern 1 and 2 (Gleason score 24) tumors (Table 4). Moderately
differentiated tumors are composed of Gleason score 5 and 6 tumors,
and, hence, predominantly include Gleason pattern 3 tumors, com-
posed of well-formed small glands, as commonly seen in PZ tumors
associated with HGPIN. A morphologically similar tumor (composed
entirely or predominantly of discreet and well-formed glands) arising
in association with mPIN in a GEM model would be classified as well
differentiated. A summary of the classification of adenocarcinoma in
GEM prostate is shown in Table 5. NE differentiation (e.g., based on
ancillary methods such as immunohistochemistry or electron micros-
copy) in an otherwise more usual adenocarcinoma is addressed below.
Poorly differentiated carcinomas, which do not show such NE differ-
entiation, may be classified as poorly differentiated adenocarcinomas
if focal gland formation is evident or if ultrastructural examination or
other ancillary techniques show focal glandular or secretory differen-
tiation. Otherwise, invasive epithelial tumors that do not show specific
differentiation as outlined in Table 3 should be classified as undiffer-
entiated carcinomas.
NE Carcinoma (Fig. 9)
Definition. NE Carcinoma [23]. NE carcinoma in GEM prostate is
an invasive malignant epithelial neoplasm that shows light micro-
scopic, immunohistochemical, and ultrastructural features indicative
Table 4 Differentiation classification of human prostate adenocarcinoma
Differentiation Gleason
score Common
gleason pattern Histologic/cytologic description
Well
differentiated 24 One Typically TZ;
a
circumscribed; fairly uniformly sized, closely spaced glands;
also BPH-like larger glands with ample clear cytoplasm (lipid), well-defined
cell borders, basally situated relatively bland nuclei
Two Typically TZ; partially circumscribed, closely spaced but more variably sized
glands with generally clear cytoplasm similar to benign prostate glands
Moderately
differentiated 56 Three Common in PZ, can be seen in TZ; in PZ, commonly associated with HGPIN;
small acinar forming, more infiltrative with greater intervening stroma and/
or infiltration between benign glands; eosinophilic cytoplasm
Moderately
poorly
differentiated
7 Four Common in PZ; can occur in TZ; characterized by gland-fusion and/or large
irregular cribriform proliferations (i.e., no longer discreet gland formation,
but still features of adeno differentiation)
Poorly
differentiated 810 Five Characterized by infiltrating cords of cells and single cells; large solid growths,
including with comedo necrosis
a
TZ, transition zone; BPH, benign prostatic hyperplasia; PZ, peripheral zone; HGPIN, high-grade prostatic intraepithelial neoplasia.
Table 5 Differentiation classification of mouse prostate adenocarcinoma
Differentiation Histologic description
Well differentiated Composed exclusively or predominantly
of discreet, well formed glands
Moderately
differentiated Gland formation clearly evident, but
focal to extensive areas are composed
of fused glands or more solid areas
Poorly differentiated Tumor is composed predominantly or
exclusively of more solid sheets or
nests, with either rare gland formation
histologically or cells demonstrated to
have secretory differentiation by
ancillary techniques
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of NE differentiation, corresponding primarily to the presence of
cytoplasmic neurosecretory-type granules.
Criteria/Explanation. The designation of a mouse tumor as an NE
carcinoma is based on the presence of characteristic histological and
cytologic properties, which can be confirmed by ancillary techniques.
NE carcinomas can take the form of solid and cribriform growth with
interspersed gland-like spaces from rosette formation. Although ro-
settes can mimic gland formation, these tumors differ from adenocar-
cinoma in that NE carcinomas do not show well-defined true glandu-
lar formation or extensive secretory differentiation. Tumor cells may
have moderate eosinophilic cytoplasm, often appreciated in areas of
rosetting. The nuclei are often oval or round and have finely granular
chromatin. Small cell carcinomas are a subtype of NE carcinoma,
which are histologically and cytologically similar or identical to the
well-known small cell carcinoma of the human lung. NE carcinoma
(not otherwise specified) and small cell carcinoma may coexist. Small
cell carcinomas are characterized by tumor cells with scant cytoplasm
(high nuclear:cytoplasmic ratio), with oval or spindled markedly
hyperchromatic nuclei, which may show nuclear molding,in which
one nucleus appears to indent into an adjacent nucleus, which con-
forms to the shape of the former.
NE carcinoma and small cell carcinoma should be immunopositive
for one or more markers of neurosecretory differentiation, such as
chromogranin (CG) and synaptophysin, at least focally. Ultrastructur-
ally, NE carcinoma cells should show dense core (NE type) secretory
granules, at least focally. The approach to the recognition and classi-
fication of NE carcinomas is outlined in Table 6.
