Can you use cytology to predict tumor behavior (Proceedings)

Cytologically, neoplasia is characterized by the presence of a homogeneous population of cells that have come from the same tissue of origin. This is best appreciated by the presence of cells with the same cytoplasmic characteristics. If a neoplasm is diagnosed, two important determinations must be attempted: 1) is the lesion benign or malignant, and 2) what is the tissue of origin.

Benign vs. Malignant

Benign neoplasia / hyperplasia is characterized by a uniform population of cells. There should be a uniform cytoplasmic and nuclear size and shape, uniform nuclear to cytoplasmic ratio, and if nucleoli are present, they are of consistent size, shape, and number among individual cells.

Malignant neoplasms will have nuclear features that are considered abnormal and indicate a cell population that is growing rapidly and uncontrollably. The following are nuclear characteristics considered to be abnormal.

     1. Anisokaryosis

     2. Pleomorphism

     3. High or variable N:C ratio

     4. Increased mitotic activity

     5. Nucleoli that vary in size, shape, and number (angular, irregularly shaped nucleoli)

     6. Coarse chromatin or uneven margination of chromatin at nuclear membrane

     7. Nuclear molding

     8. Multinucleation

If many of the cells show 3 or more of the criteria of malignancy, the tumor is malignant. In the vast majority of the cases, the cytologic criteria of malignancy are accurate predictors of the potential biological behavior of the tumor. Most tumors with malignant features will either be locally invasive or have the potential for metastasis. However, there are some circumstances, and these are the exception to the rule, where the cytologic appearance may not accurately predict the biological behavior. A practicing cytologist must be aware of the exceptions to the rule in order to be able to rely on cytology to provide meaningful, accurate information regarding the prognosis of a patient.

Specific Tumor Types and Locations

In addition to the presence of inflammation, there are specific instances where the cytologic appearance of the neoplasm does not correlate well with the biological behavior of the lesion. The location of some tumors and occasionally the specific tumor type must also be considered when using cytology to determine the malignant potential of certain neoplasms. We will discuss these specific neoplasms within each cytologic category, the mesenchymal, the epithelial, the round cell, and the neuroendocrine tumors.

I. Mesenchymal neoplasms

General Features: These tumors are composed of small to medium cells. Aspirates are usually less cellular than other tumor types, and the cells arranged more individually. Cytoplasm is wispy and often spindle-shaped with wisps of cytoplasm trailing off in 1 or 2 directions from the nucleus. Cytoplasmic borders are usually indistinct. The nuclei are typically oval to polygonal. Malignancies of mesenchymal origin are termed sarcomas.

Mesenchymal Tumors and the Criteria of Malignancy: The nuclear features of malignancy are usually reliable indicators of the malignant potential of mesenchymal tumors. The following are exceptions.

Leiomyosarcoma and myxosarcoma

Leiomyosarcomas and the myxosarcomas are uncommon tumors of smooth muscle and fibroblast origin respectively. These tumors are difficult to differentiate from their benign counterparts (leiomyoma and myxoma) based on cytologic or even histologic features alone. Since some leiomyosarcomas and myxosarcomas do not have bizarre nuclear features, histologists must often rely on invasion into surrounding tissues to determine the malignant potential of these tumors. However, if nuclear criteria of malignancy are met, the lesion is considered malignant. Leiomyosarcoma pictured to the right.

II. Epithelial neoplasms

General Features: These tumors are usually composed of large cells. Aspirates are often very cellular and the cells characteristically exfoliate in clumps or sheets. The cells are round to polygonal with very distinct cytoplasmic borders. They are often tightly adherent to each other with extensive contact between adjacent cells and clear zones between areas of cell junctions. Prominent cytoplasmic vacuolation, signet-ring, and/or acinar formation may be seen if the tumor originates from glandular epithelium. Malignancies of epithelial origin are termed carcinomas (nonglandular) or adenocarcinomas (glandular).

