Figure 7

Brandes, K., Fend, F., Monecke, S., Teifke, J.P., Breuer, W., and Hermanns, W.

(2004). Comparative morphologic and immunohistochemical investigation of

spontaneously occurring thymomas in a colony of European hamsters. Vet

Pathol 41, 346-52.

Frith, C.H., Ward, J.M., Harleman, J.H., Stromberg, P.C., Halm, S., Inoue, T., and

Wright, J.A. (2001). Hematopoietic System. In International Classification of

Rodent Tumors. The Mouse. (U. Mohr, ed.), pp 432-437. Springer-Verlag, New

York, NY.

Greaves, P. (2012). Histopathology of Preclinical Toxicity Studies, Fourth Edition.

Elsevier, Academic Press, San Diego, CA, p 128.

Hailey, J.R., Harleman, J.H., Stromberg, P., and Ward, J.M. (1993). 4.

Hematopoietic System. In: International Classification of Rodent Tumours, Part

I: The Rat, (U. Mohr, ed.), pp 17-21. International Agency for Research on

Cancer, Lyon, France.

Haseman, J.K., Hailey, J.R., and Morris, R.W. (1998). Spontaneous neoplasm

incidences in Fischer 344 rats and B6C3F1 mice in two-year carcinogenicity

studies: a National Toxicology Program update. Toxicol Pathol 26, 428-441.

Kupfer, C.F. and Beems, R.B., (1990). Thymoma, Epithelial, Rat, In. Hemopoietic

System, Monographs on Pathology of Laboratory Animals, ed. T.C. Jones,

J.M. Ward, U.M. Mohr and R.D. Hunt. Springer –Verlag, Berlin, Heidleberg,

New York. pp. 280-286.

Kupfer, C.F., Beems, R.B., and Hollanders, V.M.H. (1986). Spontaneous Pathology

of the Thymus in Aging Wistar (Cpb:Wu) Rats. Vet Pathol 23, 270-277.

Naylor, D.C., Krinke, G.J., and Ruefenacht, H.J. (1988). Primary Tumours of the

Thymus in the Rat. J. Comp. Path. Vol 99, PP 187-203.

Pearse, G. (2006). Histopathology of the Thymus. Toxicol Pathol 34, 515-547.

Rao, G. N. (1996). New diet (NTP-2000) for rats in the National Toxicology

Program toxicity and carcinogenicity studies. Fundam Appl Toxicol 32, 102-8.

Rao, G. N. (2001). Beneficial effects of NTP-2000 diet on growth, survival, and

heart and kidney diseases of Fischer 344 rats in chronic studies. Toxicol Sci

63, 245-55.

Rosai, J., and Sobin, L. H., (1999). Histological typing of tumours of the thymus.

In World Health Organisation, International Histological Classification of

tumours, 2nd ed. pp 1-36. Springer, Berlin.

Stefanski, S. A., Elwell, M.R., and Stromberg, P.C. (1990). Spleen, Lymph Nodes,

and Thymus. In Pathology of the Fischer Rat: Reference and Atlas (Boorman

G.A., Eustis, S. L., Elwell, M. R., Montgomery, C. A., MacKenzie, W. F., eds),

pp. 369-93. Academic Press, San Diego, CA.

Thymomas in Fischer 344/N Rats in the National Toxicology Program Database Gabrielle A Willson1, Rebecca R. Moore1, Hiroaki Nagai2, Rodney A. Miller1, Jerry F. Hardisty1, Neil Allison1, David E. Malarkey2

1Experimental Pathology Laboratories, Inc., Research Triangle Park, NC, USA 2Cellular and Molecular Pathology Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA

Figure 3

Thymoma is an uncommon tumor in most strains of rats and mice.

Primary tumors of the thymus of Sprague-Dawley-derived rats

(strain Tif:RAI) were reported from 7 different long-term studies

(Naylor, et al., 1988). In that study, 192 primary thymic tumors were

identified (out of 4281 animals), of which 171 appeared benign and

21 appeared malignant. In the F344/N rat spontaneously occurring

thymomas are rare, and most of those have been reported to be

benign (Stefanski, Elwell, and Stromberg, 1990; Haseman, Hailey,

and Morris, 1998). The literature contains no reports of large studies

of thymoma in F344/N rats. The purpose of this study is to describe

the morphological features, incidence, and behavior of thymoma in

F344/N rats in NTP studies.