Discussion. Invasive NE carcinoma has been observed in several
SV40 and large T antigen-based transgenic mouse models, based on
recognition of morphology typical of NE carcinomas in human pa-
thology and with subsequent confirmation by immunohistochemistry
and electron microscopy (Fig. 9, AI; Refs. 33, 38, 84). At the time of
the Bar Harbor meeting, NE differentiation in the invasive tumors in
other SV40-based models was suggested by similar light microscopic
morphological features to these documented NE carcinomas in GEM
(Fig. 9, Kand L). Classification of a mouse tumor as an NE carcinoma
is based on the presence of characteristic histological and cytologic
properties, which can be confirmed by ancillary techniques (Table 6),
and not the demonstration of focal immunostaining for traditional NE
markers in an otherwise histologically recognized adenocarcinoma.
It should be noted that NE differentiation can be seen focally in
many and maybe even most otherwise typical human prostate adeno-
carcinomas using immunohistochemistry (8, 9). The prognostic sig-
nificance of such NE differentiation, the increase in such differenti-
ation with tumor progression, and its relationship to the development
of hormone refractoriness remain a subject of controversy in human
prostate pathology (8, 9). In addition, there is a minority of human
Pcas that show characteristic light microscopic morphological fea-
tures of NE carcinomas, including small cell carcinomas, as more
typically associated with other organ sites such as the lung (8, 9). The
designation of a mouse prostate tumor as an NE carcinoma is for
precise pathological characterization. Correct tumor classification will
allow the accumulation of information regarding phenotype and tumor
behavior, genetic alterations, and treatment response. Although there
are several preliminary observations relating this phenotype to andro-
gen insensitivity (33, 38, 55, 61), there is currently insufficient data to
allow any specific biological inferences regarding this morphology in
GEM models.
Because rosette formation mimicking gland formation can be evi-
dent in NE carcinomas (Fig. 9), gland-likespaces are not incom-
patible with a diagnosis of NE carcinoma. This pattern of NE carci-
noma, somewhat reminiscent of large cell NE carcinoma in the human
lung (98), may be admixed with or appear to transition to foci with the
appearance highly reminiscent of human lung small cell carcinoma,
either classic oat cellcarcinoma or intermediate cell type (8, 9, 97,
98). In addition to hyperchromatic nuclei, which may show nuclear
molding,small cell carcinomas often show crush artifact, in which
smeared hyperchromatic material is seen in hypercellular areas and
corresponds to DNA-rich material (Azzopardi effect). Small cell
carcinoma in the human prostate may be immunopositive for PSA and
negative for NE markers such as neuron-specific enolase or negative
for PSA and positive for neuron-specific enolase. Although data are
based on relatively small numbers, patients with these tumors appear
to be hormone insensitive, have an increased incidence of visceral
(i.e., liver and lung) rather than lymph node metastases, and may
respond to established small cell chemotherapy (97). Because tumors
in human and mice with this small cell carcinoma morphology can be
seen in association with NE carcinomas with rosetting and more
cytoplasm, in the Bar Harbor Classification, small cell carcinoma is
regarded as a less-differentiated form of NE carcinoma, rather than as
a separate entity (Table 3). Although little data exist yet in GEM
regarding degree of NE differentiation in tumors with these two
Table 6 Neuroendocrine (NE) carcinoma: considerations and classification in genetically engineered mice (GEM) prostate
Summary of NE differentiation in human and GEM prostate carcinoma
The relationship between NE differentiation determined immunophenotypically and prognosis in human Pca
a
is not established.
Glandular differentiation and NE differentiation in human Pca and potentially in GEM tumors are not mutually exclusive. In human Pca:
NE differentiation may be demonstrated in usual acinar carcinoma.
Small cell carcinomas are commonly seen in association with usual acinar carcinoma or arise in patients with a prior diagnosis of more usual acinar adenocarcinoma.
Small cell carcinomas have a variable immunophenotype regarding presence of usual NE markers. Small cell carcinoma in the human is diagnosed on the basis of its
cytologic appearance.
Approach to tumors in GEM with histologic features suggestive of NE differentiation
Tumors with light microscopic appearance similar or identical to those illustrated as NE carcinoma herein (that have been substantiated by immunohistochemistry and electron
microscopy), should be regarded as potentially being NE carcinomas. NE differentiation should be confirmed by ancillary techniques.
Immunostaining should employ at least two different markers, e.g., chromogranin and synaptophysin. Positive immunostaining for either is sufficient to designate such
tumors as NE carcinoma (when morphology is as shown for NE carcinoma herein). Punctate perinuclear CK8 immunostaining is supportive but less specific.
Positive immunostaining for NE markers can be substantiated by ultrastructural examination in order to demonstrate dense core neurosecretory granules.
Negative immunostaining should be followed by electron microscopy. As NE type granules can be focal and small in number, examination should be rigorous. Consultation
with members of the MMHCC Pathology panel is encouraged. If NE type secretory granules are identified, they are sufficient to designate such tumors as NE carcinoma
(when morphology is as shown for NE carcinoma herein).
