# Presentation: 003385

## Morphologic Classification of Mouse Mammary Tumors with Some Examples in RatSabine Rehm, Dr. med. vet., ACVP DiplomateSmithKline Beecham Pharmaceuticals

## Outline

**Classification of Neoplasms**
**Classification of Rat Mammary Tumors**
**Classification of Mouse Mammary Tumors 1950-90**
**Histogenetic Classification of Mouse Mammary**
**	****	****Hyperplasia and Neoplasia:**
**	****	****	****Alveolar cell**
**	****	****	****Ductal cell**
**	****	****	****Myoepithelial cell**

**Overview**:  In the next 30 minutes, I will talk a little bit about classifications in general, then present what has been published recently for rat mammary tumors and briefly address terminology used for mice in the past 40 years or so.  The main part of my talk will deal with a classification system based on cellular composition of mouse mammary tumors with regard to alveolar, ductal or myoepithelial differentiation.

## Why Classify Neoplams?

_**Clinician:**_**  prevention, detection, treatment, prognosis**
**	****marker correlation, patient interest**
_**Academic Investigator:**_**  tumor biology**
**	****defined by investigator interests**
_**FDA Regulator**_**: hazard identification **
**	****mechanism of tumor formation****	**

**The type of classification will depend on the**
**goals to be achieved:**
**The clinician is searching for markers that will**
** ****correlate with treatment and prognosis, the **
**main objective is the well-being of the patient.**
**The academic investigator will most likely be **
**interested in tumor biology and the focus will be **
**on the interest of the investigator.**
**Then there is the FDA regulator and this is where**
** ****I am coming from.  It is important to correctly classify**
** ****lesion to allow hazard identification and mechanisms of tumor formation.**

## Morphologic Tumor Classifications

_** **__**Cellular Origin/Differentiation**_
**	****Cell type**
_**Grades of Malignancy**_
**	****Benign vs Malignant**
_**Subcategories**_
**	****Morphologic Variations**

**Morphologic Classification**:  Ideally, tumor classifications should be simple and easy to apply.  However, much will depend on the experience of the person looking at a slide, since tumors inherently will assume a confusing array of morphologies.  Basically tumors can be grouped according to cell type, such as epithelial vs mesenchymal, and according to malignancy.  This would include simple hyperplasia, atypical hyperplasia, adenoma, carcinoma in situ and various types of carcinomas.  Subcategories can be created according to distribution, subgrades of malignancy and morphologic variations .  The extent of such subdivisions may depend on the goal of the classification system or personality of the investigator, being either a lumper or a splitter

## Rat Mammary Tumors

_**Russo et al. 1989**_**	****	****    **_**van Zwieten et al. 1994**_
_**Epithelial**_
**Cystadenoma/Adenoma ****	****	****Adenoma**
**Papilloma**
**Carcinoma****	****	****	****	****Adenocarcinoma**
_**Epithelial-stromal**_
**Fibroadenoma****	****	****	****	****Fibroadenoma****	****	**
**	****	****	****	****	****Adenolipoma**
**Carcinosarcoma****	****	****	****Carcinosarcoma**
_**Stromal**_
**Fibroma****	****	****	****	****Fibroma**
**Fibrosarcoma**

**Rat Russo vs van Zwieten**:  Comparing two recently published classifications of the rat mammary gland by Russo et al and van Zwieten et al, there is agreement on the main categories of  epithelial, epithelial-stromal and stromal tumors but there are several differences.  There are benign adenomas in both classifications but papillomas only in one.  Malignant epithelial tumors on the one side are referred to as carcinomas and on the other side are called adenocarcinomas.  This difference in nomenclature is probably one we will also have to deal with in this workshop.  To me, carcinoma is suggestive of ductal differentiation, whereas adenocarcinoma implies glandular alveolar cell differentiation.  Fibroadenomas constitute the vast majority of spontaneous mammary tumors in rats.   In rats treated with chemical carcinogens, malignant tumors probably prevale and we will hear more on this subject from Dr. Russo.  Carcinosarcomas in rats are mostly derived from fibroadenomas, as evidenced by neoplasms clearly showing the transition from benign to malignant.  Neoplastic spindle cells can also be formed as an extreme dedifferentiation from epithelial cells or represent myoepithelial cells, as I will show you in a while.

