Quantitation of fixative-induced morphologic and antigenic variation in mouse and human breast cancers.

Quantitative Image Analysis (QIA) of digitized whole slide images for morphometric parameters and immunohistochemistry of breast cancer antigens was used to evaluate the technical reproducibility, biological variability, and intratumoral heterogeneity in three transplantable mouse mammary tumor models of human breast cancer. The relative preservation of structure and immunogenicity of the three mouse models and three human breast cancers was also compared when fixed with representatives of four distinct classes of fixatives. The three mouse mammary tumor cell models were an ER+/PR+ model (SSM2), a Her2+ model (NDL), and a triple negative model (MET1). The four breast cancer antigens were ER, PR, Her2, and Ki67. The fixatives included examples of (1) strong cross-linkers, (2) weak cross-linkers, (3) coagulants, and (4) combination fixatives. Each parameter was quantitatively analyzed using modified Aperio Technologies ImageScope algorithms. Careful pre-analytical adjustments to the algorithms were required to provide accurate results. The QIA permitted rigorous statistical analysis of results and grading by rank order. The analyses suggested excellent technical reproducibility and confirmed biological heterogeneity within each tumor. The strong cross-linker fixatives, such as formalin, consistently ranked higher than weak cross-linker, coagulant and combination fixatives in both the morphometric and immunohistochemical parameters.

Id-1 is not expressed in the luminal epithelial cells of mammary glands.

BACKGROUND: The family of inhibitor of differentiation/DNA binding (Id) proteins is known to regulate development in several tissues. One member of this gene family, Id-1, has been implicated in mammary development and carcinogenesis. Mammary glands contain various cell types, among which the luminal epithelial cells are primarily targeted for proliferation, differentiation and carcinogenesis. Therefore, to assess the precise significance of Id-1 in mammary biology and carcinogenesis, we examined its cellular localization in vivo using immunohistochemistry. METHODS: Extracts of whole mammary glands from wild type and Id-1 null mutant mice, and tissue sections from paraffin-embedded mouse mammary glands from various developmental stages and normal human breast were subjected to immunoblot and immunohistochemical analyses, respectively. In both these procedures, an anti-Id-1 rabbit polyclonal antibody was used for detection of Id-1. RESULTS: In immunoblot analyses, using whole mammary gland extracts, Id-1 was detected. In immunohistochemical analyses, however, Id-1 was not detected in the luminal epithelial cells of mammary glands during any stage of development, but it was detected in vascular endothelial cells. CONCLUSION: Id-1 is not expressed in the luminal epithelial cells of mammary glands.

Persistent mammary hyperplasia in FVB/N mice.

The inbred FVB/N mouse strain is widely used for creating transgenic mice. Over the past decade, persistent mammary hyperplasia has been detected in many multiparous FVB/N female mice sent to the University of California, Davis (UCD) Mutant Mouse Pathology Laboratory (MMPL) by a number of different laboratories. However, the experimental details concerning most specimens were not always available. To confirm these empiric findings, experiments were carried out to evaluate the mammary glands of FVB/N mice under controlled conditions. Persistent mammary hyperplasia that related to parity was found. Weeks after their first to fourth pregnancy, 10 FVB/N female mice from the Lawrence Berkeley National Laboratory (LBNL) colony were studied and the mammary glands were evaluated. The percentage of fat pad filled was estimated, using image analysis. Serum samples and the pituitary gland from other FVB/N mice from the LBNL were assayed for prolactin concentration. Multiparous FVB/N females consistently had persistent mammary hyperplasia. Four of seven females in the LBNL colony had hyperplasia after three pregnancies. A few foci of squamous nodules and sporadic carcinomas also were observed. Thus, some FVB/N females may have persistent mammary hyperplasia after three pregnancies without detectable pituitary abnormalities. Mammary carcinomas also may develop sporadically. These background phenotypes must be considered when interpreting the effect of genetic manipulation in FVB/N mice.

Spontaneous pituitary abnormalities and mammary hyperplasia in FVB/NCr mice: implications for mouse modeling.