In the absence of immunohistochemical or ultrastructural confirmation of NE differentiation, tumors with suggestive morphology can be designated as carcinoma with NE
differentiation or carcinoma with NE features.
Tumors with glandular differentiation (adenocarcinoma) or without histologic features of either glandular or NE differentiation (undifferentiated carcinoma) that have NE
differentiation demonstrated by immunohistochemistry or electron microscopy can be designated as adenocarcinoma (or carcinoma) with NE differentiation. The specific
ancillary techniques employed and their results should be specified.
Tumors with cytologic features typical of small cell carcinoma in human lung and human prostate should be designated as small cell carcinoma, regardless of the
immunophenotype. It is encouraged that immunohistochemistry for NE markers still be performed on these tumors in order to collect potentially useful data for future
classification.
a
Pca, prostate cancer; MMHCC, Mouse Models of Human Cancer Consortium.
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morphologies, small cell carcinomas in the human may show less
neurosecretory granules on ultrastructural examination and less ex-
tensive immunostaining for some markers corresponding to these gran-
ules. Effort should be made to distinguish these two tumor morphologies
in invasive foci or the relative contribution of each morphology in an
admixed tumor, so that any correlation with specific genetic alterations,
changes in androgen receptor expression, structure, or function, or dif-
ferential response to treatment can be potentially discerned.
If a tumor with morphology identical or similar to that shown in
Fig. 9 is encountered in a GEM model, exhaustive effort to confirm or
exclude NE differentiation should be applied (Table 6). Currently
established NE markers for which immunostains have been success-
fully applied to mouse formalin-fixed, paraffin-embedded sections
include CG (33, 38) and synaptophysin (see Protocolssections). As
more invasive prostatic adenocarcinomas are recognized in GEM
models, the specificity of these markers for unequivocally NE carci-
nomas versus possible focal expression in otherwise typical adeno-
carcinomas can be addressed. However, thus far in a model in which
CG immunostaining was noted in NE carcinoma foci, definitive
adenocarcinoma foci were negative (38), and in a model in which
synaptophysin immunostaining was noted in small cell carcinoma-like
foci, well-differentiated adenocarcinoma foci were negative (99).
28
Squamous Cell Carcinoma and Adenosquamous Carcinoma
Definitions. Squamous Cell Carcinoma [24]. Squamous cell car-
cinoma is an invasive malignant epithelial neoplasm that shows light
microscopic features of squamous differentiation, typically in the
form of keratinization and/or intercellular bridges.
Adenosquamous Carcinoma [25]. Adenosquamous carcinoma is
an invasive malignant epithelial neoplasm that shows an admixture of
foci with squamous differentiation and foci with glandular differen-
tiation.
Criteria. These lesions should be diagnosed based on the presence
of features recognized in such tumors in human and veterinary pa-
thology in prostate and other sites (8, 9). Features of adenocarcinoma
are described above.
Discussion. Primary squamous cell carcinomas are extremely rare
in the human prostate, and most typically arise from the prostatic
urethra. They have not been reported in the prostate of GEM. The
same is true for adenosquamous carcinoma.
Spindle Cell or Sarcomatoid Carcinoma
Definition. Spindle Cell Carcinoma [26]. Spindle cell or sarcoma-
toid carcinoma is a malignant spindle cell lesion in which there is
unequivocal epithelial differentiation detected by immunohistochem-
istry or by ultrastructure (8, 9). It differs from adenocarcinoma by the
absence of foci with well-formed glands.
Criteria. Such tumors should be diagnosed based on the presence
of features recognized in such tumors in human and veterinary pa-
thology. Tumors composed of foci showing glandular differentiation
and foci of spindle cell carcinoma should be classified as adenocar-
cinoma (moderately or poorly differentiated depending on extent of
glandular differentiation), with a detailed description of the less-
differentiated or sarcomatoid area, including documentation of its
epithelial nature. The latter is best demonstrated by CK immuno-
staining and/or by ultrastructural examination as in standard pathol-
ogy practice. These tumors differ from carcinosarcomas in that the
spindle cell component contains markers indicative of epithelial dif-
ferentiation. They have been referred to recently as epithelial-mesen-
chymal-transition tumors in the mouse pathology literature.
Discussion. Spindle cell or sarcomatoid carcinoma has not been
specifically reported in the prostate of GEM, although it needs to be
considered in the differential diagnosis of poorly differentiated (un-
differentiated) carcinomas, carcinosarcomas, and sarcomas, as de-
scribed below.
Undifferentiated Carcinoma
Definition. Undifferentiated Carcinoma [27]. Undifferentiated
carcinoma is an invasive malignant epithelial neoplasm (carcinoma)
that shows no glandular, NE, squamous, or spindle cell differentiation
by light microscopy.
