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Morphology of hepatocellular neoplasms in B6C3F1 mice
Cancer Letters, 9 (1980) o Elsevier/North-Holland
MORPHOLOGY MICE
319
319-325 Scientific Publishers Ltd.
OF HEPATOCELLULAR
NEOPLASMS
IN B6C3Fl
J.M. WARD
Tumor Pathology National Institutes
Branch, Carcinogenesis Testing Program, of Health, Bethesda, MD 20205 (U.S.A.)
National
Cancer Institute,
(Recieved 5 December 1979) (Revised version received 25 February 1980) (Accepted 3 March 1980)
SUMMARY
The morphology and behavior of hepatocellular neoplasms in control R6C3Fl mice were studied and classified according to cell size, tinctorial properties of the cytoplasm, pattern of growth, and other morphological features. The small tumors were composed primtiily of basophilic hepatocytes which grew in a solid adenomatous pattern. Larger solid tumors had foci of prominent trabecular formations. All mice with metastatic hepatocellular neoplasms had large areas of prominent trabecular formations in the primary liver tumors. It appears that trabecular hepatocellular carcinomas develop from adenomatous neoplasms in the liver of R6C3Fl mice. The histogenesis and morphology of hepatocellular neoplasms in R6C3Fl mice is compared with those induced by carcinogens and differences are noted.
INTRODUCTION
Hepatocellular tumors in mice have been induced by numerous carcinogens [ 12,171. A variety of terms have been proposed and used by investigators to describe these tumors [l-9,11,14-16]. Recently, a classification was developed to compromise and consolidate the many conflicting terminologies [4]. We have recently proposed that some mouse hepatic carcinogens induce hepatocellular tumors which are morphologically and biologically different from those in control mice [ 9,161. In order to differentiate ‘spontaneous’ from ‘induced’ liver tumors, this paper reports the types of hepatocellular tumors seen in control R6C3Fl mice; tumors which occur in 2030% of the male and 3-10% of the female mice used as controls in carcinogenesis tests.
320 MATERIALS
AND METHODS
Female C57RL/6N mice were mated to male C3H/HeN mice at the Charles River Rreeding Laboratories (Wilmington, MA). The Fl generation were sent to 1 of 6 National Cancer Institute contract or subcontract bioassay laboratories [18] at 4 weeks of age over the past 7 years. At the bioassay laboratory, the mice were maintained generally 5 per polycarbonate or polypropylene cage with hardwood bedding. Wayne Lab Rlox (Allied Mills, Chicago, IL) and water ad lib. The bioassay commenced when mice were 6 weeks of age. All surviving mice were sacrificed at 90-104 weeks after initiation of the bioassay. A complete necropsy was performed [lo]. The liver lesions were classified according to morphologic characteristics 141.
TABLE 1 A MORPHOLOGIC B6C3Fl MICE
CLASSIFICATION
OF HEPATOCELLULAR
Tumor type Hepatocellular adenoma Basophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Eosinophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Hepatocellular carcinoma Basophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Eosinophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Total tumors
Male
NEOPLASMS OF
Female
51
15
38 26 19 0 0 13 0 0 0 0
13 7 6 0 0 2 0 1 0 0
64
17
56 21 12 56 11 8 0 1 8 0
17 2 3 17 1 0 0 0 0 0
115
32
321 RESULTS
AND DISCUSSION
The hepatocellular tumors were classified according to cytologic features in Table 1. Small tumors, usually l-5 mm in diameter, were most commonly composed of a uniform population of basophilic hepatocytes growing in a solid pattern, with a cell size smaller than normal hepatocytes (Fig. 1). Other small nodules contained predominantly esoinophilic or vacuolated hepatocytes, or a mixture of all 3 cytoplasmic types. The eosinophilic and vacuolated cells were generally larger than normal hepatocytes. The uniform population of hepatocytes and the general difficulty in transplanting these tumors [ 5,8,19] led to their diagnoses as hepatocellular adenomas. Also, certain biologic features of these lesions such as responsiveness to peroxisomal stimulators [ 131 and lack of a-fetoprotein production [ 1,2] by neoplastic hepatocytes in the adenomas suggest their differences from carcinoma; however, they were considered to be neoplastic because of their progressive growth, resistance to iron accumulation [ 191, lack of reversibility and general morphologic features [ 14,151. Other synonyms in the literature include hyperplastic nodules [ 71, type A nodule, neoplastic nodule and liver tumor [ 111. The larger liver tumors [ 5-10 mm] frequently resembled the small tumors histologically, but also frequently had foci of vacuolated (glycogen or fat) cells, intracytoplasmic inclusions and areas of prominent trabecular formations (Fig. 2), the inclusions were
Fig. 1. Hepatocellular adenoma on right side, composed of basophilic hepatocytes solid pattern or forming single cell cords. H & E, x 130.
in a
322
Fig. 2. Hepatocellular neoplasm with prominent trabecular area on right and soli d pattern on left. H & E, X 55.
