analysis of mouse model pathology: a primer for studying

Topic Introduction

Analysis of Mouse Model Pathology: A Primer forStudying the Anatomic Pathology of GeneticallyEngineered Mice

Robert D. Cardiff,1 Claramae H. Miller, and Robert J. Munn

Center for Comparative Medicine and Center for Genomic Pathology, University of California, Davis, Davis,California 95616

This primer of pathology is intended to introduce investigators to the structure (morphology) ofcancer with an emphasis on genetically engineered mouse (GEM) models (GEMMs). We emphasizethe necessity of using the entire biological context for the interpretation of anatomic pathology.Because the primary investigator is responsible for almost all of the information and proceduresleading up to microscopic examination, they should also be responsible for documentation of exper-iments so that the microscopic interpretation can be rendered in context of the biology. The stepsinvolved in this process are outlined, discussed, and illustrated. Because GEMMs are unique experi-mental subjects, some of the more common pitfalls are discussed. Many of these errors can be avoidedwith attention to detail and continuous quality assurance.

INTRODUCTION

Once an investigator has completed the genetic modifications of a new mouse model of cancer, thenext step is to define the characteristics of that model. This article emphasizes the importance ofcontext and combining molecular phenotype (function) with morphological phenotype (structure).Investigators using animal models should integrate the biology of gross and microscopic alterationswith the associated molecular changes. However, it is important to consider basic biology! Thepathology of cancer in genetically engineered mice (GEM) has been described in detail elsewhere(Cardiff et al. 2006c). We will concentrate on the responsibilities of the investigator for assuring thatgross and microscopic pathology is placed into the context of the disease.

This “how to” article is designed to assist researchers in the planning and implementation ofrigorous pathological analyses of samples (or specimens) from their research model. All membersof the research team (principal investigator, faculty, fellow, staff, or student) are ultimately responsiblefor the quality of the pathological interpretation. Surveys reveal that about 95% of the interactionsbetween mouse and scientists are carried out by “staff” (Schofield et al. 2009). Principle investigators,senior investigators, lead scientists, and pathologists rarely see the actual mouse and usually do notperform the data collection, gross observations, sampling, or histology. These experimental activitiesare usually provided by fellows, students, or staff, many of whom may have been trained by other(untrained) persons. One of themain goals of this article is to provide greater awareness of the need fortraining in observation and preparation of both gross and microscopic specimens. Technical hints are

1Correspondence: [email protected]

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also provided and the necessity for careful observation of details is illustrated by specific examplesfrom genetically engineered mouse models (GEMMs).

PATHOBIOLOGY OF CANCER IN GENETICALLY ENGINEERED MICE

What Is Pathology?

Pathology is the study of disease. Disease is an abnormality of structure and/or function. The inter-pretation of disease requires information from all levels of organization, from environment to mol-ecule (Fig. 1). The study of disease structure is the unique domain of anatomical pathology thatprovides interpretation in the form of the “diagnosis.” The diagnosis involves putting all levels of

B C D EA

Ontology Ontology

Environment Environment

Etiology

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eP

rognosis

Experiment

Gross exam

In vivo imaging

Pathology

Cytology

IHC

Molecular pathology

Dysfunction(disease)

Diagnosticterminology

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Disease is an abnormality ofstructure and/or function.

OrganismOrganism

Organ

Tissue

Cell

Organelle

Molecule

Structure

Function

Legend

Related

1. Host2. Environment3. Topology4. Biology5. Cytology6. Molecular

OntologyData Flow

Data

FIGURE 1. The ontogeny of disease. This ontology illustrates the complex relationships among structure, function, datasources, and diagnostic terminology (words used in diagnosis). It is based on the Rosse Foundational Anatomy ontology(Rosse and Mejino 2003; Cardiff et al. 2004). It shows the information required to understand microscopic pathologywithin the context of disease. The ontological structure provides an organ-based hierarchy with levels of anatomicorganization to describe structure. The color in the boxes indicates the sources of the information: investigators (violet),imagers (yellow), and pathologists (light blue). Column A emphasizes normal relationships between organism, struc-ture, and function. Column B demonstrates the relationship between the levels of organization and diagnostic nomen-clature. Information is gathered frommany levels of structure and function (column C). The pathologist’s interpretationof the structure integrates all of the clinical, functional, and structural information (column D). Column E indicates thelevel of information used to decide the different clinical features of tumors in human medicine. The connecting linesindicate the relationships and flow of information (data) that provide context for the “diagnosis.” The diagnosis isprimarily descriptive and adheres to standard terminology. However, as pathologists integrate more sophisticateddata sources, the data must be recorded as “diagnostic modifiers.” If pathologists and scientists will adhere to this, orsimilar, ontology, computers and informatics will enable the scientific community to organize their data sets andrecognize the relationships (“map”) across species. Investigators and pathologists will need to recognize and under-stand their roles in the organizational scheme to share data sources. This is our challenge and our responsibility.

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information into the context of microscopic anatomy. Figure 1 illustrates the ontogeny of disease withemphasis on the roles played by various disciplines (Rosse and Mejino 2003; Cardiff et al. 2004). Inthe context of cancer, pathology is the study of the natural history, structure, and function ofautonomous new growths in any multicellular organism. The goal of pathology is to understandthe disease in the context of structure and function.

All cancer investigators are students of pathology, in the sense that we all study disease. As a“para”-pathologist, you should be familiar with the basic vocabulary of disease. Table 1 providesa list of these general disease processes. Those who study the molecular biology of pathology aretermed “molecular pathologists.” The discipline that is concerned with the structure of disease istermed “anatomic pathology.” Professional anatomic pathologists have unique training in, and un-derstanding of, the microscopic anatomy of cancer. The uninitiated erroneously consider pathologyas solely the study of microscopic anatomy, but the pathologist is an integrative biologist whosejob is to place structural alterations into the context of the disease (Fig. 1). With proper informationabout the background of the research animal (context and function), the anatomic pathologist iscapable of looking at a slide, which is a lesion fixed in time, to surmise its biological history. Thiscapability amazes some observers and is developed from an in-depth study of the natural history ofthe disease.

The pathologist can integrate the individual sample back into the context of the dynamic naturalhistory of the disease (Fig. 1). In the mouse arena, you and your team are the ultimate source ofinformation about the pathobiological context of your mouse model. In many cases, it is the staff(rather than the principal investigator) who observe the live mouse, perform the necropsy, and takethe initial steps in preserving and processing the samples (Schofield et al. 2009). As a result, it isincumbent upon the investigator and their staff to pinpoint areas of the gross anatomy and be ableto adequately describe the key characteristics of each lesion, as well as to know how to document thedisease, how to sample the specimen, and how to prepare it properly. The purpose of this article is toprovide youwith the basic guidelines for this task. The guidelines will be presented in the context of thepathology of cancer.