Epithelial Tumors and the Criteria of Malignancy: Unless there is inflammation present, the nuclear criteria for malignancy are reliable indicators for the vast majority of epithelial neoplasms. There are three notable exceptions, that are actually quite common tumors, A) basal cell tumors, B) perianal / anal gland tumors, and C) hepatocellular carcinomas. Although not a common neoplasm, salivary gland tumors of the dog and cat may appear cytologically benign, however, most are malignant.

Perianal / anal gland tumors in the canine

Perianal gland adenomas are benign tumors of the circumanal glands, commonly seen in intact, male dogs > 8 years of age. Perianal gland adenocarcinomas do occur, but they are much less common and may be seen in either intact or neutered male dogs. They are less frequently seen in neutered female dogs and rarely in intact females.

Cytologic features: Perianal gland adenomas are composed primarily of large, polyhedral, "hepatoid-like" cells occurring in clumps or clusters. They contain abundant basophilic cytoplasm, often with a pink hue. The N:C ratio is low and nuclei are typically round to oval with clumped chromatin and one or two large nucleoli. This gives the cell a remarkable resemblance to hepatocytes. Most perianal gland adenomas usually contain variable numbers of a second population of smaller epithelial cells with a much higher N:C ratio. These cells are called reserve cells. The distinctive population of cells allows easy identification of the tumor, however, the variability of the cell population, along with the prominent nucleoli and clumped chromatin may mimic a malignancy, even with adenomas.

Anal Sac Apocrine Gland Adenocarcinomas are epithelial tumors of the anal sac apocrine glands of the dog. Two recent studies have revealed that both female and male dogs are equally likely to develop this neoplasm (JAVMA 2003;223:825-831, JVIM 2002;16:100-104). These studies refute the previous observation that this neoplasm occurs more frequently in female dogs.

Cytologic features: This tumor appears as clumps of epithelium with very few distinct cytoplasmic borders. The appearance of free nuclei embedded in a mass of cytoplasm gives this lesion neuroendocrine-like appearance. This allows definitive identification of this tumor by finding a neuroendocrine like appearance in a cell population from a lesion located around the anus. The cells appear to have a high N:C ratio (lots of nuclei), but the cells are fairly uniform with round nuclei. There is occasionally a mild anisokaryosis and sometimes small indistinct nucleoli can be seen. However, there are usually not enough abnormalities to fulfill the cytologic criteria of malignancy.

Biological behavior and criteria of malignancy: Cytologically, this tumor will appear benign, however, the cytological appearance of this tumor does not accurately predict the biological behavior. The N:C ratio is often high, but nuclei are fairly uniform, and nucleoli when present are indistinct. Even so, it is a malignant apocrine gland tumor. The reported metastatic rate varies between studies from 36 to 96%; in most studies it is greater than 50%. This tumor is locally invasive with frequent metastasis to region lymph nodes (sublumbar/iliac). Metastasis to other internal organs and the lungs occurs less commonly.

Hepatocellular carcinomas

These tumors usually occur in older dogs and cats (>10 years) and grossly presents in three forms, a massive, a nodular and a diffuse form. The nodular form (multiple nodules in several lobes) and the diffuse form (large areas of the liver infiltrated by nonencapsulated tumor) are the most aggressive with metastasis in 100% and 93% of the reported cases respectively. The massive form presents as a large, single nodule usually affecting one lobe of the liver. Metastasis occurred in about 36% of the cases at the time of diagnosis. The massive form is the form most commonly observed in the dog.

Cytologic features: Aspirates from these tumors will contain clusters of cells with variable amounts of moderately basophilic cytoplasm which sometimes contains clear cytoplasmic vacuolation. Nuclei are typically round to oval and contain a characteristic clumped chromatin pattern and prominent, often singular nucleoli. In the nodular and diffuse forms of the disease, significant criteria for malignancy are often observed and a cytologic diagnosis of carcinoma can usually be made (Top). However, in the dog, some of the massive forms of the tumor will have minimal criteria for malignancy and require histological classification as well-differentiated carcinomas (Bottom). It is cytologically impossible to distinguish a well-differentiated carcinoma from a hepatic adenoma. However, the metastatic potential for well-differentiated carcinomas is low.