Fig. 3. Trabecular hepatocellular carcinoma with poorly differentiated and better differentiated cords. H & E, X 130.
323
of the Type 2 previously reported [6]. The morphology of hepatocytes in trabecular areas found in 53-55s of the mouse liver tumors were identical to those found in trabecular carcinomas. These trabecular foci in adenomas have been previously reported in mice [ 3,4,11,15] and may represent the early stages of trabecular carcinoma. The presence of these foci should lead to diagnosis as carcinomas. The largest tumors (greater than 1 cm in diameter) were generally composed of a variety of areas; some resembling adenomas and other larger areas of prominent trabecular formations (Fig. 3). The hepatocytes in the trabecular foci were usually basophilic and formed cords that varied from well-differentiated to poorly differentiated. Thrombosis of the sinusoids and vascular invasion by the tumor cells were sometimes seen. All 12 mice in this study (Table 1) and many other B6C3Fl mice not in this study with hepatocellular carcinomas metastatic to the lung (Fig. 4) had large areas of prominent trabecular formations in the primary liver tumors, a finding previously reported in other strains of mice [ 141. The primary tumors were usually greater than 1 cm in diameter. The naturally-occurring hepatocellular tumors in B6C3Fl mice resemble those in other strains of mice [ 5--8,111. It has been suggested that some chemicals induce hepatocellular tumors which are morphologically and biologically different from those in the controls [ 9,16,17]. Several chemicals were shown to induce only eosinophilic hepatocellular tumors [ 91 while
Fig. 4. Metastatic hepatocellular trabecular area. H & E, x 130.
carcinoma in lung. Primary tumor had a large prominent
324
others were shown to induce primarily basophilic tumors [14,16]. The chemical carcinogen may either induce a specific genetic change which results in specific phenotypic expression or may affect directly the morphology of tumor cells [ 131. The present morphological study and review of mouse liver tumors induced by more than 50 carcinogens [ 171 lead to a simple classification scheme for diagnosis of liver tumors in mice [4]. The terms adenoma and carcinoma may be used and each may be subclassified by cytologic features, pattern of development, and differentiation [4]. The utilization of this scheme, hopefully, will allow us to better study and understand liver neoplasms in mice. Using this scheme, 81 of the 147 liver neoplasms in this study would be classified as trabecular carcinomas and 66 as adenomas. Of the adenomas, 51 were of basophilic hepatocytes and 15 of eosinophilic hepatocytes. ACKNOWLEDGEMENTS
This study was supported in part by PHS Contract NOl-CP-43350 to Tracer-Jitco, Inc. The aid of Robert Nye, Jack Romine, Jean Keller, Ann Johnson, Amelia Grant, and Joan O’Brien is gratefully acknowledged. REFERENCES 1 Becker, F.F., Stillman, D. and Sell, S. (1977) Serum a-fetoprotein in a mouse strain (CBH-AvyfB) with spontaneous hepatocellular carcinomas. Cancer Res., 37, 870-872. 2 Becker, F.F. and Sell, S. (1979) a-Fetoprotein levels and hepatic alterations during chemical carcinogenesis in C57BL/6N mice. Cancer Res., 39, 3491-3494. 3 Frith, C.H. and Dooley, K. (1976) Hepatic cytologic and neoplastic changed in mice given benzidine dihydrochloride. J. Natl. Cancer Inst., 56, 679-682. 4 Frith, C.H. and Ward, J.M. A morphologic classification of proliferative and endoplastic hepatic lesions in mice. J. Environ. Pathol. Toxicol., (in press). 5 Gellatly, J.B.M. (1975) The natural history of hepatic parenchymal nodule formation in a colony of C57BL mice with reference to the effect of diet. In: Mouse Hepatic Neoplasia, Chapter 5, pp. 229-248. Editors: W.H. Butler and P.M. Newberne. Elsevier, Amsterdam. 6 Helyer, B.J. and Petrelli, M. (1978) Histochemistry and spontaneous hepatomas of CBA/H-T6T6 mice. Histochemistry and electron microscopy. J. Natl. Cancer Inst., 60,861-869. 7 Jones, G. and Butler, W.H. (1975) Morphology of spontaneous and induced neoplasia. In: Mouse Hepatic Neoplasia, Chapter 3. Editors: W.H. Butler and P.M. Newberne. Elsevier, Amsterdam. 8 Reuber, M.D. (1971) Morphologic and biologic correlation of hyperplastic and neoplastic lesions occurring “spontaneously” in C3HXY hybrid mice. Br. J. Cancer, 25, 538-543. 9 Reznik, G. and Ward, J.M. (1979) Carcinogenicity of the hair dye component 2nitro-p-phenylenediamine: induction of eosinophilic hepatocellular neoplasms in female B6C3Fl mice. Food Cosmet. Toxicol., 17, 493-500. 10 Squire, R.A. (1976) Bioassay pathology procedures. In: Guidelines for Carcinogen Bioassay in Small Rodents, pp. 51-57. Editors: J.M. Sontag, N.P. Page and U. Saffiotti. National Cancer Institute Carcinogenesis Technical Report Series NO. 1, Washington, D.C., U.S. Govt. Printing Office.