Pathology of Cancer

A neoplasia is an autonomous new growth (a tumor). A tumor is a physical (structural) entity that isidentified as a focal mass in the animal (Figs. 2 and 3). Although the origins of neoplasia may be insomatic cell mutation(s) that drivemassive alterations in themolecular and cellular biology of afflictedcells, neoplasia is a structural abnormality that can only be understood in the context of the wholeanimal. As intimated above, this morphology (structure) is the unique province of the discipline ofanatomic pathology.

TABLE 1. General disease processes

Vascular RepairArteriosclerosis Granulation tissueFatty change FibrosisPassive congestion Growth DisturbancesThrombosis HamartomaEmbolism Atrophy

Necrosis HypertrophyCoagulation necrosis HyperplasiaCaseous necrosis MetaplasiaLiquefaction necrosis Neoplasia, benignEnzymatic fat necrosis Neoplasia, malignant

InflammationAcute inflammationChronic inflammationGranulomatous inflammation

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Analysis of Mouse Model Pathology




The study of the pathobiology of cancer should begin with an understanding of the stages ofneoplastic progression: precancer, cancer, and metastasis. In principle, you should consider where thecancer starts, how it grows, and where it goes. To communicate these data elements with yourcolleagues and, specifically, the pathologist, it is important to properly collect and record the mor-phological and biological history of the disease. This information will lead to informed microscopicexamination that confirms the gross findings and leads to accurate interpretations.

The critical background information in murine pathology includes husbandry, genetic back-ground, gender, age, and experimental manipulations. In females, records of the parity and statusof the estrous cycle may be critical. Environmental factors such as cage conditions and chow ingre-dients are important. If records are not meticulously kept, critical insights into disease can be over-looked or misinterpreted. For example, subtle changes in standard chow can lead to dramatic changesin tumor kinetics (Yang et al. 2003; Liu et al. 2005). In one case, moving immunologically impairedanimals to a new vivarium resulted in changes in intestinal flora, opportunistic infections, andproliferative lesions in the gut (Borges et al. 2005). Without the environmental context, tumorscan be misinterpreted (Fig. 4).

Most nonhematogenous neoplasms can usually be identified as a focal mass. The focal area may becompletely confined to the epithelium but usually it will stand out as an atypical focus, both grossly andmicroscopically. The element of cytological atypia is the pathognomonic feature and requires micro-scopic confirmation. A neoplasm can be an entirely benign, indolentmass. Or it can bemalignant withlocal invasion and/or metastases. The ultimate outcome is the metastatic spread of the neoplasmto distant organs. Because the investigator has access to all of this information, he/she should beresponsible for knowing what to look for, how to look for it, and how to prepare it for the pathologist.

Gross examination

Unifocal Multifocal

Diffuse

The lesion is

InfarctInfarct

Granuloma

Abscess

MetastaticneoplasiaInflammation

Primaryneoplasia

Abscess

Granuloma

Fibrosis

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AcuteNeoplasia

Hypertrophy

Expansile:benign

Infiltrate:malignant

(leukemic infiltrate)

(physiological-compensatory)

Chronicpassive congestion

Followsblood supply

Followsblood supply

Cavity(thick wall)

Abnormalgrowth

Abnormalgrowth

Cavity(thick wall)

Cavity(thin wall)

Cavity(thin wall)

FIGURE 2.Gross specimen logic tree. Gross lesions can be divided into three types: unifocal, multifocal, and diffuse. Inturn, unifocal and multifocal can be further divided into those that either follow the blood supply (infarct) or have acavity with thick wall (granuloma), a thin wall (abscess), or an abnormal growth pattern (neoplasia). A diffuse diseasepattern can result in organomegaly (hypertrophy, neoplasia, or chronic passive congestion) or from inflammation(chronic or acute). The diagnosis will be made/confirmed following microscopic examination (Fig. 3).


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Hematogenous neoplasms are generally diffuse infiltrates that lead to enlarged organs (organo-megaly) (Fig. 2). Inmice, lymphomas and leukemias are recognized by enlarged lymph nodes, thymus,spleen, and/or liver. The murine spleen is a hematopoietic organ and a myeloid factory that readilyresponds to infection and other stresses. Therefore, splenomegaly will occur with cancer such aslymphoma, but also during almost any infection.

The reasons for careful observation and documentation can be illustrated by several examples.Mouse strains have different inborn, or spontaneous, disease patterns. For example, many strainsspontaneously develop leukemias (Fredrickson and Harris 2000). Others, such as strains A and FVB,are very prone to bronchioalveolar adenomas, which are frequently misinterpreted as pulmonarymetastases (Rehm et al. 1994; Mahler et al. 1996). Retired breeders of the FVB strain spontaneously

Reevaluate Reevaluate

Is lesionblue?

Is lesionred?

Do diagnosticcriteriaapply?

Mineral

Acuteinflammation

Arefibrosis/necrosis

present?

Aremononulear

cells present?

Isdisorganized

growthpresent?Do diagnostic

criteriaapply?

Malignantneoplasm

Benignneoplasm

* This algorithm continues with “focal redlesion” (not shown).

Yes

Yes

Yes

Yes

Yes

YesYes

Yes

Yes

No

No

No No

No

No

No

No

No

No

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Chronicinflammation

Lymphoma

SubclassifySubclassify

Recordimpressions

Recordimpressions

End

End

End*

Is itinfiltrative?

Is lesioncellular?

Are PMNspresent?

Focal lesion

Microscopic examination

FIGURE 3. Examining a microscopic slide. This algorithm is designed to show the thought processes used by pathol-ogists when examining microscopic slides. It will not make everyone in the laboratory eligible to take pathology boardexaminations, but we recommend that you follow the outline systematically. Step one: Look at a hematoxylin andeosin (H&E) stained slide with the naked eye. Try to orient yourself. Is it homogeneous in color? Is it mostly red? Is itmostly blue? Are different areas visible, such as an area that is pale and pink containing a mass of dark blue stainingcells? Next, look at the focal mass with the microscope. Step two: Begin scanning the entire slide using the lowestmagnification objective. Unless you are viewing hematology slides or slides containing microorganisms, oil immer-sion is never necessary. Identify the normal tissue and examine the interface between the normal and the abnormalmass (the lesion). As necessary, increase the magnification by changing objectives. Now, follow the steps indicated bythe algorithm. Identify the cells that make up the mass. Clearly, a user needs to be able to identify PMNs (polymor-phonuclear neutrophils), mononuclear cells, and disorganized growth. If not, a course in histology is recommended.






develop diffuse mammary hyperplasia and pituitary adenomas (prolactinomas) (Mahler et al.1996; Nieto et al. 2003; Radaelli et al. 2009). Afflicted FVB mice also have a higher incidence ofmammary tumors (Radaelli et al. 2009). Some strains of immunodeficientmice are prone to osteogenicsarcomas, and all immunodeficient strains are susceptible to infectious diseases (Kavirayani andForeman 2010). With accurate observation and detailed documentation, the critical differencesbetween the diseases created by genetic modification and those that are spontaneous can be identifiedand understood.