Criteria. Undifferentiated carcinomas show a nested or sheet-like
growth pattern, and their content of cytoplasm and cohesive growth
help support the diagnosis of carcinoma over other neoplasms (e.g.,
sarcoma). Epithelial nature can be confirmed by CK immunostaining
or ultrastructural examination. If features of specific differentiation
are evident only with special techniques (e.g., electron microscopy),
such tumors may be appropriately referred to as poorly differentiated
carcinoma with reference to the direction of differentiation (e.g.,
poorly differentiated carcinoma with glandular differentiation or
poorly differentiated adenocarcinoma; poorly differentiated carci-
noma with NE differentiation or poorly differentiated NE carcinoma).
Neoplastic Proliferation of the Stroma
Benign Soft Tissue Neoplasms and Sarcomas (Fig. 10)
Definitions. Benign Soft Tissue Tumor [28]. A benign soft tissue
tumor is a neoplasm that arises from or differentiates toward prostatic
stroma or specific differentiated mesenchymal tissues (such as smooth
muscle) and lacks features indicative of malignancy.
Malignant Soft Tissue Tumor [29]. A malignant soft tissue tumor
(sarcoma) is a neoplasm that arises from or differentiates toward prostatic
stroma or specific differentiated mesenchymal tissues (such as smooth
muscle) and possesses one or more features indicative of malignancy.
Criteria/Explanation. These designations are used for neoplastic
growths satisfying usual human and veterinary pathology diagnostic
criteria for benign and malignant mesenchymal neoplasms. Features
indicative of malignancy that are absent from benign tumors and
variably present in sarcomas include destructive invasive growth,
extensive necrosis, prominent mitotic activity, and markedly in-
creased cellularity, particularly if accompanied by nuclear atypia or
prominent pleomorphism. Such soft tissue neoplasms have rarely been
observed or reported in GEM. For now, if such lesions are encoun-
tered, they should be diagnosed and classified according to standard
pathology practice. A few practical considerations are offered in the
following discussion.
Discussion. Mesenchymal Neoplasms in the Human Prostate. In
the human prostate, the most common sarcoma in the pediatric pop-
ulation is rhabdomyosarcoma, usually of the embryonal type (8, 9). In
the adult human prostate, benign stromal nodules are extremely com-
mon, increasing with age (Fig. 10, Aand B). They are located
typically in the glandular poor periurethral region, and are usually
accompanied by glandular and stromal hyperplasia in the TZ as part
of BPH. Benign circumscribed lesions with histology and immuno-
phenotype of smooth muscle, and hence classifiable as leiomyomas,
occur uncommonly (Fig. 10, Cand D; Refs. 8, 9). Rare proliferations
of the specialized prostate stroma occur that demonstrate increased
cellularity, atypia, and mitoses (8). Some stromal proliferations in the
human prostate appear to have an associated epithelial component,
imparting a biphasic appearance. The epithelial or glandular spaces
28
N. Greenberg, personal communication.
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can exhibit a leaf-likegrowth pattern, quite similar to the phyllodes
tumor more typical in the human breast (8, 9). Such lesions in the
human prostate are best regarded as low-grade and, less commonly,
high-grade malignant neoplasms (rather than hyperplasias), as they
have occasionally demonstrated local recurrence and even metastases
(8, 9). The most common sarcoma in the adult human prostate is
leiomyosarcoma (8, 9). Criteria for distinguishing potentially malig-
nant from benign smooth muscle neoplasms include the presence of
infiltration, nuclear atypia, significant pleomorphism, necrosis, and
appreciable mitotic activity (8, 9).
Mesenchymal Lesions in the GEM Prostate. Stromal hypercellu-
larity is a prominent feature of the prostate in some GEM models (Fig.
5, AC). It is a prominent feature of the AP and DP/DLP of the faster
growing LPB-Tag lines (Ref. 37; Fig. 5A). The stromal hypercellu-
larity is diffuse in these lobes (accompanying the marked epithelial
proliferation), generally increases with age, and the cells and sur-
rounding extracellular collagenous stroma have morphology in keep-
ing with prostate stroma. Similar, but potentially lesser, stromal hy-
percellularity has been observed in the prostate of TRAMP mice (Ref. 83;
Fig. 5B).
11
Similar extensive or focal stromal hypercellularity has also
been observed in the smooth muscle wall of the seminal vesicle in
LPB-Tag and TRAMP mice.
29
In the prostate, these changes are too
diffuse and too morphologically similar to normal mouse prostate stroma
to represent desmoplasia. Whether they occur as a paracrine response to
neoplastic epithelial cells or as a more direct consequence of transgene
expression in these cells remains to be further resolved. When not
associated with a distinct circumscribed or destructive growth pattern
suggestive of neoplasia, such lesions should be described as hyperplasia,
either focal or diffuse, and with or without atypia, as described above.
We have noted in the background of more diffuse stromal hyper-
cellularity in some LPB-Tag mice, somewhat discreet foci that stand
out as more cellular, and which show increased pleomorphism and
mitoses.
