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Histopathology of the Thymus

AstraZeneca, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK

Correspondence: Address correspondence to: Gail Pearse, AstraZeneca, 23F22A, Mere-side, Alderley Park, Macclesfield, Cheshire, SK10 4TG, UK; e-mail:Gail.Pearse{at}astrazeneca.com

	Abstract

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The thymus is a primary lymphoid organ that manifests dynamic physiological changes as animals age in addition to being exquisitely sensitive to stress and toxic insult. It is typically the first lymphoid tissue to respond to immunotoxic xenobiotics, with the first change being loss of cortical lymphocytes by apoptosis. This is followed by removal of the apoptotic cellular debris and, in the absence of recovery, may lead to loss of the corticomedullary demarcation and organ atrophy. Nonneoplastic proliferative changes include focal lymphoid hyperplasia and proliferation of medullary epithelial cells, often with formation of ribbons, cords, or tubules. Thymomas are relatively rare tumors that exhibit a wide spectrum of morphologic types but do not metastasize. Thymic lymphomas are common in some mouse strains and can become leukemic with hematogenous spread throughout the body.

Key Words: Epithelial hyperplasia • apoptosis • thymoma • lymphoma • atrophy

	Introduction

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The immune status of the thymus, as reflected in the histological appearance, and/or changes in the relative organ weight, varies according to factors such as the age and genetic background, the adequacy of nutrition, the stress levels, and hormonal status of the animal, in addition to, and potentially interfering with, the interpretation of exposure to xenobiotics. According to Kuper: "The primary limitation of histopathology is that a tissue section represents a static time point in a dynamic process. Therefore, the dynamics of the immune system should be carefully considered in the histopathologic assessment of immunotoxicity" (Kuper et al., 2002). In addition to the thymus not being a static histologic entity, the interchangeable use of interpretive and descriptive terms has led to overlapping terminology for the same morphological change, potentially causing confusion and poor communication. For example, when there is a decrease in thymic size and cellularity, it is always appropriate to use descriptive terminology such as "reduced numbers of cortical lymphocytes" and "increased numbers of tingible body macrophages." However, similar or even identical changes may be diagnosed as "atrophy" or "involution," especially in the evaluation of tissues from older animals in chronic studies. Normal development, histology, and function of the thymus have been reported previously (Kuper et al., 1992; Pearse, 2006). The use of standardized descriptive nomenclature with respect to thymic pathology is addressed elsewhere (Haley et al., 2005; Elmore, 2006). This paper focuses on morphological features of nonproliferative and proliferative lesions of the rodent thymus.

	Nonproliferative Morphological Changes in the Thymus

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Factors Effecting Thymic Cellularity
A variety of factors and conditions result in an alteration in the cellular density and cellular composition of the thymus. Most commonly recognized is a decrease in lymphyocytes resulting from a range of background physiological influences and the immunosuppressive effect of xenobiotics.

Normal age-associated decreases in cellularity are termed involution, whereas induced reductions such as from inadequate nutrition, stress, or toxicity represent thymic atrophy. The histological appearance of the thymus under these varied conditions is similar, since the end point is the reduction in cortical lymphocytes, and shrinkage of the thymic lobules (Schuurman et al., 1994). Increased numbers of apoptotic bodies and tingible body macrophages are followed by lymphocyte depletion and increased prominence of interlobular septae and eventually an inverse of the normal histological picture, where the medulla has a higher cellular density than the cortex. Factors such as stress and toxicity can simultaneously be superimposed on the normal ageing process of lymphocyte reduction. Consequently, the distinction between atrophy and involution in older animals can be problematic. In such situations, the best course of action is to (1) assess changes in cell density and compartment size, (2) take into consideration comparison with concurrent untreated controls, (3) factor in the temporal sequence of events, (4) dose-response findings, (5) and the totality of clinical findings including histological changes in other tissue and from there deduce the appropriateness of interpreting the change atrophy versus involution.