FIGURE 4. Pitfalls illustrated. These images illustrate a focal intestinal mass caused by roundworms (A,B), liver withEMH (C ) and lymphoma (D), and an adenosquamous carcinoma before proper processing (E) and after reprocessingusing appropriate techniques (F ). The intestinal mass is shown as a screen capture of whole-slide imaging (WSI)viewed with the Aperio ImageScope web browser. (A) The low-power view with a large tumor mass bulging intothe lumen. Scale bar, 4 mm. This was originally mistaken as an intestinal adenocarcinoma. However, close scrutinyrevealed that the mass was composed of macrophages surrounding roundworms. Their hard, chitinous exoskeletonsare easily visualized as boat-shaped blue profiles at higher magnification (B). Scale bar, 50 µm. The livers of micefrequently exhibit extra-medullary hematopoiesis (EMH) that differs from lymphomatous infiltrates in the size andnature of the nest and composition of the cells within the nest. EMHs tend to be smaller clusters with a mixture of celltypes (C). Scale bar, 50 µm. Lymphomatous infiltrates present with more irregular profiles and a monomorphous cellpopulation (D). Scale bar, 50 µm. The inadequately prepared adenosquamous carcinoma was poorly dehydrated andembedded in high-temperature paraffin (E), which proved impossible to cut thin without fracturing the tissue. Scalebar, 100 µm. The tumor was removed from the paraffin block and rehydrated and reprocessed. As a result, thereprocessed tissue could be cut at 5 µm in thickness and the fine details adequately viewed (F ). Scale bar, 100 µm.To view the corresponding WSI for this figure, see the URLs provided in Table 3.


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Gross and Microscopic Tissue Examination

The gross examination beginswith observation of the live animal and endswithnecropsy. The behaviorand general condition of the animal can indicate a disease state. Necropsy techniques are providedin Protocol: Limited Mouse Necropsy (Cardiff et al. 2014a) and have been described and docu-mented online (http://tvmouse.compmed.ucdavis.edu and http://eulep.pdn.cam.ac.uk/Necropsy_of_the_Mouse/printable .php). In addition, a detailed atlas of normal mouse and human anatomy andhistology has recently been published that includes an extensive illustrated section on necropsy (Kno-blaugh et al. 2012; Treuting andDintzis et al. 2012).One point that needs emphasis is the collection andexamination of all tissues. This section will concentrate on the gross andmicroscopic characteristics ofcancer in mice and the differential diagnosis of tumors (Figs. 2 and 3, Tables 1 and 2).

As stated, neoplastic epithelial diseases are focal, space-occupying masses. This is their key grossfeature. However, not all focal masses are malignant tumors. Abscesses, infarcts, granulomas, andbenign neoplasms also present as focal masses (Figs. 2 and 5). Careful observation can sometimesdistinguish between gross features of malignant neoplasms and other focal lesions. Figure 2 shows thelogic tree that pathologists use to distinguish among various gross focal lesions. Malignancy is gen-erally invasive and attached to adjacent tissue. A specific warning here: Many malignant tumors inmice have expansile, pushing borders and do not adhere tightly to adjacent tissue, but will stillmetastasize (Cardiff et al. 2006c) (Fig. 5).

Gross differential diagnosis of tumors is described in Figure 2. In contrast to malignant tumors,abscesses are cystic with thin walls and are filled with a yellowish liquid (pus). Some chronic abscesseshave a fibrotic host response and may adhere to adjacent tissues. The purulent fluid of an abscess canmimic the necrotic center of a neoplasm but is generally less viscous. Granulomas are the focal resultof granulomatous inflammation, which is a type of chronic inflammation dominated by monocytesand their derivatives such as multinucleated giant cells. Mice rarely form grossly visible granulomas.Infarcts are relatively rare in mice but can be confusing. They are the result of vascular occlusion andtypically follow the blood supply. Therefore, one needs to know the vascular patterns of the organunder study (Fig. 5).

The gross observations, however, are just “impressions” and the diagnosis must be confirmed bymicroscopic examination. The logic used to distinguish between the basic lesions upon microscopicexamination is represented in algorithmic format with key features in Figure 3. All too frequently,morphology is sacrificed for molecular studies. Without a histological sample of the tissue to verifythat it is a tumor, the data on an expression microarray can be misinterpreted and misleading.

Another limitation to gross-only diagnoses is that some mouse models have tumors with differentphenotypes (see discussion below). Even worse, this heterogeneity can occur within the same neo-plastic mass. Once again, the gross impressions need to be confirmed microscopically to avoidmisinterpretation of the molecular data.

Natural History of Cancer and How to Identify Precancers

Carcinomas arise from precursor lesions, which are variously referred to as carcinoma in situ, pre-neoplasms, precancers, and/or premalignancies (Cardiff and Borowsky 2010, 2011). All forms ofepithelial precancers are characterized as focal areas of atypical cells with an increased risk of malig-nant transformation (Cardiff et al. 2006a). This implies that they are not yet composed of malignantcells but require another molecular hit. They can be observed at the subgross level, using a dissectingmicroscope, as small foci in the organ without evidence of invasion. Although difficult to see in solid

TABLE 2. Microscopic key features: Focal lesions

Infarct Coagulation necrosis (granulation tissue)Granuloma Multinucleated giant and epithelioid cells (monocytes)Abscess Liquefaction necrosis and polymorphonuclear (PMN) leukocytesNeoplasm, benign Pushing margins, well differentiatedNeoplasm, malignant Invasive margins, poorly differentiated






organs, they can be found by experienced observers in the mammary gland, skin, lung, gut, andprostate. In human medicine, their biological potential is judged by their relationship to malignancy.The documentation may require years of observation and extensive epidemiological evidence toestablish a relationship. This is essentially guilt by association; evidence is rarely obtained by directobservation because, in human medicine, the diagnostic tissue has been removed and processed. Inthe inbred mouse, however, experimental evidence can be obtained using orthotopic and ectopictransplantation (Cardiff and Borowsky 2010, 2011). This is known as “test by transplantation”(Cardiff and Borowsky 2010, 2011). Basically, if the focal lesion/tumor mass will not grow as a

FIGURE 5. Pathologic processes. These images of H&E stained slides show the key microscopic features of the majorprocesses associated with focal lesions. A (abscess) shows the center of an abscess with liquefactive necrosis andcollections of polymorphonuclear leukocytes (PMNs). Note the loss of tissue detail. Scale bar, 100 µm. B (infarct)shows a hepatic infarct with coagulation necrosis. Note the bright pink cytoplasm with loss of nuclei. Scale bar, 100µm. C (granuloma) shows a granuloma with an infiltrate of predominantly monocytic cells. Note the numerousmultinucleated giant cells. Scale bar, 100 µm. D (benign neoplasm) shows a mammary adenomyoepitheliomawithin a duct-like space. Note the well-differentiated glands with a spindle cell stroma. Scale bar, 200 µm. E (malig-nant neoplasm) shows a Myc-induced (Tm(cMyc)) adenocarcinoma with poorly differentiated glands invadingthrough a dense fibrous stroma. Scale bar, 100 µm. Contrast this invasive growth pattern with the expansile marginof the tumor in F . F (malignant neoplasm) shows the solid nodular profile of a Tg(MMTV-cNeu)-induced tumor with aperipheral palisade of basal cells and an expansile margin. Although not invasive, these expansile tumors can result intumor emboli (see Fig. 6). Scale bar, 50 µm. To view the corresponding WSI for this figure, see the URLs provided inTable 3.