13
The natural history of such lesions in this or other models
29
S. B. Shappell, R. Barrios, R. Herbert, et al., unpublished observations.
Fig. 10. Mesenchymal neoplasms in human prostate and genetically engineered mice. Aand B, benign stromal nodules in human prostate. A, low power showing circumscribed nature
of stromal nodules (), in this case an incidental finding in a radical prostatectomy for prostate cancer. The characteristic suburothelial location is indicated, with the prostatic urethra
shown at top left (arrowhead). Stromal nodules are typically located in the periurethral stroma, where there is little or no gland tissue (e.g., penetrated only by periurethral ducts, arrows),
interior to the more typical areas of benign prostatic hyperplasia glandular and stromal hyperplasia, with which they are often associated. Proximity to the urethral can lead to obstructive
symptoms with larger nodules. B, high power showing moderately increased cellularity, with oval to spindle cells similar to normal prostate stroma, often in a somewhat myxomatous
or edematous stroma (pale blue) in contrast to more dense usual collagenous stroma. Small vessels are a common conspicuous component (), as are scattered lymphocytes
(arrowheads). Cellular atypia, pleomorphism, and mitotic activity are not appreciated. Cand D, leiomyoma (benign smooth muscle tumor) in human prostate. The lesion was a
circumscribed 1.3-cm mass protruding from the prostate base noted at the time of radical prostatectomy for prostate cancer. Larger tumors can be symptomatic, but these lesions are
uncommon in the prostate. The neoplasm has the same histological features as similar tumors in other sites, such as the uterine myometrium or soft tissues. Compared with the stromal
nodule in Aand B, note the more defined fascicular growth, with interlacing bundles of cells typically intersecting at right angles, such that some are cut longitudinally and others in
cross-section (e.g., in D). This example is moderately cellular, but at high magnification (D), the cells are generally uniform, and show typical oval or cigar-shapedround-ended
nuclei. Such cells are positive on immunostaining for common smooth muscle markers (e.g., smooth muscle actin, desmin). Given the relative rarity of such neoplasms in the prostate,
criteria to identify tumors capable of behaving in a malignant fashion are not as defined as in the uterus. However, infiltrative growth and increased cellularity, and especially necrosis,
marked nuclear atypia and pleomorphism, and any appreciable mitotic activity are compatible with leiomyosarcoma. Eand F, sarcoma consistent with leiomyosarcoma in genetically
engineered mice prostate. The tumor is from an LPB-Tag 12T7s mouse that was castrated at 24 weeks and sacrificed 24 weeks later. Atrophic PIN foci, some with intraluminal
phyllodes-likeprojections, are noted at right (arrowheads). Even at low magnification (E), the marked hypercellularity of the spindle cell tumor () is evident, as is extensive necrosis
(ⴱⴱ). At high magnification (F), the tumor is composed of interlacing fascicles of markedly atypical spindle cells. There is marked hypercellularity, and the tumor cells show prominent
nuclear atypia, including hyperchromasia and pleomorphism. Mitotic figures were numerous. The differential diagnosis includes a poorly differentiated spindle cell or sarcomatoid
carcinoma (this mouse had a neuroendocrine carcinoma elsewhere in the prostate) or some other sarcoma, such as malignant fibrous histiocytoma. On immunostaining, this sarcoma
would be negative for cytokeratin and strongly positive for smooth muscle markers, such as smooth muscle actin.
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has not been examined. Neoplasms properly classified as sarcomas
have rarely been noted in the prostate of GEM, so that their true
incidence in different models or under different experimental manip-
ulations remains to be clarified. In addition to poorly differentiated
carcinomas with histological features suggestive of NE differentiation
emerging in castrated animals from fast-growing LPB-Tag lines, foci
of increased stromal hypercellularity with atypia similar to that de-
scribed above and lesions compatible with frank sarcomas are occa-
sionally noted (55). One lesion in a 12T7s mouse castrated at 25
weeks and maintained for an additional 25 weeks developed a sar-
coma with histological features compatible with leiomyosarcoma. The
tumor is composed of highly atypical and mitotically active spindle
cells organized in well-formed intersecting fascicles typical of
leiomyosarcoma (Fig. 10, Eand F). As the mouse had other foci
involved by poorly differentiated carcinoma with NE differentiation,
consideration could be given to a designation of carcinosarcoma as
described below. Some of the specific sarcoma subtypes are included
in the Bar Harbor Classification for completeness and anticipation of
the possible spectrum of neoplasms that may occur in the GEM
prostate. However, to our knowledge, tumors such as rhabdomyosar-
coma, chondrosarcoma, and osteosarcoma have not been observed. If
encountered, they should be diagnosed based on standard practices in
veterinary and human pathology.