Age Associated Effects/Involution
The age of the animal plays a large part in the level of cellularity of the thymus and its overall histological appearance. Physiological involution reflects the change in function of the thymus from lymphocyte production to recirculation. In the intact rodent, involution is a normal, gradual, and, irreversible aging change that begins at puberty and is considered to be associated with increased circulating levels of sex steroids. Gonadectomy will delay involution in both sexes (Grossman, 1985). Greenstein et al. (1987) showed that orchidectomy restored the thymus and raised the total white cell count in 18-month-old rats in which the thymus had virtually disappeared. Similar thymic regeneration was achieved in intact old rats with subcutaneous implants of lutenizing hormone releasing hormone (LHRH).

Histologically, involution is characterized by a reduction in the size of the thymus with a decrease in cortical lymphocytes, thinning and irregularity of the cortex, and loss of corticomedullary demarcation (Figures 1 and 2). At the corticomedullary junction, there is an increase in perivascular spaces, and perivascular B lymphocyte and plasma cell populations, which may form lymphofollicular structures with prominent germinal centers. There is infiltration by adipose tissue in the connective tissue capsule and septae (Figure 3). In the medulla, epithelial cells become progressively more prominent. The microscopic changes in epithelial cells that accompany involution can show considerable pleomorphic variation in distribution, architectural arrangement, and cytological appearance. Undoubtedly, there is some degree of cell proliferation as the epithelial cells are sometimes arranged in cords or ribbons and may form tubules or cysts lined by cuboidal to squamous epithelium. Epithelial changes are more prominent in females than in males and in rats (Figure 3A) more so than mice.

View larger version (577K): [in this window] [in a new window]   Figure 1 A thymus from a normal 31-week old Sprague–Dawley rat (Figure 1A and 1C) shows a distinct dark-staining cortex surrounding and enclosing the paler staining medulla. In contrast, physiological thymic involution (Figure 1B and 1D) from a 2-year-old untreated Sprague–Dawley rat shows a markedly smaller thymus with a few patchy areas of dark-staining cortex and increase in adipocytes in capsular and septal areas. Extensive tubule formation by epithelial cells is prominent in the medulla (Figure 1D).

View larger version (570K): [in this window] [in a new window]   Figure 2 The thymus from a 7-week-old untreated B6C3F1 mouse (Figure 2 A and 2C) had a distinct cortex enclosing the paler staining medulla. While the thymus in this example of physiological involution from an untreated 2-year-old B6C3F1 mouse (Figure 2B and 2D) is smaller than that of the younger mouse, the distinction between cortex and medulla is still apparent, although the cortical thickness is reduced. There are patchy areas throughout the cortex where cortical lymphocyte density is reduced. There is also a slight increase of small lymphocytes in the medulla (Figure 2D).

View larger version (330K): [in this window] [in a new window]   Figure 3 The involution varies between species, strain, and sex (Kuper et al., 1992). An example of physiological involution in a rat (Figure 3A) shows reduction in cortical thickness, patchy area of cortex without darkly staining small lymphocytes, and some epithelial tubule formation (arrow) in the medulla. Increased numbers of adipocytes are present in the capsule and extending into the lobular septa and also into the cortex. A similar pattern of physiological involution is present in this aged dog (Figure 3B). Adipocytes populate in widened interlobular septa. Photomicrographs courtesy of Dr. Michael Leach.

View larger version (164K): [in this window] [in a new window]   Figure 4 Physiological involution in this 2-year-old male B6C3F1 mouse shows extensive reduction in the cortex and the presence of a cyst filled with homogeneous pale staining protein. Although one cannot rule out the possibility that the cyst was present from birth (see Pearse, 2006), increased cyst formation accompanies physiological involution in some mouse strains.

