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The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting

Oncogene volume 19, pages 968–988 (2000)Cite this article

Abstract

NIH sponsored a meeting of medical and veterinary pathologists with mammary gland expertise in Annapolis in March 1999. Rapid development of mouse mammary models has accentuated the need for definitions of the mammary lesions in genetically engineered mice (GEM) and to assess their usefulness as models of human breast disease. The panel of nine pathologists independently reviewed material representing over 90% of the published systems. The GEM tumors were found to have: (1) phenotypes similar to those of non-GEM; (2) signature phenotypes specific to the transgene; and (3) some morphological similarities to the human disease. The current mouse mammary and human breast tumor classifications describe the majority of GEM lesions but unique morphologic lesions are found in many GEM. Since little information is available on the natural history of GEM lesions, a simple morphologic nomenclature is proposed that allows direct comparisons between models. Future progress requires rigorous application of guidelines covering pathologic examination of the mammary gland and the whole animal. Since the phenotype of the lesions is an essential component of their molecular pathology, funding agencies should adopt policies ensuring careful morphological evaluation of any funded research involving animal models. A pathologist should be part of each research team.

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Acknowledgements

The Annapolis meeting was sponsored by the NIH Breast Cancer Think Tank and the NCI Cancer Genome Anatomy Project. The organizers and panelists would like to thank Ms Susan Greenhut for her assistance in organizing the meeting, slides, and logistics for the meetings. We would also like to thank Jai Evans and Ulrike Wagner for their excellent technical assistance throughout the course of this project. The organizers and panelists gratefully acknowledge the following investigators who graciously provided information, blocks and slides to the slide sets: Ari Elson, Priscilla Furth, Jeffrey Green, Hans Weiher, Glenn Merlino, A Graessmann, Izumi Nakashima, Chris Graham, Daniel Medina, Glenn Radice, Eugene Lukanidin, Mark Sternlicht, Barbara Knowles, Dimitrina Pravtcheva, Gilbert Smith, Sonia Jakowlew, Eric Sandgren, Barry Davis, Lothar Hennighausen, Robert Cardiff, Ann Harrington, Jake T Liang, Emmett Schmidt, Philip Leder, Harold Varmus, Robert Coffey, Rob Callahan, Bill Muller, Roy Jensen, Jeffrey Rosen, Hal Moses and Jose Russo. This project has been funded in part with Federal funds from the National Cancer Institute, NIH, under contract no. NO1-CO-56000 and a grant from CGAP.

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Author notes

  1. Robert D Cardiff and Jeffrey E Green: RD Cardiff assisted in the organization of the pathology workshop and prepared the manuscript. JE Green was primarily responsible for the workshop concept and organization. All other authors contributed equally to the manuscript and are, thus, listed alphabetically

Authors and Affiliations

  1. U.C.D. Center for Comparative Medicine, County Road 98 and Hutchison Drive, University of California, Davis, Davis, 95616, CA, USA

    Robert D Cardiff

  2. Pathology/Histotechnology Laboratory, SAIC Frederick, NCI-FCRDC, P.O. Box B, Building 539, Frederick, 21702-1201, MD, USA

    Miriam R Anver

  3. Institute of Cancer Research, The Breakthrough Toby Robins Breast Cancer Research Centre, 237 Fulham Road, London, SW3 6JB, England

    Barry A Gusterson

  4. Laboratory of Genetics and Physiology, NIDDK, NIH, Building 8, Room 101, Bethesda, 20892, MD, USA

    Lothar Hennighausen

  5. Department of Pathology, 4918 TVC, Vanderbilt University Medical Center, 22nd Avenue South and Pierce Avenue, Nashville, 37232-5310, TN, USA

    Roy A Jensen

  6. Laboratory of Pathology, NCI, NIH, Building 10, Room 2N212, Bethesda, 20892, MD, USA

    Maria J Merino

  7. SmithKline Beecham Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA, USA

    Sabine Rehm

  8. Breast Cancer Research Laboratory, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, 19111, PA, USA

    Jose Russo

  9. Department of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306-6000, USA

    Fattaneh A Tavassoli

  10. Laboratory of Cell Regulation and Carcinogenesis, NCI, NIH, Building 41, Room C629, 41 Library Drive, Bethesda, 20892, MD, USA

    Lalage M Wakefield & Jeffrey E Green

  11. Veterinary and Tumor Pathology Section, Office of Laboratory Animal Resources, National Cancer Institute, Frederick, 21702-1201, MD, USA

    Jerrold M Ward

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The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organization imply endorsement by the US Government

IDEALIZED MOUSE WORKUP FOR MAMMARY TUMORIGENESIS STUDIES

IDEALIZED MOUSE WORKUP FOR MAMMARY TUMORIGENESIS STUDIES

The following outline is provided as a guide for maximizing the information obtained from your mouse. Since considerable information can be obtained from analysis of microscopic changes in the mammary gland, it is particularly important to give the pathologist representative ``uninvolved'' mammary tissue in addition to gross mammary lesions. This can give insight into the preneoplastic processes occurring in your model.