Distinction of Carcinoma and Sarcoma. When a poorly differen-
tiated neoplasm with oval or spindle cells is encountered in the prostate
and/or as a metastatic lesion, designation as carcinoma should be sup-
ported if necessary by either convincing CK (typically a pan-CK or CK8,
not HMWCK such as CK5 or CK14) immunostaining or by ultrastruc-
tural examination. Spindle cell neoplasms may also be small cell carci-
nomas as described above, which can have weak CK immunostaining.
Inclusion of appropriate NE markers (CG or synaptophysin) can address
this potential differentiation (along with electron microscopy). Vimentin
immunostaining is also theoretically useful in this setting, as strong
vimentin and absent CK staining support a sarcoma. However, few if any
successful mouse vimentin immunostaining protocols have been de-
scribed. If the differential diagnosis includes a specific sarcoma, antibod-
ies for characteristic differentiation markers, such as smooth muscle actin
for possible smooth muscle tumor, can be added. Sarcomatoid (or spindle
cell) carcinoma defined above can have focal or weak vimentin immu-
nostaining (in addition to CK immunostaining).
Neoplastic Proliferations of Stroma and Epithelium
Carcinosarcoma
Definition. Carcinosarcoma [30]. Carcinosarcoma is a truly bi-
phasic malignant lesion in which areas of unequivocal carcinoma and
unequivocal sarcoma are both present.
Criteria/Explanation. Any detected lesions in GEM should be
diagnosed according to similar principles in human prostate pathology
(8, 9). The epithelial component may be poorly differentiated adeno-
carcinoma, or show morphology of another carcinoma, such as squa-
mous cell carcinoma. These tumors differ from the sarcomatoid car-
cinomas in that the spindle cell component does not demonstrate
epithelial markers. The sarcoma may be a sarcoma, not otherwise
specified, show features of leiomyosarcoma, or show heterologous
differentiation, such as a rhabdomyosarcoma or osteosarcoma (8, 9).
Discussion. Carcinosarcomas are rare, highly aggressive tumors in
the human prostate. Most have been observed with disease progres-
sion after hormonal and/or radiation treatment in patients with previ-
ously diagnosed more typical prostate adenocarcinoma (8, 9). Al-
though radiation treatment could in theory contribute to the tumor
dedifferentiation,these tumors can develop without the history of
such pretreatment, precluding definitive association. Epithelial-
mesenchymal transformation could underly the development of any
such biphasic lesion observed in GEM prostates.
Pathology of Disorders of the Periurethral Glands and BUGs
(Table 3; Fig. 11)
In general, definitions and diagnostic criteria are similar to those
described above for the homologous lesions in the prostate. Pertinent
classification guidelines and discussion based on observed lesions in
existing GEM models are offered below.
Developmental Disorders of the Periurethral Glands and
BUGs. Given the relationship between the embryonic develop-
ment of the prostate and these other ductular/glandular derivatives
of the urogenital sinus and their shared androgen dependence,
genetic manipulations resulting in abnormal prostate development
may be expected to also give rise to related abnormalities in these
other male accessory glands. As such, agenesis and hypoplasia
should be defined and these designations applied as described
above for the prostate. For example, abnormal development of
secretory differentiation compatible with hypoplasia in the BUGs
was noted in Nkx3.1 knockout (Nkx3.1 /) mice, with reduced
mucin-producing epithelium accompanying a corresponding in-
crease in ductular epithelium (54).
Hyperplastic and Neoplastic Proliferations of the Periurethral
Glands and BUGs. Hyperplasia and Atypical Hyperplasia. Epithe-
lial and/or stromal hyperplasias should be described as for prostate,
with attention to their focal or diffuse nature and presence or absence
of atypia. Description of extent can include involvement of multiple
discreet periurethral or BUG gland lobules as well as that within any
given focus. Presence or absence of bilaterality of involvement should
be noted for the BUGs. Thus far, observed epithelial proliferations in
these sites have been accompanied by cytologic atypia, similar to the
prostate lesions in their corresponding mouse models, and stromal
proliferation has not been appreciated. Possible precursor lesions in
which atypical epithelium is noted in periurethral glands or BUGs
without obvious stromal invasion can be designated as atypical hy-
perplasia (Fig. 11, A, B, and H). These lesions show nuclear enlarge-
ment and hyperchromasia with frequent mitotic figures and apoptotic
bodies. These foci typically appear to conform to the normal glandular
architecture, the smallest of which appear to be localized in the ducts
of these accessory glands. More expansile forms may be seen with
progression, with an apparent increase in slightly irregular new ductu-
lar or glandular profiles, but still without extension into surrounding
stroma (41). More extreme forms with a nodular or micropapillary
growth pattern have been reported as atypical nodular hyperplasia
(41). These lesions (without frank invasion) should be classified as
atypical hyperplasia, and terms such as nodular or micropapillary can
be added as modifiers, along with other descriptions, such as in-
creased nuclear atypia, to document apparent progression of the lesion
in any given model.