Two hundred seventy-seven thymomas recorded in chronic studies in

the NTP archives database were reviewed. No thymomas were

recorded in any of the subchronic studies. Thymomas in F344/N rats

in this survey were rare occurrences (<0.2% incidence). No

occurrences of this tumor were found to be associated with

treatment. Thymomas occurred more commonly in male rats

(156/277 [56.3%]) than in female rats (121/277 [43.7%]). Benign

thymomas comprised 84.8% (235/277) of the thymomas reviewed,

and the remaining 15.2% (42/277) were diagnosed as malignant

thymomas based upon metastasis, unequivocal tissue invasion, or

cytological malignant features (Table 1). In male rats 27/156 (17.3%)

thymomas were malignant, and in female rats 15/121 (12.4%) were

malignant. Metastases were observed in 14 of the 42 animals with

malignant thymomas, and in all 14 cases metastatic lesions were

observed in the lung. Metastases also were present in the lymph

node of 1 animal and in the liver of another animal. All of the

malignant thymomas that metastasized had features of the main

tumor mass and no malignant epithelial neoplasms were present in

other organs. Seventeen of the remaining 28 malignant thymomas

that exhibited no metastases were diagnosed due to their

unequivocal tissue invasion, and 11 cases were diagnosed based

upon cytological features, such as pleomorphism, cellular atypia,

karyomegaly, anisokaryosis, prominent nucleoli, and increased

mitoses. These cytologic features typically associated with

malignancy seemed to have little to no relationship to a propensity for

invasion or metastasis.

Thymomas in Fischer 344/N rats were morphologically

heterogeneous but could be divided into 6 general categories

according to morphologic features (Table 2). Most tumors displayed

a mixture of more than one morphologic pattern, and these were

grouped according to the predominant pattern. The most common

morphologic pattern consisted of epithelial cells arranged in cords

and tubules and was present in 155/277 (56.0%) of the tumors in this

review (Figure 1). This was followed by thymomas with a

predominantly spindloid pattern, which occurred with a frequency of

79/277 (28.5%) (Figures 2 and 3). A papillary pattern was observed

in 17/277 (6.1%) of the tumors and was frequently associated with

cystic features (Figure 4). A squamous epithelial pattern was present

in 12/277 (4.3%) of the tumors (Figure 5). A myoid pattern was

present in 11/277 (4.0%) of the tumors (Figure 6A). The myoid cell is

a striated muscle cell which exhibits expression of desmin (Figures

6B and 6C). While it is possible that myoid cells within the thymomas

in this series represented invasion of pre-existing muscle, myoid

differentiation of neoplastic cells could not be ruled out. A

neuroendocrine pattern was observed in only 3/277 (1.1%) of the

tumors (Figure 7). No particular pattern was unequivocally correlated

with a metastatic or invasive propensity.

Four benign thymomas (4/228; 1.8%), 11 malignant thymomas

without metastasis (11/28; 39.3%), and 11 malignant thymomas with

metastasis (11/14; 78.6 %) were considered to be the cause of death.

The survival days of animals with malignant thymomas were less

than those of animals with benign thymomas (Table 3).

Figure 2

Results

Thymoma is defined as the neoplastic proliferation of thymic

epithelial cells. By virtue of the cellular composition of the thymus,

thymomas contain varying numbers of non-neoplastic lymphocytes.

Various classification schemes have been described in veterinary

medicine, and most are based upon histomorphological features.

One such scheme creates divisions based upon the relative

proportions of lymphocytes and epithelial cells, thus specifying tumor

types as either epithelial type, mixed epithelial and lymphoid cell

type, or “pure” lymphoid type (containing scattered epithelial cells to

distinguish it from well-differentiated lymphoma) (Greaves, 2012).