Stress
Stress causes elevated circulating levels of glucocorticosteroids mediated by the hypothalamus-pituitary-adrenal axis. The thymus is the most sensitive of the lymphoid tissues to changes in adrenocortical hormone levels and a decrease in thymic weight occurs due to loss of cortical lymphocytes. The splenic white pulp and lymph nodes can be similarly affected, although to a lesser degree. The initial response of apoptosis of cortical lymphocytes can be seen within hours of treatment with the synthetic glucocorticoid dexamethasone, followed by removal of apoptotic debris by macrophages (Figure 5). Such changes due to acute debilitating disease are common and may be prominent in animals sacrificed in a moribund condition. Known environmental stressors are social ranking within gang-housing systems, immobilization, as well as excessive changes in temperature or humidity and restriction of access to food and water (Gamallo et al., 1986; Kioukia-Fougia et al., 2002; Dal-Zotto et al., 2003; Engler and Stefanski, 2003). These changes are usually reversible on removal of the stressor.

View larger version (578K): [in this window] [in a new window]   Figure 5 Stress associated thymic changes begin with apoptosis of cortical lymphocytes. Depending upon the degree of stress, the loss of thymic lymphocytes is generally reversible if the stress is removed. If the stress is removed, apoptosisceases and macrophages remove the apoptotic debris. A Sprague–Dawley rat was treated with dexamethasone to mimic corticosteroid induced thymic lesions. Collections of hyperchromic and slightly shrunken lymphocytes undergoing apoptosis can be seen distributed throughout the cortex 6 hours after treatment (Figure 5A, 5B, and 5C). The apoptotic lymphocytes are just beginning to be phagocytized by tingible body macrophages (Figure 5C, arrows).

Immunotoxicity
Immunotoxicity refers to the potentially harmful effects that physical, chemical, or other agents have on the immune system (Koller, 1987). The dynamics of the immune system, with its ongoing cellular proliferation and differentiation, lymphocyte trafficking, and gene amplification, make it highly susceptible to toxic insults particularly in the thymus and bone marrow where rapid cell turnover occurs. Since generation of T cells by the thymus is particularly important early in life, immunotoxicants may show their effects in particular, and in lower doses, during the prenatal and the early postnatal period (Schuurman et al., 1994).

The consequences of immune dysfunction are immunosuppression or immunoenhancement, and either type of immunomodulation can provoke hypersensitivity or autoimmunity. Immunotoxic reactions manifest themselves most commonly as immunosuppression (Gopinath, 1996) characterized by selective or generalized depression of the lymphoid organs. However, there may be dramatic species differences in response to given immunotoxicants (Haley, 2003).

The most sensitive indicator of immunosuppression, particularly in short-term studies, is a decrease in relative thymus weight, which may or may not have a corresponding detectible morphological finding of decreased cellularity in the cortex, and less commonly the medulla or both. However, high doses of an immunotoxicant can cause overt toxicity or a decrease in food and/or water consumption and severe stress resulting in nonspecific (secondary) inhibitory effects on the immune system. A decrease in thymus:body weight ratio, therefore, cannot be used as stand-alone criterion of immunosuppression. In some cases a dose-response relationship as well as changes in other lymphoid tissues may be of some help in deciding whether thymic atrophy is a direct effect of immunosupression or nonspecific response to stress (Greaves, 2000).

The thymus is especially sensitive to exposure to immune system toxicants, and often there is a clear dose-associated decrease in size of the thymus secondary to apoptosis of cortical lymphocytes. The histological changes present are dependent upon the dose of the immunotoxicant and when, in the dynamic process, the thymus is examined (Figures 6–9). Following apoptosis of cortical lymphocytes and their removal by macrophages, a decrease in cortical cellularity, characterized by thinning and loss of the cortex and blurring of normal corticomedullary demarcation are seen. At a sufficiently high dose of an immunotoxicant, there may be degeneration of epithelial cells (Figure 7) and epithelial cell proliferation with development of glandular structures containing eosinophilic material (Figure 10).

View larger version (431K): [in this window] [in a new window]   Figure 6 Dioxin is a known immunotoxic agent and when given to rats it leads to loss of thymic cortical lymphocytes. After 31 weeks of treatment with a low dose of dioxin, this female Sprague–Dawley rat showed early cortical atrophy characterized by loss of cortical lymphocytes, especially in the middle to inner cortex (Figure 6B). In contrast, the age-matched control (Figure 6A) shows a distinct cortex with a uniformly dense population of lymphocytes. While a conventional diagnosis of atrophy was given for the thymic change, this type of lesion might more appropriately be characterized using the enhanced histopathology descriptions (Haley et al., 2005; Elmore, 2006).