Carcinoma. Invasive carcinomas in the periurethral glands and
BUGs should in general be classified according to the same guidelines
described above for the prostate. Carcinomas with focal and ill-
defined, but definitive, glandular formation have been observed, in-
cluding lesions associated with prominent stromal fibrosis and re-
ferred to as scirrhous carcinoma.
30
Such a lesion would be classified
as poorly differentiated adenocarcinoma in the current classification
scheme, based on the criteria outlined above for prostate (Table 5). In
addition, invasive tumors with histological and cytologic features
typical of NE carcinoma have been observed in multiple models (Fig.
11, CG). As described above for prostate, such tumors should be
30
J. M. Ward, personal communication.
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Fig. 11. Neoplastic involvement of periurethral and bulbourethral glands in genetically engineered mice. Aand 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 Eand Fto 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 temptingto
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.
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tentatively classified as NE carcinoma, but efforts should be made to
confirm the diagnosis by immunohistochemistry and/or electron mi-
croscopy, as outlined in Table 6.
Discussion. The relationship of the periurethral glands and BUGs
to the prostate embryologically, their regulation by androgens, the
documented and possible expression of transgenes in these tissues
with prostate selective or prostate specificpromoters, and the ob-
served occurrence of neoplasms in these sites in GEM all contributed
to the decision to include a classification of their neoplastic prolifer-
ations in the Bar Harbor Classification (Table 3). Neoplasms arising
in the corresponding human sites are extremely rare. However, in situ
and invasive tumors involving these tissues have been observed in
multiple transgenic mouse models generated with androgen-depen-
dent and even androgen-independent potentially prostate-specific pro-
moters (Fig. 11). Whether tumors arise in these sites or secondarily
involve them is not always clear. However, origin of tumors in the
periurethral glands and BUGs has been described in some models,
along with likely precursor lesions. Tumors arising in the prostate can
extend to involve the periurethral region, either by growth along
prostatic ducts to where they enter the urethra or by direct invasion
into periurethral stroma. Conversely, tumors arising in the periurethral
glands can extend into prostate lobes, potentially mimicking a primary
prostate tumor. Finally, neoplasms may originate in both the prostate,
and the periurethral glands and/or BUGs (either synchronously or
metachronously), and either or both could give rise to metastases. As
reported and unreported examples of periurethral gland and BUG
carcinomas have morphological characteristics quite similar or iden-
tical to those arising in the prostate in given models, the latter also
raises issues of determining the primary site for metastases.
Periurethral gland and BUG neoplasms have been best character-
ized in the C3(1)-SV40 mouse model (Ref. 41; Fig. 11, AD). In fact,
most metastases in this model may come from periurethral gland and
BUG tumors.
31
Invasive tumors, essentially uniformly with NE dif-
ferentiation, have also been noted in the periurethral glands and BUGs
of intact LPB-Tag 12T-10, and castrated and even intact faster-
growing LPB-Tag lines (Refs. 38, 55; Fig. 11, Eand F). Similar
tumors were noted to involve the periurethral region of TRAMP mice
from multiple institutions (Table 1) based on slides reviewed by the
Pathology Panel (Fig. 11G).
32
However, the potential origin or iso-
lated occurrence of such tumors in the periurethral glands in this
model versus their simultaneous occurrence with tumors of similar
morphology in the prostate has not been described in detail. The
combined experience in different models with androgen-dependent
promoters would certainly support the possibility of neoplastic trans-
formation due to transgene expression in these androgen-dependent
other male accessory glands, and transgene expression in these sites
has been documented with C3(1)-SV40 and LPB-Tag models (38,
41). Interestingly, atypical hyperplastic lesions have been noted in the
ducts of periurethral glands in CR (2)-SV40 mice (Fig. 11H).
12
As
this androgen-independent promoter appears to selectively target NE
cells in the prostate (33), this suggests possible targeting of a normal
likely very minor NE component in these other male accessory gland
sites. However, these lesions do not appear to progress in this model,
and involvement of the periurethral region by more extensive NE
carcinomas appears to be by direct extension from unequivocal pros-
tate tumors.
12
Although tumors originating in the homologous sites in the human
are rare, given the relationship of the development of these sites to that
of the prostate and the potential similar androgen dependence or
independence of tumors arising therein, if mouse models of Pca have
relevance to human Pca, tumors in these other related sites may share
at least some of that relevance (41, 100). Pathways regulating cell
proliferation, apoptosis, invasion, and angiogenesis, and the role of
hormones and stromal interactions may be closely related in tumors of
the prostate and other male accessory glands in the mouse. Addition-
ally, promoters that can selectively target transgenes to the prostate
versus these other male accessory glands may be developed. Knowl-
edge of how to grossly procure these sites for pathological analysis
and how to classify neoplasms therein is essential for understanding
the natural history of current models, characterizing future models,
and analyzing selectivity of new promoters. Techniques for tissue
sampling are described below in the Protocols section.