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transplant in an ectopic site, it is not considered to be a malignant neoplasm. If it does grow in anorthotopic site where it eventually becomes a tumor, it is considered a precancer or a neoplasm withmalignant potential.

Microscopically, a precancer is characterized by a focal area of dysplastic cells that is confinedwithin the basement membrane or normal tissue limits (Fig. 6) (Cardiff et al. 2006a). The key featuresare the abnormal growth and dysplastic cytology (Fig. 6). Detailed descriptions of specific precancerscan be found elsewhere. Fundamentally, these lesions will stand out from the background of normaltissue because their cells are darker staining (hyperchromatic) and are larger and more irregular(pleomorphic) than normal. In addition, normal tissue organization is disrupted. The nuclei arelarger with a higher mitotic rate. Unfortunately, it is impossible to predict biological behaviorbased on cytological criteria (Medina 1996; Cardiff et al. 2006b). As with any pathological lesion, a

FIGURE 6. Precancer, tumor heterogeneity, and metastasis. These are examples of precancer (A,B), tumor heteroge-neity (C,D), and tumor emboli and colonization in the lung (E,F ). (A) Low-grade PIN (prostate intraepithelial neoplasm)in a Tm(Pten−/−) male. Multilayered areas of atypical cells (center) stand out from the glands with normal cells (11 and1 o’clock). Scale bar, 50 µm. (B) A small zone of hyperchromatic, dysplastic cells occupying a crypt in the smallintestine of an APC-MIN mouse. Scale bar, 100 µm. (C ,D) Serial sections from a transplanted tumor cell line derivedfrom a Tm(Stat−/−) mammary tumor stained for CK19 (C ) and CK5 (D). Scale bar (C ,D), 100 µm. Note the branchingvascular space at right center outlined by CK19-positive (C ) but CK5-negative (D) cells. (E,F ) Pulmonary involvementwith tumor emboli within vascular spaces (E) that are inside the vessels. In contrast, the tumor metastasizes into thelung with colonization of the lung parenchyma (F ). Scale bar (E,F ), 100 µm. To view the corresponding WSI for thisfigure, see the URLs provided in Table 3.






differential diagnosis must be considered. Occasionally, a systemic condition can lead to widespreadatypical hyperplasia. In these cases, such as some inflammatory bowel diseases, the entire epitheliummight seem to be dysplastic but there are no focal lesions. Some focal lesions are the result of injuryand do not progress to malignancy. The inflammatory nodules in postlactational mammary glands areexamples of such lesions (Raafat et al. 2012).

Malignancy

Malignant neoplasia is life-threatening. In its early stages, the neoplasm invades the surrounding tissueor the vasculature (Fig. 6). The ultimate stage is its spread (metastasis) to distant sites. The very earlieststage involves microinvasive disease. The criteria for microinvasion include extension through thebasement membrane by distinctively dysplastic cells and a host response to the invasive cells (Shappellet al. 2004). Unfortunately, most examples lack one or more of these criteria and even experiencedpathologists have difficulty interpreting the histology. In these cases, the better part of valor is torecognize that criteria are not met so that the lesions are designated “suspicious” or “probable” formicroinvasion (Shappell et al. 2004). This cautionary approach is particularly important in epitheliallesions of mice. The intestinal herniation phenomenon is one example in which specific knowledge ofmouse anatomy is critical and caution is necessary. The mucosa of mice has a tendency to herniatethrough themuscularwall ofmost tubular organs, such as intestines. In inflammatory conditions of thebowel, reactive epithelium may bulge through the muscularis. The condition is reversible if theinflammation is treated and regresses. If the pathologist is not aware of this tendency, they can“overcall” microinvasion and cause a futile search for more convincing evidence of malignancy. Thetest by transplantation can provide the definitive experimental proof if the histopathology is equivocal(Cardiff and Borowsky 2010, 2011).

Metastases

Distant spread of the neoplasm (metastasis/es) is the endpoint of the neoplastic process.Here again, themouse has a number of peculiarities that will be highlighted below. In contrast tomost human cancers,slower growing tumors, not the highly proliferative tumors, are more likely to result in metastasis inmice. Also, in contrast tomost human carcinomas, which primarily spread to local lymph nodes and tospecific organs, the vast majority of mouse cancers metastasize to the lung through the hematogenousroute and do not invade lymphatic channels (Fig. 6). With diligent searches, metastases may be foundin regional lymph nodes, liver, and other organs. However, metastases to these other tissues tend to berare in the mouse.

Another issue is the large proportion of metastatic tumors that arrive in the lung as tumor emboli(Fig. 6) (Cardiff et al. 2006c). Frequently, these emboli arrive carrying their own coating of endothelialcells (Oshima et al. 2004; Cardiff et al. 2006c). The tumor emboli can clog up the major pulmonaryvessels without ever invading or colonizing the lung. As a result, experienced pathologists keep a recordof different types of emboli. For example, it is conceivable that small clusters of cells arrive at the lungonly to be trapped in the peripheral vasculature but do not invade through the vessel wall. They mayalso grow (enlarge) without colonizing the lung. A number of investigators have recorded a transienttime period when tumor cells will be trapped in the lung or liver before they break loose and travel tocolonize a secondary organ (Fidler and Poste 1982; Poste 1982; Poste and Nicolson 1983; Morris et al.1994). Again, these types of tumor microemboli should be recognized and enumerated (Siegel et al.2003). Most authorities insist that the diagnosis of pulmonary metastasis be reserved only for thosetumors that colonize the lung parenchyma. Figure 6 illustrates tumor emboli and metastatic coloni-zation in the lung.

Bronchioalveolar adenoma/carcinoma (BAC) (Rehm et al. 1994) is another entity that frequentlycauses confusion. These tumors are very common in older females of some strains, such as Strain Aand FVB. Many uninitiated observers will misinterpret these lesions as metastatic. Most BAC arepapillary or lepidinal and do not resemble the primary tumor. Further, they are associated with thebronchi and not the vasculature.