View larger version (210K): [in this window] [in a new window]   Figure 9 The thymus from a male B6C3F1 mouse treated with cyclophosphamide in a 28-day study is reduced in size with a blurring of the corticomedullary demarcation (Figure 9A and 9B). There is a minimal to mild decrease in lymphocytes in the cortex and a moderate increase in small lymphocytes in the medulla. Cyclophosphamide is frequently used as a positive immunotoxic control in functional immunotoxicity studies. These thymic changes plus changes in other lymphoid tissues and a complete blood cell count should provide a good snapshot of the immunomodulatory effects present at the 28-day interval.

View larger version (1059K): [in this window] [in a new window]   Figure 7 The thymus from a male F344 rat collected after 4 daily doses of cyclophosphamide (Figure 7B–7F) shows dramatic changes in cellular density compared to the concurrent control (Figure 7A). By the fourth day, there is almost complete loss of cortical lymphocytes and degenerative changes in the remaining epithelial and stromal cells are characterized by cytoplasmic vacuolization. In contrast to the control (Figure 7A), there is an increase in small lymphocytes in the medulla. This type of immunotoxic change would most appropriately be described using the enhanced histopathology guidelines (Haley et al., 2005; Elmore, 2006). Such descriptive terminology would provide a more appropriate and complete characterization of the change in contrast to using a diagnostic term such as cortical atrophy.

View larger version (281K): [in this window] [in a new window]   Figure 10 Severe thymic atrophy was produced in this female Sprague-Dawley rat treated with dioxin for 53 weeks (Figure 10A). There is loss of cortical lymphocytes and an increase in adipocytes in the superficial cortex in most severely affected areas. The proliferating epithelial cells in the medulla have formed prominent glandular structures containing eosinophilic material (Figure 10B).

For interpretation of immunopathology, it is important to remember that the thymus is not a fixed histological entity. The level of cellularity will vary according to a number of background factors. Not all effects on the thymus seen in regulatory studies are due to the immunomodulatory effects of the test material and the use of age-matched control animals is critical. To assist with the evaluation of thymic weight data, it is recommended that an historical database should be developed and maintained for each species and strain used and should include the age, weight and sex of the animal from which the data are collected (Haley, 2003).

For a more in-depth discussion of immunotoxicology, the following references should be consulted: De Waal et al. (1997); Kuper et al. (1995); Schuurman et al. (1991); Luster et al. (1988); Koller (1987); Dean and Thurmond (1987); and Vos et al. (1998).

Other Nonproliferative Lesions of the Thymus
Other non-proliferative lesions are uncommon. Occasional developmental anomalies resulting in ectopic thyroid, parathyroid, or thymic tissue as well as intrathyroid cysts have been reported (Pearse, 2006). Inflammatory and vascular lesions are rare in the rodent thymus. Primary inflammation by extension from adjacent tissues is always possible. Multifocal dystrophic mineralization (Figure 11A) secondary to renal or parathyroid disease may be observed. Hemorrhage (Figure 11B and 11C) and fibrosis (Figure 11D) have been seen occasionally with the former, possibly a consequence of esophageal perforation during gavage administration of a test agent.

View larger version (805K): [in this window] [in a new window]   Figure 11 Examples of miscellaneous lesions in the thymus include multifocal mineralization in an aged mouse (Figure 11A) possibly associated with secondary hyperparathyroid effects from chronic renal disease, localized (Figure 11B) and diffuse (Figure 11C) hemorrhage in Sprague–Dawley rats, and focal fibrosis (Figure 11D) in a mouse. The etiology of the hemorrhagic and fibrotic lesions is unknown in these cases but could be sequela of esophageal rupture during gavage administration of test agents or vehicles. Photomicrographs courtesy of Dr. Michael Leach.