## Classification Rat vs Mouse Mammary Tumors

_**Epithelial   **_**                                                 Rat          Mouse**
**	****Benign****	****	****	****          ********              ******
**	****Malignant ****	****	****                      ********              ******** **
_**Epithelial-myoepithelial**_** (B/M) ****	****          ******** ****	****    ******
_**Epithelial-stromal**_
**	****Benign (fibroadenoma)                  ********                ?**
**	****Malignant (carcinosarcoma)         ********                ?**
_**Stromal**_
_**	**_**Benign (fibroma)                             ********              ******
**	****Malignant (fibrosarcoma)              ********              ******

**Rat vs Mouse**:  Now let us compare mammary tumors of rats and mice.  In both pure epithelial malignant neoplasms outweigh benign adenomas, based on local invasion and cytologic appearance.  Metastases, will depend on the model. Rodents are usually killed because of tumor size and skin erosions with associated bleeding.  In rats, there is no special category for tumors with myoepithelial cells, although these tumors have been reported to occur in rats.  Old mice spontaneously develop benign adenomyoepitheliomas, and malignant myoepitheliomas can reliably be induced by DMBA.  As mentioned, epithelial-stromal or pure stromal tumors are common in rats, but rare in mice.  However, in the transgenic slide set, there are probably some than can be called fibroadenomas.  Many carcinosarcomas diagnosed to date in mice, probably represent malignant adenomyoepitheliomas.

## Mouse Mammary Tumor Classification 1950-1990

_**Morphological and Alphabetical**_
**	****Adenocacrcinomas**
**Type A  ****	****Type B****	****Type A/B****	****   Type C**
**Type Y  ****	****Type L****	**** Pale cell****	**** Fissured **
**Adenoacanthoma, Organoid/Molluscoid Tumor,**
**Carcinoma type P, Carcinosarcoma, Fibroadenoma,**
**Mixed Tumor**

**1950-1990**:  Previous classifications of mouse mammary tumors represented a combination of morphologic structures and the alphabet.  It is not a systematic evaluation, it is difficult to understand, there are many oldfashioned names such as adenoacanthoma or moluscoid tumor and there is considerable overlap.  This is not a useful approach.

## Determining Cellular Differentiation 

_**Stem - Alveolar  - Ductal - Myoepithelial -  Fibroblastic - Multiple Cell Types**_
**Fixation: formalin 10% neutral buffered**
**Processing artifacts**
**Initial emphasis: small, early lesions **
**Cellular markers/Additional techniques**
**DDX: salivary/lacrimal glands, dermal elements, clitoral gland, lung tumors**

**Cellular differentiation**:  How do we obtain a useful system?  At first we can try to go on the path of determining the cellular differentiation of a tumor.  There are clearly limits to this, but at least we can give it a try.  In the transgenic slide set, there were good examples of early and late lesions, but there are also slides with problems.  Initially, the best fixation for H&E slides is formalin and beware of processors that do not perform properly.  The tissue is permanently damaged. It is very helpful to first concentrate on early lesions that are not yet visible macroscopicallyLocalization of initial lesions will often enable us to determine the origin of larger neoplasms.  Initially, cells may still retain many differentiated features or markers that will help characterize the cell types.  Normal mammary tissue can be expected to be present in the same section, serving as an internal positive control for markers.  In larger tumors, the origin is obscured due to invasive growth, cells have dedifferentiated losing markers or tissue is lost altogether because of widespread necrosis.  One also has to keep in mind, that not all sc nodules arise from the mammary gland.  In the slide collection there appears to be a tumor of the parotid gland – or tumors can arise from the skin.  When it comes to assessing pulmonary metastases, one needs remember that mice have a reputation for developing lung tumors and we a couple of those in the slide set too.

## Normal Mouse Mammary Gland

## Mouse Mammary TumorsRehm & Liebelt 1996

_**Alveolar differentiation**_**:  **
**    ****Adenoma**
**    ****Adenocarcinoma**** **
**    **

## Alveolar Hyperplasia

**Focal /Multifocal/ Diffuse **
**Atypical**
**Increased serum prolactin levels**
**Cytoplasmic lipid droplets & secretions**
**Squamous metaplasia**

## AdenomaFibroadenoma

## Adenocarcinoma

## TUMOR  GROWTH   PATTERNS

**TUMOR  GROWTH   PATTERNS**

**Tumor growth patterns:**  This cartoon summarizes different growth patterns mouse mammary adenocarcinomas can assume.  Variations range from the well-differentiated acinar pattern to tubular, papillary, comedo and solid, forming sheets, trabeculae or nests.  A single tumor can display one or even all of these features.  Acinar structures can be absent and then it becomes difficult to classify the tumor.  In such instances it may be useful to have an additional category, such as carcinoma NOS, meaning not otherwise specified.