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Since the mouse liver is a common site for extramedullary hematopoiesis (EMH), these foci aresometimes mistaken for metastases or diagnosed as lymphoma. On H&E staining, EMH foci canusually be identified by their small size and round, well-circumscribed profile. The dominant cell typeis myeloid and its derivatives create a morphologically diverse, mixed cell population. In contrast,most lymphomas are monomorphous (single-cell type) and form larger, more irregular foci (Fig. 4).Sometimes, these lesions can be differentiated from each other by using immunohistochemistry todetermine whether the cells are from the same or mixed lineages (Fig. 7).

Neoplastic Variation, Heterogeneity, and Reproducibility

The study of GEM pathobiology has led to the recognition of gene- and pathway-specific tumorphenotypes (Rosner et al. 2002) and oncogene addiction (Cardiff et al. 2011; Couto et al. 2012). Gene-related morphology reflects the molecular biology of gene addiction. These phenomena are consistentwith our current concepts of tumor clonality. Further, the loss of the inciting oncogenic transgene isassociated with a distinctive change in the tumor morphology. As tumor biologists, we need to be

FIGURE 7. Immunohistochemistry. An illustration of the molecular heterogeneity of specific antigens using immuno-histochemistry in an epithelial–mesenchymal transition (EMT) tumor fromaTm(cMyc)mammary gland.Note the tumorhas a mixture of solid nests/glands and spindle cell elements that are typical of EMT phenotype tumors. Also note thatboth the epithelial nests/glands and spindle cells contain CK8/18 (A), N-cadherin (D), and TGF-β (F ). The spindle cellscontain vimentin (E) but not CK19 (B) and a relatively small amount of E-cadherin (C ). E-cadherin and CK19 areprimarily found in the epithelial component. The dual location of the intermediate filaments is considered diagnosticof EMT tumors. Scale bars, 100 µm. To view the corresponding WSI for this figure, see the URLs provided in Table 3.






aware that even clonal tumors can be variable. This biological and morphological variation andheterogeneity of all tumors should be recognized, documented, and understood (Fig. 7). This isprobably one of the most confounding aspects of tumor pathology. Although the pathologist canpredict outcome on the basis of the current appearance of the neoplasm, the prediction is frustratinglyimprecise. The goal of molecular pathology is to increase the prognostic and predictive precision ofthe diagnosis. It is envisioned that molecular clues will be more predictive than morphological clues.To some extent, these clues have been transforming oncology.

Science technology has becomemore sensitive and sophisticated. The search has intensified to findthe key characteristics of malignant neoplasms that can be used to prevent, treat, and hopefully curecancer. Huge national and international programs are dedicated to cancer genomics and biomarkers.In part, this search has been driven by the success of the reductionist approach of molecular biologyand, in part, by the dazzling power and unexplored potential of current and emerging technologies.The stumbling block to the realization of this vision can be found in the amazing variation betweenand within neoplasms.

Some authorities argue that every tumor is different and each has infinite potential for variationwith a complexity that can never be completely understood (Berman 2004). The clonal origin of mosttumors would argue against this rather nihilistic hypothesis of infinite variation. The current enthu-siasm for cancer stem cells also argues for a common origin (Damonte et al. 2007; Garbe et al. 2012).The issues are the speed at which subclones evolve and whether subclones retain the characteristics ofthe primary clone.

In support of the primacy of the original clone, one can cite the remarkable genotype-specificphenotypes of the strong initiating oncogenes used to create GEM (Rosner et al. 2002). Activation ofmost transgenic oncogenes results in very specific tumor morphology (phenotypes) (Cardiff et al.2006c). An experienced comparative pathologist can recognize tumors associated with many geneticmanipulations. For example, it is easy to recognize changes induced by Ras, Myc,Wnt, PI3 kinase, andErbB2 activation; inactivation of Pten or p53 in themammary gland; activation of Ras, pAkt, and SV40in the prostate; and activation of Ras or T antigen in the lungs (Cardiff et al. 2006c). This phenomenonhas been discussed under the heading of oncogene addiction and non-oncogene addiction (NOA)(Cardiff et al. 2011; Couto et al. 2012). These types of morphological observations reinforce theconcepts of clonal origin and the potential for customized medicine. However optimistic, these con-cepts are limited when the variability and heterogeneity of malignant neoplasms are recognized.

In spite of the similarities emphasized in oncogene and NOA, each signature phenotype hasvariation between tumors and even within tumors in the same animal. For example, although Tg(Wnt) tumors may share certain features such as the retention of the myoepithelium, they may alsohave as many as five different tumor phenotypes in the same animal (Rosner et al. 2002). Myc-derivedtumors have the same large, hyperchromatic blue cell phenotype but their tumors will vary in thedetails of their histological organization (Andrechek et al. 2009; Leung et al. 2011). The biologicalpotential of these variations has not been studied nor have expression microarray studies beencorrelated with the microscopic tumor pathology.

The situation becomes more complex when discussing GEM tumors that have escaped oncogeneaddiction. These recurrent or persistent tumors generally have lost the expression of the activatingoncogene. The most extensively documented histological studies of escape from addiction involvedthe phenomenon of epithelial-to-mesenchymal transition (EMT) (Cardiff 2010). The tumors thatundergo EMT become increasingly less differentiated and end up as spindle cell tumors (Fig. 7)(Radaelli et al. 2009). They can be differentiated from sarcomas by the presence of intermediatefilaments characteristic of both mesenchyme (vimentin) and epithelium (keratin) (Damonte et al.2007). Interestingly, this EMT-type transition is accompanied by an increase in local invasion and lossof metastatic potential (Cardiff 2010). The EMT tumors, regardless of the initiating oncogene, seem tobe a common final pathway for diverse oncogenes during neoplastic progression.

Mouse tumorigenesis seems to be particularly prone to EMT. The term EMT was not introduceduntil 1989, but, in retrospect, mouse mammary EMT has been observed from the very beginnings ofexperimental mouse pathology. It was originally attributed to transplantation artifact (Cardiff 2010).


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When in vitro culturing of tumors was developed, many of the cultured cells became spindle-shapedand the EMT phenomenon was thought to be a result of tissue culture. With the development ofevidence for oncogene addiction, based largely on GEM, escape from oncogene addiction was foundto be a result of EMT. Although EMT has been extensively studied in the context of the mammarygland, EMT occurs in the context of other organ systems, such as skin and prostate (Lacher et al. 2006;Ding et al. 2012).

The lesson to be learned when dealing with tumors of all types is that they are heterogeneous. Theinvestigator cannot assume, because two tumors arose in a molecularly defined animal, that thephenotype or biological history is known. In addition, investigators need to be aware of transplan-tation and tissue culture and their effect upon tumor variability. Both are very valuable techniques, yetthey have a profound effect upon the critical characteristics of a tumor.

TECHNICAL PREPARATION FOR STUDY OF GEM

The investigators and their pathologist need to know the details of the mouse model being used(Fig. 1). Presumably, the environment, experiment, gross examination, in vivo imaging, and molec-ular biology are all documented andmade available to the pathologist. Ideally, the documentation is ina well-organized format in a database with controlled, searchable terminologies and common dataelements (Cardiff et al. 2004) (Fig. 8). If any one of these elements is neglected, the analysis of thepathology may be incomplete. Complete documentation involves integration of diverse sources ofinformation. In modern times, it means using electronic formats with the use of proper nomenclatureand controlled vocabularies (Cardiff et al. 2004). The final form of documentation is the presentationfor publication.