	Proliferative Morphological Changes in the Thymus

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View larger version (361K): [in this window] [in a new window]   Figure 12 Focal epithelial hyperplasia is present at the septum separating thymic lobules in this control female F344 rat in a 3-month toxicity study (Figure 12A and 12B). No other areas of epithelial hyperplasia were noted in the thymus from this rat nor were any evident in any of the other controls in this study. The change was graded as minimal severity and is considered a sporadic lesion.

View larger version (896K): [in this window] [in a new window]   Figure 13 There is marked thymic atrophy in this female Sprague–Dawley rat treated with dioxin for 31 weeks (Figure 13A–13C). There is moderate epithelial hyperplasia with tubule formation (Figure 13B and 13C). Tubules contain eosinophilic homogeneous and granular proteinaceous material. There is also marked to severe loss of cortical lymphocytes, minimal to mild increase in lymphocytes in the medulla, and loss of the corticomedullary demarcation.

View larger version (436K): [in this window] [in a new window]   Figure 14 This unusual example of epithelial hyperplasia from the National Toxicology Program data base was diagnosed as focal oncocytic hyperplasia (Figure 14A–14C). The thymus is from a 2-year-old untreated male B6C3F1 mouse. A small cluster of eosinophilic cells with round leptochromatic nuclei and copious granular cytoplasm (Figure 14C) is morphologically similar to proliferative oncocytes seen at other locations in rodents. The patchy loss of cortical lymphocytes evident in the upper left of Figure 14A is reflective of the physiological involution underway in this mouse.

View larger version (1101K): [in this window] [in a new window]   Figure 15 This example of medullary hyperplasia (Figure 15A and 15B) from a control male F344 rat at the end of a 2-year cancer study shows a patchy area of epithelial hyperplasia in the medulla (Figure 15A, arrows). The proliferating epithelial cells have formed irregular tubules and cords which are surrounding small numbers of lymphocytes in this higher magnification of Figure 15A (Figure 15B). In this thymus there is physiological involution with decrease in cortical thickness, reduction in the number of cortical lymphocytes, increase in medullary lymphocytes, and loss of a sharply demarcated corticomedullary boundary. Increased numbers of adipocytes are present at the edge of a septum (left side of Figure 15A).

View larger version (1161K): [in this window] [in a new window]   Figure 16 These examples of lymphoid hyperplasia are from mice (Figure 16A–16F). In Figure 16A and 16B, there is a circumscribed nodule of hyperplastic small and large lymphocytes in the medulla. The thymus in this case is showing decreased thickness of the cortex with patchy loss of lymphocytes. In a similar example with more advanced physiological involution, a larger patch of focally proliferating lymphocytes extends from the medulla to the cortex (Figure 16C and 16D). A third example of localized lymphoid hyperplasia is extending from the medulla into the adjacent cortex in this 12-week-old mouse (Figure 16E and 16F) and is comprised of small lymphocytes. Figure 16A and 16B courtesy of Dr. Michael Leach. Figure 16C and 16D courtesy of Drs. Charles Frith and Jerrold Ward.

Neoplasia
Thymoma
This neoplasm of thymic epithelial cells is characterized by a variable admixed population of lymphoid cells (Rosai and Levine, 1976). Neoplastic epithelial cells may be localized or dispersed among lymphoid cells (Figures 17 and 18). It is likely that the gradation between epithelial hyperplasia and thymoma may present diagnostic challenges, especially for the more diffuse proliferative lesions. While it has been reported that thymomas in rodents can be divided into 3 main histological groups, depending on the relative preponderance of epithelial and lymphoid cells (Greaves, 2000), the many morphological variations seem to defy predetermined categories.