Others suggest that thymomas are divided into two types, consisting

of tumors with and without medullary differentiation (Kuper, Beems,

and Hollanders, 1986; Kuper and Beems, 1990). Tumors exhibiting

lobular, medullary differentiation have pale staining areas containing

fewer lymphocytes, fibrous trabeculae, and may contain epithelial

cells forming cords, tubules, and cysts. Thymomas without medullary

differentiation consist of a mixture of small lymphocytes and epithelial

cells without a lobular architecture. While epithelial cell morphology

can be quite variable, cellular atypia has been found to be rare

(Kuper and Beems, 1990). Epithelial cells of thymomas can form

several morphological patterns, which have been described by one

author as epidermoid (non-keratinizing squamous epithelium);

squamoid (with keratinization); papillary; ribbons, cords, or tubules;

spindloid; endocrine-like; neuroendocrine; and myoid (Pearse, 2006).

Thymoma is an uncommon tumor in most strains of rats and mice.

Primary tumors of the thymus of Sprague-Dawley-derived rats

(strain: Tif:RAI) were reported from 7 different long-term or lifespan

studies (Naylor, et al., 1988). In that study, 192 primary thymic

tumors were identified (out of 4281 thymuses), of which 171 were

classified as benign and 21 were called malignant. In the F344/N rat

spontaneously occurring thymomas are rare, and most of those have

been reported to be benign (Stefanski, Elwell, and Stromberg, 1990;

Haseman, Hailey, and Morris, 1998). The purpose of this study is to

describe the morphological features, incidences, and behavior of

thymoma in Fischer 344/N rats in NTP studies.

Introduction

Figure 1

Figure 4

Thymomas are neoplasms of thymic epithelial cell origin which may

contain variable numbers of non-neoplastic lymphocytes. Thymomas

appear grossly as a firm, smooth mass in the anterior mediastinum,

and compression of the surrounding tissues may or may not be

present. These tumors are rare in humans and most lab animals. In

the Fischer 344/N rat, thymomas are rare and are said to occur with

no apparent sex difference (Stefanski, Elwell, and Stromberg, 1990),

although in this review more thymomas were observed in male rats.

There is no meaningful established classification system of

thymomas in veterinary medicine, whereas in human medicine there

is the widely used classification system established by the World

Health Organization (WHO) (Rosai and Sobin, 1999). Nevertheless,

the WHO system has been adapted for use in one of the largest

hamster thymoma papers to date (Brandes et al., 2004).

Diagnostic paradigms have been described for rat thymomas,

including those dividing them according to the proportions of

epithelial and lymphoid cells (Greaves, 2012), as well as divisions

based upon thymomas with and without medullary differentiation

(Kuper and Beems, 1990; Kuper, Beems, and Hollanders, 1986).

While understandable and useable, these paradigms do not seem to

add significantly to our ability to interpret the rat thymoma in relation

to toxicologic or biological significance.

The thymomas in this review were variable in microscopic

appearance and were separated into 6 categories based upon

morphological patterns. Although this could be done with some

consistency, there was no apparent difference in biological behavior

between the categories. Since separating thymomas into several

sub-classifications appears to have limited, if any, scientific relevance

in terms of biological behavior, it seems adequate to designate them

only as benign or malignant until new information is obtained.

Classifying thymomas as benign or malignant traditionally has been

based upon cytologic features and biological behavior such as

invasion and metastasis as discussed in two volumes of International

Classification of Rodent Tumors (Hailey et al., 1993; Frith et al.,

2001). In this review there was little evidence that cytologic features

typically associated with malignancy were necessarily correlated with

invasion or metastasis

Discussion

References

The NTP studies evaluated for this review ended at 104 to 105

weeks for chronic studies and 13 weeks for subchronic studies.

Complete necropsies were performed on all animals. All gross

lesions and approximately 43 tissues were collected, fixed in 10%

neutral buffered formalin, stained with H&E, and examined. Thymic

tissue and/or thymic masses were collected from all rats on each

study.