Great concern and demand has been expressed for developing “standards” for anatomic pathology(histopathology). Most demands focus on the person interpreting the slides, the anatomic pathologist.However, as has been outlined above, pathologists are limited by the quality and quantity of theinformation about each mouse and the quality of the preparations presented to them (Fig. 4). Asstatedabove, thepathologicalanalysisbeginswith thepersonexamining themouse. Inmost institutions,the pathologist is a specimen’s “last stop.”Therefore, the investigator has primary responsibility for theinitial observations, descriptions, dissections, and processing of the specimen. The investigator is re-sponsible for understanding the normal gross anatomy and identifying abnormalities. Thus, we haveoutlined the basic information the investigator should know about cancer and neoplastic progression.Next,wewill dealwith technical preparations (necropsy and tissuefixation) and choosing a pathologist.

The Necropsy

The investigator should be very familiar with murine gross anatomy and, specifically, the anatomy ofthe organ of interest. Reference guides are available for dissection of specific organs. The recentComparative Anatomy and Histology: A Mouse and Human Atlas is a good place to start (Treutingand Dintzis 2012). If it is already known that the animal has a tumor, plan the dissection in advanceand be prepared to triage portions of the tumor to appropriate sources using the proper fixative for thetest being performed. General instructions for sampling and preparing murine organs have beenpublished (Ruehl-Fehlert et al. 2003; Kittel et al. 2004; Morawietz et al. 2004). Refer also to Protocol:Limited Mouse Necropsy (Cardiff et al. 2014a).

Fixation

Fixation stops postmortemchanges that degrade tissue andallowsoptimal preservationofmorphologicand cytological detail as well as nucleic acid integrity. Following death, tissues soon undergo autolysis,and if organisms from the gastrointestinal, urinary, or respiratory tracts are present, their colonizationcan soon cause putrefaction. Placing the tissue into a fixative stops these postmortem changes. SeeProtocol:MouseTissueFixation (Cardiff et al. 2014b) for abasic tissuefixationprocedure andguidance






on choosing an appropriate fixative, the timing and duration of fixation, sample storage, andquality issues.

Handling Tissues after Fixation

Fixed tissues are transported to the appropriate facility for the procedures required by the experiment.That facility can be your own or an “outside” laboratory. Regardless, if you are primarily interested ingenetic studies, there should always be a portion of the tissue set aside for H&E staining. This willprovide an answer to “What was the tissue that was evaluated?” If other, esoteric evaluations arerequested, it is important to check with the histology laboratory performing the procedures to becertain all requirements are met. Needless to say, this should be done before the necropsy. Protocolsfor routine H&E staining and also for immunohistochemical staining are given in Protocol: ManualHematoxylin and Eosin Staining of Mouse Tissue Sections (Cardiff et al. 2014c) and Protocol:Manual Immunohistochemistry Staining of Mouse Tissues Using the Avidin–Biotin Complex(ABC) Technique (Cardiff et al. 2014d).

Interpretation and Choosing a Pathologist

To obtain proper interpretation of your specimens, a pathologist (preferably one with experience inGEM pathology) needs to view your records and slides. The microscopic examination is something

Study hierarchy

Studies Study-1.0 Pten

1.0 Pten knockoutExperiments (1,…, n)

Cohorts Experimental (KO) Control (WT)

Subcohorts (1,…, n)

Specimens(1,…, n)

Samples (1,…, n)

Slides(1,…, n) Slide 1.1 Slide 2.1 Slide 3.1 Slide 4.1

Prostate Prostate Prostate Prostate

Mouse 1 Mouse 2 Mouse 3 Mouse 4

Treated Nontreated Treated Nontreated

Example

FIGURE 8. Study hierarchy. This figure depicts the application of the ontogeny for experimental design. The ontogeny,labeled Study hierarchy, follows Aristotelian rules. In most clinical settings, the organism, be it a mouse or human, isthe subject (red arrow) and carries the universal identifier. The Specimen (red box) (or specimen) is the equivalent ofOrganism in Ontology C (violet box) of Figure 1. The Slides are the children of a Sample, the Samples are children of aSpecimen, the Specimens are children of a Subcohort, the Subcohorts are children of a Cohort, the Cohorts arechildren of an Experiment, and the Experiments are children of a Study. In practice, Slides belong to an organ(Sample) (e.g., prostate) in an animal (Specimen) (e.g., Mouse 1,…, n), all Specimens belong to a Subcohort(treated or untreated), Subcohorts belong to a Cohort (Experimental or Control), Cohorts belong to an Experiment(e.g., Pten KO) and all Experiments belong to a Study (e.g., Pten). By using these simple rules, the investigator andcomputer can track the origin and distribution of multiple levels of information while maintaining order in relationshipto each animal and their samples. This ontogeny is also consistent with the anatomic ontogeny in Figure 1.


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anatomic pathologists actually do control. You should approach the pathologist with realistic expec-tations. The wise investigator identifies and works with their pathologist before the project starts.Seeking advice after the project is completed is too late for most remedial action. The pathologist’s jobis to integrate the microscopic observations with all other sources of information about the disease inthe animal. The diagnostic interpretation is provided in context.

What attributes are required of a pathologist? Basically, two kinds of pathologists exist: diagnosticand discovery pathologists. The diagnostic pathologist will verify that you do, or do not, have a tumorand attach a name (diagnosis) to your slide and move on to the next slide. This superficial behavior iscurrently used for high-throughput clinical diagnoses and, in investigative pathology, has led to anynumber of errors in the literature (Fig. 4). Pseudopathology is particularly rampant when the pathol-ogist is not trained in comparative pathology. Examples are numerous and include mistaking neuro-endocrine tumors for adenocarcinoma and mistaking the rodent preputial glands for teratomas andmouse nipples for “premalignant” papillomas (Barthold et al. 2007; Cardiff et al. 2008; Ince et al. 2008).

If the investigator is looking solely for a “name” (diagnosis), satisfaction will be achieved if thename fits their bias. If it does not, some investigators will try to find another pathologist who tells themwhat they want to hear. This behavior is called “shopping for a pathologist.” Or an investigator canignore the pathologist and apply his or her own preconceived nomenclature. This is called “do-it-yourself” (DIY) pathology (Ince et al. 2008). These types of behavior end in well-documented scien-tific gaffes (Couto and Cardiff 2008; Ince et al. 2008).