## Mouse Mammary TumorsRehm & Liebelt 1996

_**Alveolar differentiation**_**:  **
**    ****Adenoma**
**    ****Adenocarcinoma**** **
**    ****Adenosquamous carcinoma**

## Mouse Mammary TumorsRehm & Liebelt 1996

_**Alveolar differentiation**_**:  **
**    ****Adenoma**
**    ****Adenocarcinoma **
**    ****Adenosquamous carcinoma**
_**Ductal differentiation**_**:**
**    ****Ductal papilloma/carcinoma**** **

## Ductal Hyperplasia

**Intraductal**
**Ductal (budding/branching)**
**Focal/Multifocal/ Diffuse**
**Atypical**

**DUCTAL HYPERPL**:
- Obvious intraductal hyperplasia is easily recognized, whereas, ductal hyperplasia that occurs through budding and branching, may not and certainly needs to be distinguished from alveolar hyperplasia.

## Ductal Carcinoma

**DUCTAL CARCINOMA**:
- It seems logical to call the neoplasm that arises from an intraductal hyperplasia, an intraductal carcinoma.  However, since ductal tumors could also form by branching and budding, the term intraductal may be too limiting.  It may be easier to call these lesions simply ductal carcinomas.

## Mouse Mammary TumorsRehm & Liebelt 1996

_**Alveolar differentiation**_**:  **
**    ****Adenoma**
**    ****Adenocarcinoma **
**    ****Adenosquamous carcinoma**
_**Ductal differentiation**_**:**
**    ****Ductal papilloma/carcinoma**** **
**    ****Ductal sqaumous carcinoma**

**DUCTAL SQUAMOUS CARCINOMA**:
- Similar to the adenosquamous carcinoma, there is a ductal squamous carcinoma.
**Ductal squamous carcinomas** can be induced with DMBA.  They are rather peculiar tumors since they have this radiating growth pattern.  At first sight, one may think of a skin tumor which is not unusual since the mammary gland is an adenexal gland of the skin. Therefore, it is not surprising to be reminded of this fact in neoplasms.  These lesions, however, have no contact with the skin.

## Mouse Mammary TumorsRehm & Liebelt 1996

_**Alveolar differentiation**_**:  **
**    ****Adenoma**
**    ****Adenocarcinoma **
**    ****Adenosquamous carcinoma**
_**Ductal differentiation**_**:**
**    ****Ductal papilloma/carcinoma **
**    ****Ductal sqaumous carcinoma**
_**Myoepithelial differentiation**_**:**
**   ****Adenomyoepithelioma (Benign/Malignant)**

**ADENOMYOEPITHELIOMA: **The third category of mouse mammary tumors shows myoepithelial differentiation.  Similar tumors are known to occur in humans and I suspect there are several in the transgenic slide set.  This is my special field of interest where I was able to do some experimental work at NCI.

## Mouse DMBA-induced Mammary Tumors

_**Study Design:**_
**Strains: B6D2F1 or D2B6F1   ****(C57BL x DBA)**
**Age at start: 8 weeks**
**Compound: 7,12-dimethylbenz(********)anthrazene**
**Dose: 1 mg/mouse**
**Dosing: 1/week for 4 weeks**

## Mouse DMBA-induced Mammary Tumors

_**Results:**_
**6-8 weeks after last treatment:  **
**ductal squamous carcinomas**
**6-8 months after last treatment:**
**adenomyoepitheliomas**

## Adenomyoepithelioma

## Transplantation of Adenomyoepitheliomas and Clonally Selected Malignant Myoepithelium

_**Hypothesis**_**:**
**1.  Cellular differentiation potential of**
**myoepithelial cells (cartilage/bone)**
**2. Increase capability to metastasize**

## Classification of Mouse Mammary Tumors

_**Epithelial, benign**_**:**
**	****	****Adenoma/ Papilloma/Fibroadenoma**
_**Epithelial, malignant**_**:**
**  ****	****	****Adenocarcinoma**
**  ****	****	****Adenosquamous carcinoma**
**  ****	****	****Ductal carcinoma**
**  ****	****	****Ductal sqaumous carcinoma**
_**Myoepithelial tumors**_**:**
** ****	****	**** ****Adenomyoepithelioma (Benign/Malignant)  **
_**Carcinoma, NOS**_

**Final Classifcation**:  In summary, it can be concluded that most proliferative lesions of the mouse mammary gland can be classified into benign epithelial, some epithelial-stromal, and malignant epithelial processes.  These lesions can show alveolar or ductal differentiation with or without a dominant squamous component.  Very rarely neoplasms may also show both alveolar and ductal differentiation.  Thirdly, there is a less common category composed of myoepithelium and various other cellular elements.  And if you have no idea on the differentiation of the neoplasm, there is always carcinoma,NOS