Basic Protocols in Characterization of Prostate Lesions in GEM
Necropsy of Newly Established Models and Prostate Dissection
Ages for Sampling. To fully characterize a GEM model for prostatic
disease, the suggested sampling should include time points that mark
the sexual maturity and reproductive milestones of the mouse, in
addition to the time points determined by the investigator, based on
previous findings, published reports, natural history, and so forth. The
recommended initial/minimal sampling periods include: (a) day 1, at
time of birth; (b) week 3, at time of weaning; (c) week 6, at time of
reaching sexual maturity; and (d) week 40, at time of end of repro-
ductive capability. Described time courses of phenotype development
for published models, such as those listed in Table 1 and others, may
also be helpful.
Examination and Dissection of GEM Prostates. At necropsy,
macroscopic examination of the prostate (and/or its individual lobes)
should be carried out with relevant data recorded, such as: (a) size; (b)
weight;; (c) descriptive features (e.g., enlargement and atrophy); (d)
tumor, if identified (with documentation of location, size, extent of
local invasion or characteristics of other local effects, such as bladder
obstruction, seminal vesicle distension, and so forth); (e) necrosis; and
(f) presence of visible metastases in distant organs.
Prostate Dissection and Tissue Submission. The method of pros-
tate dissection and tissue submission for histology depends in part on
the intended use of the tissues, particularly whether ancillary studies
such as gene expression analyses are being performed. Histological
examination and tissue-based analyses such as immunohistochemistry
and in situ hybridization are routinely performed on standard forma-
lin-fixed, paraffin-embedded sections. Caution should be observed
regarding the duration of exposure of tissues to 10% buffered forma-
lin, which is adequate for most if not all analyses. If tissues are not to
be processed right away, tissues or cassettes with tissues in them
should be switched to 50 or 70% ethanol after 46 h. Prolonged
exposure to formalin can reduce tissue antigenicity, compromising
some subsequent immunohistochemical or in situ hybridization
analyses.
Separate identification of individual prostate lobes (grossly or in
microscopic sections) and characterization of pathology in specific
lobes is important for thorough model characterization. For analyses
only involving histopathologic assessment and possible subsequent
immunohistochemical and/or in situ hybridization studies on paraffin
sections, the prostate and associated organs can be submitted en
blocas described below and in more detail on cited websites. For
protocols involving submission of snap-frozen tissues, the individual
lobes of the prostate can be dissected with the aid of a dissecting
microscope (31). Lobe dissection is accomplished after removal of the
entire genitourinary bloc (prostate lobes, seminal vesicles, ampullary
glands, proximal ductus deferens, bladder, and proximal urethra) after
transection of the urethra. The individual prostate lobes can be
weighed and representative portions of each can be snap frozen, and
31
J. M. Ward, unpublished observations.
32
R. Barrios, R. Herbert, unpublished observations.
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the remaining tissue fixed and processed for standard paraffin sec-
tions. High quality RNA can be obtained from tissues procured in this
manner. Representative portions of both right and left lobes or an
entire lobe from one side can be snap frozen, and the remaining tissue
from specifically designated lobes can then be submitted in its entirety
in individual tissue cassettes. If such protocols are used, sections of
seminal vesicles and the remaining genitourinary bloc should be
submitted (in a similar fashion as described below and shown in Fig.
12), to allow for sampling of the ampullary and periurethral glands.
After a single transverse cut of this portion of tissue through the
urethra (with or without prostate lobes attached), both transected
surfaces can be embedded down so that initial sections from the block
are at the cut surface (Fig. 12). Pathological alteration of the prostate
and associated organs can distort the normal anatomy here and can
modify the level of this initial cut somewhat. If, for example, the
periurethral glands are of interest and are not well represented in the
initial histological section, additional cuts into the block allow for
examination of levels thus extending both proximally and distally
along the urethra. The anatomy of the mouse prostate and techniques
of dissection are described in more detail on the web.
33
The extent to which paraffin blocks are sectioned for H&E-stained
slides depends on the nature of the study being conducted and the
focality of lesions being detected. For example, in the use of already
characterized models that have documented progression to fairly
extensive lesions in a reasonably reproducible time course and in
which the effects of genetic manipulations or pharmacological inter-
ventions are being examined, and pathology is an end point, one slide
per prostate block per animal may be sufficient if adequate numbers
of animals are included. The ability to achieve statistical significance
may be impacted by the magnitude of the biological effect being
investigated and the focality of the lesions. Several examples with
pathology as the assessed end point have been reported, and an idea
of the required mouse numbers can be gleaned from past studies. Even
33
Internet address: http://ccm.ucdavis.edu/mmhcc/prostate/prostateindex.htm.
Fig. 12. Technique for histological examination
of the prostate with en bloc submission. A, exposed
intact prostate, bladder, and seminal vesicles [gen-
itourinary (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 section-
ing through the urethra to include both dorsolateral