The NTP historical databases were reviewed for the diagnosis of

benign thymoma, malignant thymoma, and thymoma. The

databases contained data on ~190,000 Fischer 344/N rats (~175,000

rats from chronic studies and ~15,000 rats used in subchronic

studies). All sections of thymomas were evaluated for unique

morphologic features, patterns of growth, and the presence or

absence of invasion and/or metastasis that could be used to

categorize them as benign or malignant. Tabulation of data was

designed to determine thymoma occurrences in male and female

rats, classification of tumors as benign or malignant, and to

characterize general morphologic patterns that may relate to

determining malignancy.

The classification of the neoplasms as benign or malignant was

based upon tumor expansion and invasion of surrounding tissues, as

well as presence of metastatic lesions. Thymomas were classified as

benign when they were confined to the mediastinal space, discrete,

exhibited an expansile non-invasive growth pattern, and there was no

evidence of metastasis. Extension into the mediastinal fat was

considered a benign feature as long as there was connection to the

main tumor mass and as long as the other benign tumor criteria were

met. Malignant thymomas were characterized by implantation on

serosal surfaces, metastasis to other organs, invasion of adjacent

tissues (body wall, diaphragm, or thoracic organs), and/or infiltration

of mediastinal fat with nests of neoplastic cells but no apparent

connection with the main tumor mass.

Methods

Abstract

Figure 6

Figure 5

Figure 1A. Benign thymoma from a male F344/N rat from a 2-year NTP

study. (H&E)

Figure 1B. Higher magnification of 1A, the neoplastic epithelial cells form

cords and tubules. (H&E)

Figure 2. Spindloid pattern in a malignant thymoma from a female F344/N

rat from a two-year NTP study. (H&E)

Figure 3. Lung metastasis from a malignant thymoma in a male F344/N rat

from a 2-year study. Sheets of elongated neoplastic cells form a spindloid

pattern. (H&E)

Figure 4. Benign thymoma is from a male F344/N rat from a 2-year study.

Neoplastic epithelial cells form papillary projections, which protrude into

cystic spaces. There are small clusters of non-neoplastic lymphocytes

interspersed with the neoplastic cells. (H&E)

Figure 5. Malignant thymoma is from a male F344/N rat from a 2-year

study. The neoplastic epithelial cells form a squamous pattern. (H&E)

Figure 6A. Malignant thymoma is from a female F344/N rat from a 2-year

study. The neoplastic epithelial cells form a myoid pattern. (H&E)

Figure 6B. Immunohistochemical staining of neoplasm from 6A showing

expression of desmin in myoid cells. (Desmin)

Figure 6C. Immunohistochemical staining of neoplasm from 6A showing

expression of desmin with prominent cross striations in myoid cells.

(Desmin)

Figure 7. Benign thymoma is from a female F344/N rat from a 2-year study.

Packets of elongated neoplastic cells form a neuroendocrine pattern.

(H&E)

Benign

Malignant

without metastasis with metastasis

Number of animals 228 a 28 14

Mean survival days b 713 ± 4 609 ± 27 506 ± 42

Cause of death: thymoma 4 11 11

Sacrifice type

Natural death 16 8 8

Moribund sacrifice 48 9 6

Terminal sacrifice 164 11 0

a Seven animals were not included in this table because there were 2 accidental deaths

and 5 animals were sacrificed for interim necropsy.

b Study days on which animals were sacrificed. Value shown is group mean ± S.E.

Morphologic pattern Benign Malignant Total

Cords/Tubules 139 16 155

Spindloid 66 13 79

Papillary 17 0 17

Squamous 5 7 12

Myoid 6 5 11

Neuroendocrine 2 1 3

Table 2. The morphologic patterns of thymomas

Benign Malignant Total

Male 129 27 (10*) 156

Female 106 15 (4*) 121

Total 235 42 (14*) 277

Table 1. The incidence of thymomas in male and

female Fischer 344/N Rats

Table 3. Correlation of the malignancy of thymoma with

survival days, cause of death and sacrifice type

*The number within parentheses denotes the number of malignant thymomas with metastasis.

A B

C

A B

Grateful thanks for assistance with this poster to Karen Cimon, Kim

Pernicka, Maureen Puccini and Emily Singletary, of EPL and Lois

Wyrick of Image Associates.

Acknowledgments