View larger version (651K): [in this window] [in a new window]   Figure 17 The thymoma in this figure is from a female B6C3F1 mouse treated with tricresyl phosphate in a 2-year cancer study and is characterized by a sharply defined proliferation of epithelial cells (Figure 17A and 17B). This neoplasm stands out as an expansive growth in an otherwise involuted thymus and has remained within the thymic capsule. A sharply demarcated thymoma with an irregular border is seen in this untreated female F344 rat from a 2-year cancer study (Figure 17C and 17D). The sheets of epithelial cells impinge upon and have begun to interdigitate with the cortex. A focal nodule of lymphoid hyperplasia is also present (Figure 17C, arrow).

View larger version (695K): [in this window] [in a new window]   Figure 18 Some thymomas grow with generalized involvement of the affected thymic lobule, with variable proportions of lymphocytes, as in this example from a male B6C3F1 mouse treated with a high dose of 1-chloro-2-propanol for 2 years (Figure 18A and 18B). Irregular islands and nests of small lymphocytes are surrounded by bands of somewhat sarcomatous appearing epithelial cells. Another example with generalized involvement of the thymus is seen in this vehicle control male B6C3F1 from a 2-year cancer study (Figure 18C and 18D). Thymoma cells with large nuclei (Figure 18D) are intermixed within a population of small and large lymphocytes.

View larger version (689K): [in this window] [in a new window]   Figure 19 Thymomas with epidermoid features represent one of the cellular forms seen in rodents. A malignant thymoma from an untreated female F344 rat in a 2-year cancer study (Figure 19A–19C) is characterized by a proliferation of a pleomorphic population of epithelial cells forming nests and sheets surrounding patches and nests of small lymphocytes. The epithelial cells have irregularly shaped nuclei with prominent nucleoli (Figure 19C). This thymoma was considered malignant base upon invasion of adjacent adipose tissue (Figure 19A, arrow). Epidermoid features of another malignant thymoma from a female F344 rat administered a low dose of diphenylhydramine HCl for 2 years are evident in Figure 19D. Thymomas typically do not metastasize and malignancy is based upon extension beyond the capsular surface.

View larger version (583K): [in this window] [in a new window]   Figure 20 True thymic carcinomas with squamous differentiation are occasionally seen as in this example from an untreated male F344 rat from a 2-year cancer study (Figure 20A–20C). Ribbons, bands, and nests of squamous epithelial cells with minimal keratinization (Figure 20C, arrow) are present in this example. It was originally diagnosed as thymoma, squamous cell type, but a diagnosis of thymic carcinoma or thymic squamous cell carcinoma would also be acceptable if the existence of a primary carcinoma elsewhere in the body was ruled out.

View larger version (828K): [in this window] [in a new window]   Figure 21 This papillary and cystic malignant thymoma is from a male F344 rat exposed to ozone for 2 years (Figure 21A–21C). Malignancy is based on expansion beyond the thymic capsule (Figure 21A, arrows). In addition to the papillary growth pattern and cystic cavities filled with homogeneous eosinophilic proteinaceous fluid, there is a glandular formation partially lined by columnar epithelial cells and filled with degenerating exfoliated cells (Figure 21C, arrow heads), and areas with squamous differentiation of neoplastic epithelial cells (Figure 21C, arrows) are also present.

View larger version (682K): [in this window] [in a new window]   Figure 22 Thymomas consisting of ribbon and tubular growth patterns represent one of the tumor types. Ribbons of proliferating epithelial cells separated by more mesenchymal appearing stromal cells are seen in this thymoma from a female F344 rat treated with 2, 6-toluenediamine for 2 years (Figure 22A and 22B). Although not evident at these magnifications, mast cells were prominent in the stromal cells between ribbons of epithelium. Both ribbon and tubule formations are present in a thymoma from a female F344 rat treated with codeine for 2 years (Figure 22C). In the original description of this thymoma, the growth pattern was labeled as trabecular with tubule formation. An irregular tubular pattern of a thymoma is present in this example from a female F344 rat exposed to a high dose of Stoddard solvent for 2 years (Figure 22D).

