The term cellular atypia is used to describe the morphological features expressed by a cell undergoing malignant neoplastic transformation. These features, alone or in combination, form the morphological basis that enables a cytopathologist to distinguish a malignant neoplastic process from phenomena of retroplasia, metaplasia and dysplasia. The morphological analysis can be divided into consideration of the features of cytoplasmic atypia, nuclear atypia, atypia of the arrangement of cells andatypia of extracellular material. However, when conducting a morphological evaluation, it is essential to accept that the none of these features of atypia may be sufficient to determine the malignant nature of a cell and that it is often indispensable to have signalment information, the history and clinical details on the pathological process under investigation in order to be able to express an opinion on the existence of malignant neoplastic transformation.
FEATURES OF ATYPIA RELATIVE TO CELL ARRANGEMENT
Number of cells (Fig. 1). The number of exfoliated cells depends on the type of tissue. Epithelial parenchymal organs, such as the liver, pancreas and prostate, usually shed a large number of cells, while mesenchymal organs, such as muscles, connective tissue and bone, exfoliate a very small number of cells or none at all. It is, therefore, obvious that the presence of a high number of cells in samples from lesions of these latter organs is an indication not only of cellular proliferation, but also of the loss of cohesiveness in the tissue, in other words, features of malignant neoplastic transformation.
Disorganized architecture (Fig. 2). The geometric orderliness of the organization of individual cells indicates respect for tissue order and, therefore, benignity; examples of geometric orderliness are the linear palisades of ciliated cells of respiratory epithelium and the honeycomb arrangement of prostate cells. Cells from malignant neoplastic tissues are, in contrast, found in disorganized aggregates, in which the cells are crowded on each other without a clear morphological pattern.
Disaggregation (Fig. 3). A frequent finding in the context of malignant neoplasia is ‘discohesiveness’ or loss of adhesion between cells. This loss of adhesion leads to the presence of irregular strips of cells, from which groups of a few cells or even single cells can detach. This phenomenon is mediated by the loss of intercytoplasmic mechanisms of adhesion, such as gap junctions or desmosomes.
Cannibalism (Fig. 4). Sometimes two adjacent neoplastic cells can show such plasticity that one is completely surrounded by another, in appearance resembling phagocytosis between cells of the same type, so that the phenomenon is named ‘cannibalism’.
Presence of cells not compatible with the tissue biopsied (Fig. 5). This feature of atypia refers to the presence of elements in a tissue which no inflammatory, regressive, proplastic, dysplastic or metaplastic process could justify; in particular it refers to the presence of epithelial cells in a lymph node. The presence of such cells in this site, regardless of the morphological features of the cells themselves, is the demonstration of ongoing metastatic invasion.
FEATURES OF NUCLEAR ATYPIA
Nuclear enlargement (Fig. 6). The nuclei of malignant cells tend to be larger than those of normal cells of the same lineage. This feature becomes significant only if it is possible to compare, contemporaneously, neoplastic cells and normal cells from areas of tissue not involved by the neoplastic process.
Increased nucleus/cytoplasm (N/C) ratio (Fig. 7). This criterion of atypia is significant only if the ratio between the diameters of the nucleus and cytoplasm of normal cells is known. In fact there are perfectly normal cells, such as the keratinocytes of the basal layers of the epidermis and mature lymphocytes, which have very little cytoplasm and are almost entirely filled by a nucleus. In other cases, such as keratinising cells, the nucleus disappears or is reduced to a small, pyknotic mass. During neoplastic transformation, as exemplified in squamous cell carcinoma, the keratinising cytoplasm contains a sometimes bulky nucleus, whose size contributes to producing an abnormal N/C ratio.
Pleomorphic shape and size (Fig. 8). The nuclei of normal cells, with the exception of those of polymorphonuclear granulocytes and of megakaryocytes, have a roundish or ovoid contour. Neoplastic transformation causes irregularity of the contour, which can become indented (small indentures of the contour), cleaved (single deep indentures of the contour), or lobed (emergence of irregular protuberances of the contour).
Multiple nuclei (Fig. 9). Only in exceptional cases do normal cells have multiple nuclei; the exceptions are osteoclasts, which typically have multiple nuclei of regular shape and size, concentrated at one pole of the cytoplasm. Multiple nuclei are present during non-neoplastic phenomena, such as the transformation of macrophages during pyogranulomatous inflammation, when the coalescence of several cells leads to the development of so-called foreign body giant cells. Reactive hyperplasia, a common defence response of cells to various inflammatory stimuli, can also cause the presence of multiple nuclei: in all these cases, however, the nuclei have a regular shape and size, while the multiple nuclei found in cells undergoing malignant neoplastic transformation can be of different sizes and with irregular contours. An excellent example of this is represented by the multinucleated cells present in the exfoliate of histiocytic sarcoma.