The second type of anatomic pathologist is the one who should be sought for your unique study:the discovery pathologist. Discovery pathologists will not only name the disease but will want to putthe disease into biological context. They are interested in the study of disease and want to use yourmice to learn more. Our experience has been that most true discovery pathologists are eager to learnabout the biological process in the experimental mice they are studying. However, it is most beneficialif the investigator engages them early in the process and not use them as the final step in theexperiment. Because the mouse models are intended for the study of human disease, the investigatorshould choose comparative pathologists with sufficient knowledge of both species to avoid errors.Medically trained pathologists should be able to recognize the spontaneous tumors in your mousestrain and compare them with the gene-induced tumors. Ideally, they should also be “genomicpathologists” who are well-versed in modern molecular biology and nomenclature. Most important,the pathologist should engage the investigator in the learning experience.

Unfortunately, trained comparative pathologists are relatively rare. Various training programs arebeing devised to fill this need but a shortage remains (Barthold et al. 2007; Cardiff et al. 2008; Warrenet al. 2009). Workshop training courses are available (Sundberg et al. 2012). A group of comparativepathologists has banded together under the banner of the Center for Genomic Pathology (http://ctrgenpath.net) (Cardiff et al. 2008; Couto and Cardiff 2008). They can either provide the investigatorwith assistance or direct you to the appropriate resources. The Center for Genomic Pathology hasdeveloped online courses aimed at graduate students, fellows, and new investigators. In cooperationwith UC Davis Extension, CGP has seminar-type interactive programs for Principle Investigators(http://www.extensiondlc.net/).

HANDLING EXPERIMENTAL PATHOLOGY DATA

Data Organization and Integration

Little has been written to guide the laboratory documentation of experimental studies of GEMMs. Thedocumentation is generally considered the purview of the individual laboratory and, as it should be,the responsibility of the principle investigator. Although this all seems logical, in an unpublishedsurvey carried out in 2005 for the National Cancer Institute’s Mouse Models of Human CancersConsortium, the principle investigators of five prestigious laboratories were asked to show theirdocumentation for recent publications. None of the five were able to show the primary data for






the published experiments. The primary data were said to exist in a collaborator’s laboratory or in thenow-missing laptop of a former fellow. None could show evidence that technical data provided byanother university actually belonged to a specific mouse. The best the investigators could do was toshow PowerPoint presentations, which were representations of the data but not the actual data.

Moreover, the scientific community has expanded. Core laboratories are called on to providespecific data sets. Collaborative studies are encouraged. In modern times, data need to be collected inelectronic format and coordinated in a common, accessible server. The principle investigator musttake responsibility for the collection, integration, and integrity of the data. The following sections willprovide some suggestions of how the neophyte can conceptualize, organize, and document their data.

A logical hierarchy for recording pathology data has been developed and successfully applied(Fig. 8). The hierarchy organizes data according to Study, Experiments, Cohorts, Specimens, andSlides. In this experimental hierarchy, each Slide belongs to a Sample, each Sample belongs to ananimal (Specimen), each animal belongs to a Cohort and subcohort, each Cohort belongs to anExperiment, and each Experiment belongs to a Study. This hierarchy can be divided into subcatego-ries. For example, Experiments frequently involve experimental and control animals. Each is consid-ered a separate group or Cohort. The experimental groups may contain multiple Cohorts and eachCohort multiple subcohorts (such as dosage or other experimental manipulation).

When data concerning a given animal arrive from another laboratory, the data need to be iden-tified or tagged by the identifier from each laboratory. For the pathologist, the animal’s samples can bedivided into separate blocks for processing. Further, different slides from each block may be stainedusing different techniques. A common identifier must be used for all of the slides, samples, andspecimens.

We use a laboratory accession number that identifies the animal to track all data. The computeralso records the animal ID provided by the investigator which links the slide to the Specimen, Cohort,Experiment, and Study. When the slide is recorded by whole slide image (WSI; see below) or as a“point-of-interest” still image, a standard image file format is used that includes accession number-block-photograph-stain-magnification-camera-photographer-subject. For example, file name EX12-0420-8-A-HE-x20-Ax1-RJM-MIN denotes image A from block 8, on a H&E-stained slide from ananimal with accession number EX12-0420, captured with the 20× objective on a Zeiss AxioCam byoperator RJM, showing an image of a mammary intraepithelial neoplasm (MIN).

We have developed and continue to maintain a database that is customized with the appropriatehierarchies and data fields. Some fields use a required (controlled) vocabulary but others permit freetext. Because we have many submitters, the entries need to be audited throughout the experiment toensure data integrity. The auditors must address whether the data are searchable. To the extent that wecan control entries frommultiple sources, the data are searchable. We have 22 years of data from wellover 500 laboratories and submitters. This article is a demonstration of the power of such archives:Almost all of the examples were identified by searches of our database.

Nomenclature: Understanding “Pathology-Speak”

In this modern era of electronic capture, storage, and retrieval, controlled vocabularies become ab-solutely essential. Considerable effort has been given to controlled vocabularies and synoptic reportingin health care and in experimental pathology (Cardiff et al. 2004; Schofield et al. 2009; Schofield et al.2010a,b, 2011; Gaudet et al. 2011; Hoehndorf et al. 2011). Ontologies of disease are useful in thisexercise (Fig. 1). However, most research laboratories do not have immediate access to these systems.As research becomes increasingly more complex and sophisticated, the comparative pathology of allspecies, let alone human and mouse, will inform us. Ultimately, the investigators who can relate theirmodels to the rest of comparative pathobiology will have the most impact. Therefore, it is incumbentfor the neophyte to learn proper nomenclature and use synoptic reporting.

A survey of the current terminologies will reveal that some discrepancies still exist betweenhuman and mouse nomenclature (Rudmann et al. 2012). The major discrepancies involve thecurrent separation between structure and function. The standard mouse nomenclatures are not


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supported by molecular data. Thus, they are based almost completely on descriptive morphology.The human classifications are almost completely based on gene expression profiles with relativelyfew attempts to match to the morphology. The genetically modified mouse is the one modelsystem in which structure and function have been matched (Cardiff et al. 2006; Rudmann et al. 2012).

Nonetheless, the pathology community has worked hard over the past two decades to providecontrolled vocabularies and standard nomenclatures (Schofield et al. 2010a,b). A huge internationalcooperative sponsored by the Society of Toxicological Pathology periodically reviews, updates and“harmonizes” diagnostic nomenclature (Renne et al. 2009; Thoolen et al. 2010; Rudmann et al. 2012).Numerous consensus reports and harmonized vocabularies have been published to assure the scien-tific community that we are using similar, if not the same, vocabulary (Cardiff et al. 2000; Kogan et al.2002; Nikitin et al. 2004). Comparative anatomy monographs and atlases are also available as refer-ences (Holland 2004; Bissell et al. 2011; Treuting and Dintzis 2012).

At the laboratory level, a difficult problem is nomenclature, because of the human tendency topersonalize data elements. The worst offenders choose to enter their data independently, whileignoring the rules enforced by the chosen hierarchy. Because we work with people around theworld, we have encountered different levels of data organization as well as complete lack of dataorganization. Inside the individual laboratory one finds varying degrees of compliance. As a result,someone from each laboratory should be responsible for vocabulary compliance and data integrity.Failure to meet this responsibility can result in the loss of valuable data.