View larger version (683K): [in this window] [in a new window]   Figure 23 Mesenchymal or spindeloid forms of thymoma have been seen in rodents. A benign thymoma with a sarcomatous growth pattern consisting of spindeloid cells with abundant pale staining eosinophilic cytoplasm occupied all but a few islands of residual lymphocytes and epithelial cells in a 2-year-old untreated female F344 rat (Figure 23A–23C). A different example of a sarcomatous growth pattern characterized by high nuclear density and scant cytoplasm is present in this thymoma from an untreated male F344 rat at the end of a 2-year cancer study (Figure 23D).

View larger version (197K): [in this window] [in a new window]   Figure 24 The thymoma from this low dose female F344 rat in a 2-year study has an endocrine growth pattern (Figure 24). Small nests of epithelial cells are separated by thin bands of connective tissue.

View larger version (691K): [in this window] [in a new window]   Figure 25 A thymic neuroendocrine tumor from a high dose female F344 rat treated with triethanolamine for 2 years has a neuroendocrine growth pattern consisting of packets of elongated epithelial cells (Figure 25A and 25B). This tumor was immunopositive for chromagranin A, with cytoplasmic staining consistent with neuroepithelial differentiation.

View larger version (1997K): [in this window] [in a new window]   Figure 26 Myoid thymomas occur in humans and have occasionally been seen in rodents. This thymoma with rhabdomyosarcomatoid features is from a vehicle control female F344 rat in a 2-year cancer study (Figure 26A–26D). Islands and short trabeculae of epithelial cells are surrounded by proliferation of undifferentiated mesenchymal cells with occasional areas where cytological features are characteristic of muscle fibers (Figure 26A and 26D, arrows). There is osteoid formation in one area of this thymoma (Figure 26C). The mesenchymal cells in this thymoma were immunopostive for desmin (Figure 26E and 26F) and cross-striations characteristic of skeletal muscle were present in occasional muscle fibers (Figure 26F, arrow).

View larger version (493K): [in this window] [in a new window]   Figure 27 Benign thymomas are typically confined within the lobular capsule. In this example from a vehicle control female F344 rat in a 2-year study, the thymoma occupies multiple lobules but is contained within the thickened capsule (Figure 27A–27C). The neoplastic epithelial cells are forming irregular trabeculae and, in some areas, have a papillary and cystic growth pattern (Figure 27A, arrow).

View larger version (644K): [in this window] [in a new window]   Figure 28 In this example of a malignant thymoma from an untreated female B6C3F1 2-year-old mouse, there is capsular invasion and multiple areas of necrosis (Figure 28A, arrows). The neoplastic epithelial cells are growing in irregular bands and trabeculae with occasional tubule formation (Figure 28B–28D). Infiltrating neutrophils are present in areas of necrosis (Figure 28C) and the malignant epithelial cells have squamoid features (Figure 28C and 28D).

View larger version (626K): [in this window] [in a new window]   Figure 29 This example of a malignant thymoma from a treated male F344 rat in a 2-year electromagnetic field study is comprised of sheets of epithelial cells that have extended beyond the thymus and invaded adjacent skeletal muscle. This degree of spread beyond the tissues immediately adjacent to the thymus is unusual.

Thymic Lymphoma
Neoplasms of the thymus of mice are most commonly T-cell lymphomas of thymic lymphocyte origin. T-cell lymphoma can occur spontaneously in young mice <3 months of age (Frith et al., 1985). In B6C3F1 mice spontaneously occurring thymic lymphomas are rare but can readily be induced by chemicals, viruses, irradiation, and in some types of tumor suppressor gene knockout mice. (Dunnick et al., 1997; Ward, 2006).

Most spontaneous and induced lymphoblastic lymphomas in the mouse arise unilaterally in the thymus (Frith et al., 1985). The initial gross lesion is a reduction in thymic size (thymic atrophy). Cortical atrophy is characterized by loss of cortical lymphocytes with thinning of the cortex. There is maintenance of normal lobular architecture. Subsequent to cortical atrophy, a preneoplastic stage of thymic lymphoma called atypical hyperplasia has been described in chemically treated B6C3F1 and p53-deficient mice (Dunnick et al., 1997). Atypical hyperplasia may be unilateral (Figure 30) or bilateral. There is a diffuse change with loss of the normal corticomedullary demarcation. Normal architecture is replaced by sheets of large, atypical lymphocytes and fewer admixed small lymphocytes (Figure 31). This preneoplastic lesion can be differentiated from lymphoma by the heterogenous cell population, variable mitotic index, and failure of lymphocytes to extend beyond the capsule of the thymus.