Naked nuclei (Fig. 10). If the procedures for mounting a cell preparation are carried out well, the integrity of cells not affected by malignant neoplastic transformation is generally maintained. The presence of naked nuclei is a manifestation of cytoplasmic fragility which can be a characteristic of some neoplasms; the integral naked nuclei are exposed and dispersed in the background, sometimes surrounding intact cells. This morphological finding can be of diagnostic significance in particular during the evaluation of well-differentiated hepatocarcinomas and some neuroendocrine tumours.
Moulding (Fig. 11). This morphological feature of atypia is represented by deformation of one nucleus by a directly adjacent nucleus resting on it. This feature is an indicator of the loss of contact inhibition that occurs in malignant transformation.
Crowding (Fig. 12). This is another consequence of a loss of contact inhibition, which leads to cells amassing in three-dimensional layers, with resulting pseudostratification and apparent stacking of the nuclei.
Thickened membrane outline (Fig. 13). The nuclear membrane is not usually visible with normal cytological techniques and can be seen only by electron microscopy; ultrastructural examination of the nucleus shows a thin line of chromatin that adheres intimately to the membrane; in reality, staining techniques can be used to visualise this chromatin and, indirectly, the nuclear membrane. In malignant cells, there is an increase in the chromatin that attaches to the membrane, sometimes with such an irregular and coarse distribution as to render evident the thickened membrane.
Hyperchromasia (Fig. 14). As a result of malignant transformation, the density of the nuclear DNA increases and, consequently, the intensity of the nuclear colouring. This feature is very subjective and often depends on the method and duration of staining; it is, therefore, advisable to use it only when it is possible to compare preparations from the suspected neoplastic areas with those from presumed normal areas, stained at the same time with the same technique.
Irregular chromatin (Fig. 15). The so-called “euchromatin”, the morphological expression of thin, regular filaments of chromatin which are microscopically visible as finely granular or lightly packed nuclear elements, is replaced, during malignant neoplastic transformation, by chromatin which is irregularly distributed, sometimes in clumps or twisted filaments.
Prominent multiple nucleoli (Fig. 16). The nucleus usually contains a single, small nucleolus; in malignant cells the nucleoli become prominent, acidophilic and, often, multiple.
Mitotic figures (Fig. 17). It is always difficult to determine whether mitosis is typical or atypical from cytological studies; in general a typical mitotic figure is symmetrical, albeit irregular, whereas an atypical mitosis is irregular and asymmetrical. It should be emphasized that a large number of mitoses can be seen in some normal tissues, such as lymph nodes and testicles, without this representing a criterion for diagnosing atypia. Generally speaking it is better not to calculate the so-called “mitotic index” on the basis of cytological observations, because this parameter expresses the number of mitoses in tissue sections in a microscope field at a high magnification (400X); the unit of measure of the mitotic index is number of mitoses/high power field (hpf).
FEATURES OF CYTOPLASMIC ATYPIA
Anisocytosis (Fig. 18). While normal cells of the same type have roughly the same shape and size, atypical cells have the same general appearance, but their shape and size differ.
Abnormal chromatism (hyperchromatism) (Fig. 19). The DNA of neoplastic cells undergoes changes that lead to activation of abnormal metabolic processes, including increased production of RNA and, consequently, of proteins, resulting in more intense colouration of the cytoplasm.
Abnormal cytoplasmic products (Figs. 20 and 21). In the same way as described above, a cell’s productive capacity can be altered following changes to the DNA, which regulates cytoplasmic metabolic activity. Secretory cells, such as the cells of the gastric mucosa, can undergo changes that lead to the formation of a large vacuole that displaces the nucleus to the periphery of the cell, giving this latter a characteristic appearance summarised by the term “signet-ring shaped cell”. Neoplastic mast cells, which typically have compact metachromatic granulation, can express fewer cytoplasmic granules or granules that are less clearly visible with standard stains as the lack of morphological differentiation increases. Neoplastic plasma cells accumulate cytoplasmic immunoglobulins in an abnormal manner, giving rise to a dense peripheral eosinophil chromatism and the name “flame cells”. Neoplastic melanocytes can also undergo alterations induced by malignant transformation: in fact, morphological transformation of cytoplasmic melanosomes is common, with these normally rod-shaped organelles taking on a finely granular appearance or sometimes even decreasing to the point of being no longer detectable, as in the forms of so-called amelanotic melanoma.
BACKGROUND OF THE PREPARATION
Necrosis (Fig. 22). The background of cytological preparations of benign neoplastic processes is usually clear, lacking debris and acellular material; however, it may show irregular basophilic chromatism, caused by the presence of dispersed, amorphous, proteinaceous material or necrotic debris derived from the processes of cell death mediated by impaired vascularisation.
Suggested readings
- McGavin MD, Zachary JF. Pathologic Basis of Veterinary Diseases. IV edition. St. Louis, Missouri, Mosby Elsevier, 2007.
- Meuten DJ. Tumors in Domestic Animals. Iowa State Press, 2002
- Raskin RE, Meyer DJ. Canine and Feline Cytology, a Color Atlas and Interpretation Guide. St Louis, Missouri, Saunders, 2001.