Presenting the Images

Almost all pathology is now represented in publications as microscopic images, and the legends usediagnostic terminology rather than descriptions. Unfortunately, this mode has a number of short-comings. Any experienced pathologist can determine the relative knowledge of the person capturingthe images with a simple glance. The photographer should know the rudiments of proper microscopeadjustment. The most common error is the peripheral vignette in the image. When present, it isobvious the photographer does not know microscopy. Anyone unfamiliar with Köhler illuminationshould learn to use it for optimum adjustment of the microscope.

Naïve investigators who have been misled by less-experienced pathologists have presentedus with images labeled as “teratomas” that were in fact preputial glands (Ince et al. 2008), “prema-lignant papillomas” of the skin that were in fact nipples (Coste et al. 2007), “adenocarcinomas ofthe prostate” that were in fact neuroendocrine tumors (Couto and Cardiff 2008), “adenomas”with invasive margins, “colonic cancers” that were, in fact, submucosal granulomas associatedwith parasitic infestations (Borges et al. 2005), and the list goes on. The point here is that theimages were not only misleading, but the diagnosis was incorrect. The critical consideration in allof these examples is whether or not the images showed the appropriate field in their small thumbnailimages.

Often manuscripts present multiple thumbnail images that seldom are informative. This is knownas “postage stamp” pathology. These images are highly selective and may not necessarily represent theoverall process. Second, the images are too small for any legitimate pathologist to make a diagnosis orverify the written interpretations in the text. Unfortunately, the microscopic images found in thisarticle are good examples of this frustrating practice (Figs. 4, 6, and 7). When the images are used torepresent immunohistochemistry, one can generally only determine whether the cells are brown ornot; they rarely contain positive and negative controls that indicate sensitivity and specificity of thestain. Pathologists who are experienced in the field or have participated in Consensus Workshopsfrequently find that the published images do not accurately reflect what is actually on the correspond-ing slides.

Whole-Slide Imaging (WSI)

Many, if not most, institutions now have instruments that can digitize entire microscopic slides athigh resolution so that they can be viewed at almost any magnification at any point on the slide,






anywhere in the world. These are invaluable instruments for storing and annotating microscopicimages. They are now used on a daily basis for education and conferencing. They provide the viewerwith a comprehensive view of the entire slide from remote locations. WSI should also be used torepresent pathology in manuscripts. At the very least, these images should be made available for thereviewers, who could prevent the egregious errors that currently plague publications. However,publishers have been very slow to adopt WSI and have yet to come up with a reasonable businessplan. Until the time comes that publishers can profit from the current technology, they will continueto publish “faux” pathology. Beware of what you see in the hard-copy publication! Although wecannot show you the WSI directly, Table 3 provides URLs to WSI of Figures 4–7.

SUMMARY AND CONCLUSION

This primer of pathology emphasizes the necessity of using biological context for the interpretationof anatomic pathology. The steps involved are outlined, discussed, and illustrated. Some of thepitfalls are discussed and illustrated. These errors can be avoided with attention to detail and con-tinuous quality assurance. This primer is not a comprehensive review of pathology but shouldstimulate you to increase your awareness and knowledge of what pathologists can do and howthey do it.

TABLE 3. Links to whole slide images (WSI) for selected figures

Figure no. Description URL

1 None2 None3 None4A Polypoid GI mass, low magnification (granuloma) http://bit.ly/RrK0WB4B Worms in GI mass, High magnification (Granuloma) http://bit.ly/RrK0WB4C Extramedullary hematopoiesis, liver http://bit.ly/U8Lj984D Leukemic infiltrate, liver http://bit.ly/W6JpHA4E Mammary adenosquamous Ca, original http://bit.ly/Xd824p4F Mammary adenosquamous Ca, recut http://bit.ly/TVhs785A Abscess http://bit.ly/128HMxj5B Infarct, liver (in lower right of WSI) http://bit.ly/SRPsiq5C Granuloma http://bit.ly/SRRj6X5D Benign myoepithelioma http://bit.ly/12lGZYY5E Invasive margin, adenoca http://bit.ly/Te0OgH5F Expansile margin, carcinoma http://bit.ly/12nZYCr6A PIN, prostate http://bit.ly/Z0IjT86B GIN, small bowel http://bit.ly/Z3zOH66C Heterogeneity, keratin 19 http://bit.ly/W6Jxqz6D Heterogeneity, keratin 5 http://bit.ly/T9LSjC6E Tumor emboli, lung http://bit.ly/UHNezR6F Metastatic colonization, lung http://bit.ly/SdcBip7A Keratin 8/18, mammary EMT tumor http://bit.ly/TVgTtZ7B Keratin 19, mammary EMT tumor http://bit.ly/Z0RRh87C E-cadherin, mammary EMT tumor http://bit.ly/VCLPv07D N-cadherin, mammary EMT tumor http://bit.ly/TPB09p7E Vimentin, mammary EMT tumor http://bit.ly/UUnAMO7F TGF-β, mammary EMT tumor http://bit.ly/TVgR5b8 None

If you are viewing an electronic version of this article, simply click on the bit.ly link. If you are viewing a hard copy of thisarticle, type the URL into the subject line of your browser. When prompted, type in “guest” for the name and password.You might be asked to enter the name and password a second time. If you see an error message such as “Unable to openhttp://bit.ly./..”, simply click OK and try opening the link again. Your browser should navigate to the WSI. Once the WSIopens in WebScope from Aperio, you can click and drag the image to move it around. To change the magnification, clickon one of the magnifications in the “Zoom” box in the upper, left corner.

GI, gastrointestinal; WSI, whole-slide imaging; PIN, prostatic intraepithelial neoplasia; GIN, gastrointestinal intraepithe-lial neoplasia; EMT, epithelial–mesenchymal transition; TGF-β, transforming growth factor β.


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RELATED INFORMATION

To guide investigators in smaller, self-sufficient laboratories, we have provided several Protocol:Limited Mouse Necropsy (Cardiff et al. 2014a), Protocol: Mouse Tissue Fixation (Cardiff et al.2014b), Protocol: Manual Hematoxylin and Eosin Staining of Mouse Tissue Sections (Cardiff et al.2014c), and Protocol:Manual Immunohistochemistry Staining of Mouse Tissues Using the Avidin–Biotin Complex (ABC) Technique (Cardiff et al. 2014d).

ACKNOWLEDGMENTS

The research described here was supported by grants U01 CA141582, U01 CA141541 and U01CA105490-01 from the National Cancer Institute’s Mouse Models of Human Cancers Consortium.The authors also appreciate the discussions and review by Drs. A.D. Borowsky and J.A. Engelberg andsupport from Mr. Arishneel Ram.

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