PROCEDURE:

1. For any newly generated mouse model, the founder mice and a select number of offspring (ideally no less than 5) should be subjected to a complete necropsy in order to get a complete description of the phenotype. This will allow identification of any additional phenotype changes that might impact on mammary tumorigenesis studies. A guideline for performing complete necropsies can be found at http://www.ncifcrf.gov/vetpath/necropsy.html.

2. For mice in a mammary tumorigenesis study, it is preferable to have a minimum of 10 mice/group. The use of appropriate controls is critical. There should be an equal number of age-matched, non-engineered or untreated controls, of identical genetic background and parity. Note that mammary gland morphology and the incidence of spontaneous mammary tumors varies widely between different genetic backgrounds. This is a particularly important issue when working in a hybrid background. The pathologist should be supplied with the information about the mice found in the Pathology Request Form.

3. For a mammary tumorigenesis study, the mouse should be opened and examined grossly, with any lesions or gross changes noted on the necropsy sheet. Examine all mammary glands grossly and note positions and size of lesions on a diagram of the glands at: URL http://www.ncifcrf.gov/vetpath/necropsy.html. Assign a unique post-mortem number to the mouse.

4. To maximise the amount of information obtainable from each mouse, the tissues indicated in Table 7 should be harvested for each mouse. Tissues of immediate interest should be processed to paraffin block as quickly as possible (within 48h for formalin) as this gives best results for immuno-histochemistry. Organs that are not immediately useful can be fixed and saved for later use (see #8 below). Correct identification of all mice is critical. It is useful to include the identifier (e.g. ears if mice are ear-notched) to allow subsequent cross-checking of mouse ID with PM number.

Table 7 Summary of pathology workup
Full size table

5. Mammary masses: For masses >0.5cm, it may be desirable to snap freeze half for molecular analysis and fix half for histology (see notes about fixatives below). For smaller masses, fix all for histology. If possible include some uninvolved gland, and harvest the contralateral gland for both histology and molecular analysis.

6. Whole mounts: To visualize the morphology of the ductal tree, it is desirable to whole mount some glands from a select subset of animals. The #4 and #9 (abdominal glands) are generally best for whole mounting because of the presence of the lymph node for orientation. A protocol for mammary whole mounts can be found at http://www-mp.ucdavis.edu/tgmice/Histolab.html. Lesions identified at whole mount can subsequently be sectioned for histology.

7. Trimming and sectioning mammary glands: For trimming mammary glands, remove mammary gland from skin and put on paper (rough DRY brown paper towels work well). Press gland on towel and flatten with forceps. Alternatively, the gland can be spread on a glass slide for better visualization. You can trim off some fat at this point. It is helpful to have a magnifying glass present when trimming muscle away from glands (esp. for thoracic glands). Glands are then fixed (see below).

8. Fixatives:

a. If the study will require use of specific antibodies for immunohistochemistry, it is critical to research the optimal fixative for the antibody of interest in advance. Remember that the mammary epithelium is embedded in excessive amounts of fat that either needs to be trimmed or may require special defatting procedures.

b. For many applications, 10% neutral buffered formalin can be used. Agitate tissues in a 10x-fold excess volume of fixative overnight at room temperature and then change to fresh neutral buffered formalin for storage. For best results with immunohistochemistry, PROCESS TO BLOCK WITHIN 48h. When saving tissues in fixative, double bag all untrimmed tissue with formalin. Recheck bags after 6 mo. and top up with formalin if necessary.

c. For in situ hybridization, best results are usually obtained with 4% paraformaldehyde as fixative. Fix for 3-5d at 4°C.

d. The study and grading of nuclear atypia is better with some acidic or heavy metal fixatives (Bouin's, Zenker's, B-5 ect). Tissue stored in formalin can be post-fixed with these fixatives to improve nuclear detail.

9. Sectioning and staining. When cutting sections from mammary gland blocks, cut longitudinally (“fried egg morphology”. 5u sections are optimal. Hematoxylin/eosin staining is usually optimal for histology. A very light hematoxylin counterstain is generally used alone for immunohistochemistry. However, other nuclear counterstains may be better for specific purposes. Sometimes diagnosis will be aided by additional special stains (e.g. immunohistochemical staining for smooth muscle actin).

Figure A1
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Annapolis Pathology Request Form for Studies of the Mouse Mammary Gland

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Figure A2
figure 7

Annapolis Pathology Reporting Form

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Figure A3
figure 8

Annapolis Pathology Reporting Form Microscopic Description

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Cardiff, R., Anver, M., Gusterson, B. et al. The mammary pathology of genetically engineered mice: the consensus report and recommendations from the Annapolis meeting. Oncogene 19, 968–988 (2000). https://doi.org/10.1038/sj.onc.1203277

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