Round cell neoplasms

General Features: Round cell tumors as a group share the common cytologic characteristics of being composed of individually arranged, round cells which usually exfoliate in moderate to large numbers. The cells contain discrete cytoplasmic borders but are not very adherent to each other and contain no cell junctions between cells. There is discrepancy in the literature concerning the number of round cell tumors. Some cytologists consider only 5 types of round cell neoplasms, many however, include melanomas in this group, giving a total of 6.

Round cell Tumors and the Criteria for Malignancy: Unlike with most epithelial and mesenchymal tumors, the cytologic criteria of malignancy are not reliable indicators of the biological behavior of round cell tumors. Fortunately, tumors from this group can usually be definitively identified cytologically. The biological behavior can then be considered based on the tumor type, the location, and sometimes the cellular differentiation. The 6 specific round cell tumors will be discussed below, however, other detailed cytologic description of these tumors can be found in the sections indicated.

Mast cell tumor

These tumors are all considered potentially malignant and the saying goes "NEVER TRUST A MAST CELL TUMOR". The potential for metastasis and invasiveness is evaluated using 2 criteria, the cytologic differentiation of the tumor cells, and the anatomic location of the tumor.

Cytologic classification of mast cell tumors

          1. Well differentiated - cytoplasmic granules are large and plentiful and often occlude visualization of the nucleus in intact cells. Nuclei are uniform in size and round to oval in shape (Top).

          2. Moderately differentiated - cytoplasmic granules are in low to moderate in number but usually do not occlude visualization of the nucleus. Anisokaryosis is moderate and there is a variable N:C ratio. Mitotic figures may be seen in low numbers (Center).

          3. Poorly differentiated - cytoplasmic granules are sparse to absent and small. Anisokaryosis and variable N:C ratio is frequently observed. Nuclei are irregular in shape, and mitotic figures are seen in moderate numbers. Eosinophilic inflammation may be the key to recognition of an undifferentiated mast cell tumor (Bottom).

The less differentiated the cells are, the greater the potential for invasion and metastasis, regardless of the anatomic location.

Location of cutaneous mast cell tumors

In the dog, cutaneous mast cell tumors that are located in the perineal or inguinal regions frequently metastasize, usually to popliteal or abdominal lymph nodes. In addition, mast cell tumors located around the muzzle, in the oral or nasal cavity, or at mucocutaneous junctions frequently metastasize to regional lymph nodes. In these locations the biological behavior of the tumor cannot be accurately predicted by the cytologic differentiation of the mast cells. Even well differentiated tumors can and often do metastasize.

Melanoma

As mentioned, this neoplasm is often included in the group of round cell tumors. It is similar in some respects to the mast cell tumor in that the differentiated cells are composed of many cytoplasmic granules, and the biological behavior of the lesion depends heavily of the anatomic location in addition to the cellular differentiation. Melanomas located in the oral cavity or on the digits are considered malignant regardless of the cellular differentiation. Melanocytes contain dark green to black cytoplasmic granules whereas mast cell granules are purple. In addition, melanomas do not become infiltrated with eosinophils.

IV. Neuroendocrine neoplasms

Knowledge of the malignant potential for each tumor type is important because the cytologic criteria for malignancy are not easily interpreted with these neoplasms. If criteria of malignancy are present, the tumors are likely to metastasize or locally invade surrounding structures. However, nuclei from these tumors often do not display sufficient criteria of malignancy, even when the potential for metastasis or invasion is likely. The most frequently encountered neuroendocrine neoplasm is the thyroid tumor.

Canine thyroid tumors

These tumors are easily identified by the location along with a neuroendocrine appearance plus the presence of colloid (pink amorphous material) and/or tyrosine granules (dark cytoplasmic pigment). Most thyroid tumors will be composed of a fairly uniform population of cells. They will display few if any criteria of malignancy. However, in the dog, > 85% of the lesions are adenocarcinomas.

Feline thyroid tumors

Thyroid tumors in the cat are cytologically identical to those seen in the dog. However, the vast majority of tumors in the cat are benign adenomas or adenomatous hyperplasia.