The FVB/N mouse strain is widely used in the generation of transgenic mouse models. We have observed that mammary glands of wild-type virgin female FVB/NCr mice frequently have the morphologic and histologic appearance of a gland during pregnancy. By 13 months of age, the mammary glands of more than 40% of the mice examined had lobuloalveolar hyperplasia that was characterized by the presence of secretory alveoli and distended ducts apparently containing secretory material. The prevalence of this phenotype further increased with age. The mammary phenotype was highly correlated with the presence of proliferative, prolactin-secreting lesions in the pituitary gland. In mice aged 18 to 23 months, hyperplasia of the pars distalis was seen in 11 of 21 mice (52%), and a further 4 of 21 mice (19%) had pituitary adenomas. Pituitary hyperplasia was already evident in some mice as young as nine months. The pituitary phenotype was also associated with high prevalence (4/6 mice) of spontaneous mammary tumors in aged multiparous, but not virgin FVB/NCr mice. This high prevalence of pituitary abnormalities and their effects on the mammary gland have important consequences for the interpretation of new phenotypes generated in transgenic models using this mouse substrain.

Structured reporting in anatomic pathology for coclinical trials: the caELMIR model.

Electronic media, with their tremendous potential for storing, retrieving, and integrating data, are an essential part of modern collaborative multidisciplinary science. Structured reporting is a fundamental aspect of keeping accurate, searchable electronic records. This discussion on structured reporting in anatomic pathology for pre- and coclinical trials in animal models provides background information for scientists who are not familiar with structured reporting. Practical examples are provided using a working database system for preclinical research-caELMIR (Cancer Electronic Laboratory Management Information and Retrieval)-developed by the U.S. National Cancer Institute's (NCI's) Mouse Models of Human Cancers Consortium (MMHCC).

Validation: the new challenge for pathology.

Modern pathologists have been challenged to "validate" mouse models of human cancer. Validation requires matching of morphological attributes of the model to human disease. Computers can assist in the validation process. However, adequate controlled, computer-readable vocabularies that can match terms do not currently exist in mouse pathology. Further, current standard diagnostic terminologies do not include the new concepts discussed here such as pathway pathology and mammary intraepithelial neoplasia. The terminologies must be revised and improved to meet the challenge. Human medicine has traditionally used "guilt-by-association" to validate interpretations of disease. Experimental pathology uses experimental verification exemplified by "test-by-transplantation." Genetically Engineered Mice (GEM) develop unique tumor phenotypes bringing new structural-functional insights and reevaluation of concepts. Novel GEM-related tumors appear in all organ systems but mouse models of human breast cancer are prototypes. For example, mammary tumors induced by Mouse Mammary Tumor Virus (MMTV), chemical, radiation or other carcinogenic stimuli have limited phenotypes. These "spontaneous" or induced mammary tumors have never resembled human breast cancers. GEM tumors created with genes associated with human cancer are strikingly different. GEM tumors have unique histological phenotypes. Depending on the genes, the tumors may: 1) resemble MMTV-induced tumors, 2) display "signature" phenotypes, and 3) mimic human breast cancers. The phenotypes can be placed into structural and functional clusters with shared characteristics leading to the concepts of Pathway Pathology: tumor phenotype reflects the genotype.

Prostatic intraepithelial neoplasia in genetically engineered mice.

Several mouse models of human prostate cancer were studied to identify and characterize potential precursor lesions containing foci of atypical epithelial cells. These lesions exhibit a sequence of changes suggesting progressive evolution toward malignancy. Based on these observations, a grading system is proposed to classify prostatic intraepithelial neoplasia (PIN) in genetically engineered mice (GEM). Four grades of GEM PIN are proposed based on their architecture, differentiation pattern, and degree of cytological atypia. PIN I lesions have one or two layers of atypical cells. PIN II has two or more layers of atypical cells. PIN III has large, pleomorphic nuclei with prominent nucleoli and the cells tend to involve the entire lumen with expansion of the duct outlines. PIN IV lesions contain atypical cells that fill the lumen and bulge focally into, and frequently compromise, the fibromuscular sheath. Within the same cohorts, the lower grade PINs first appear earlier than the higher grades. Morphometric and immunohistochemical analyses confirm progressive change. Although the malignant potential of PIN IV in mice has not been proven, GEM PIN is similar to human PIN. This PIN classification system is a first step toward a systematic evaluation of the biological potential of these lesions in GEM.