View larger version (479K): [in this window] [in a new window]   Figure 30 This example of atypical hyperplasia (Figure 30B and 30D) is from a heterozygous p53+/– female mouse treated with phenolphthalene for 13 weeks. A wild-type C57BL/6 control thymus is shown in Figure 30A and 30C). In the treated mouse there is replacement of normal cortical and medullary cells with sheets of large atypical lymphocytes along with occasional small lymphocytes and cells undergoing apoptosis (Figure 30D).

View larger version (684K): [in this window] [in a new window]   Figure 31 This example of unilateral atypical hyperplasia (Figure 31A–31D) in a phenolphthalene-treated heterozygous p53+/– mouse shows loss of a distinct cortex and medulla in the affected lobe with replacement by mostly large lymphocytes (Figure 31D). There is a focus of nodular lymphoid hyperplasia on the left side of Figure 31B and 31C.

View larger version (950K): [in this window] [in a new window]   Figure 32 Thymic proliferative change in this heterozygous female p53+/– treated with phenolphthalene in a prechronic study has progressed to lymphoma (Figure 32A–32C). The relatively uniform population of neoplastic lymphoblasts has invaded the capsule (Figure 32A) and adjacent adipose tissue (Figure 32B). Occasional tingible body macrophages have phagocytosized apoptotic lymphoblasts (Figure 32C, arrows).

View larger version (563K): [in this window] [in a new window]   Figure 34 A high magnification of a lymphoma in a heterozygous p53+/– mouse shows the morphological features of the malignant lymphoblasts. There are multiple mitoses (arrows) and occasional tingible body macrophages (arrow heads).

View larger version (1228K): [in this window] [in a new window]   Figure 35 Mononuclear cell leukemia (large granule lymphocyte leukemia) is a common spontaneous malignancy in F344 rats. It originates in the spleen and can be seen in virtually all tissues of the body. This example of mononuclear cell leukemia is from a female F344 rat treated with a high dose of benzophenone for 2 years (Figure 35A–35C). The malignant large granule (NK) lymphocytes spread to various tissues through the vascular system and malignant cells can often be seen within blood vessels in the affected organs. In this affected thymus, the leukemic lymphocytes are more loosely arranged than the more dense normal thymic lymphocytes. Leukemic infiltrates are often accompanied by erythrocytes (Figure 35C).

View larger version (1120K): [in this window] [in a new window]   Figure 8 This example of treatment-induced thymic atrophy was present in a Tg.AC hemizygous mouse on an FVB/N background. Sodium bromate was given in the drinking water for 26 weeks. The thymus is significantly reduced in size (Figure 8A) and the corticomedullary demarcation is obscured (Figure 8B). There is a marked decrease in cortical lymphocytes and an increase in small lymphocytes in the medulla (Figure 8A and 8B). As evidence that the toxic process is still active, there is ongoing apoptotic cell death occurring in the cortex (Figure 8C).

View larger version (887K): [in this window] [in a new window]   Figure 33 This thymic lymphoma is from a female p53+/– heterozygous mouse that received a high dose of phenolphthalenin in a subchronic study. There is complete obliteration of the normal thymic architecture and invasion into adjacent adipose tissue (Figure 33A). The neoplasm consists of a uniform proliferation of lymphoblasts with only a few small lymphocytes (present at the right edge of Figure 33B around a blood vessel). Mitotic figures are present (Figure 33B, arrows).

	Footnotes

 
This research was supported in part by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences.

	References

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Toxicologic Pathology, Vol. 34, No. 5, 515-547 (2006)
DOI: 10.1080/01926230600978458


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