325 11 Stewart, H.L. (1975) Comparative aspects of certain cancers. In: Cancer - A Comprehensive Treatise, Vol. 4, pp. 320-329. Editor: F.F. Becker, Plenum Press. 12 Tomatis, L., Partensky, C. and Montesano, R. (1973) The predictive value of mouse liver tumor induction in carcinogenicity testing - a literature survey. Int. J. Cancer, 12, l-20. 13 Tsukada, H., Gotoh, M., Mochizuki, Y. and Furutkawa, K. (1979) Changes in peroxisomes in preneoplastic liver and hepatoma of mice induced by a-benzene hexachloride. Cancer Res., 39, 1628-1634. 14 Vesselinovitch, S.D., Michailovich, N. and I&o, K.V.N. (1978) Morphology and metastatic nature of induced hepatic nodular lesions in C57BL XC3HFl mice. Cancer Res., 38, 2003-2010. 15 Ward, J.M. and Vlahakis, G. (1978) Evaluation of hepatocellular neoplasms in mice. J. Natl. Cancer Inst., 61, 807-811. 16 Ward, J.M., Bernal, E., Buratto, B., Goodman, D.G., Strandberg, J.D. and Schueler, R. (1979) Histopathology of neoplastic and non-neoplastic lesions in mice fed-diets containing tetrachlorvinphos. J. Natl. Cancer Inst., 63, 111-118. 17 Ward, J.M., Griesemer, R.A. and Weisburger, J.H. (1979) The mouse liver tumor as an endpoint in carcinogenesis tests. Toxicol. Appl. Pharmacol., 51, 389-397. 18 Ward, J.M., Goodman, D.G., Squire, R.A., Chu, K.C. and Linhart, M.S. (1979) Neoplastic and non-neoplastic lesions in aging (C57BL/6N X C3H/HeN) Fl (B6C3Fl) mice. J. Natl. Cancer Inst., 63, 849-854. 19 Williams, G.M., Hirota, N. and Rice, J.M. (1979) The resistance of spontaneous mouse hepatocellular neoplasms to iron accumulation during rapid iron loading by parenteral administration and their transplantability. Am. J. Pathol., 94, 65-74.
MORPHOLOGY MICE
319
319-325 Scientific Publishers Ltd.
OF HEPATOCELLULAR
NEOPLASMS
IN B6C3Fl
J.M. WARD
Tumor Pathology National Institutes
Branch, Carcinogenesis Testing Program, of Health, Bethesda, MD 20205 (U.S.A.)
National
Cancer Institute,
(Recieved 5 December 1979) (Revised version received 25 February 1980) (Accepted 3 March 1980)
SUMMARY
The morphology and behavior of hepatocellular neoplasms in control R6C3Fl mice were studied and classified according to cell size, tinctorial properties of the cytoplasm, pattern of growth, and other morphological features. The small tumors were composed primtiily of basophilic hepatocytes which grew in a solid adenomatous pattern. Larger solid tumors had foci of prominent trabecular formations. All mice with metastatic hepatocellular neoplasms had large areas of prominent trabecular formations in the primary liver tumors. It appears that trabecular hepatocellular carcinomas develop from adenomatous neoplasms in the liver of R6C3Fl mice. The histogenesis and morphology of hepatocellular neoplasms in R6C3Fl mice is compared with those induced by carcinogens and differences are noted.
INTRODUCTION
Hepatocellular tumors in mice have been induced by numerous carcinogens [ 12,171. A variety of terms have been proposed and used by investigators to describe these tumors [l-9,11,14-16]. Recently, a classification was developed to compromise and consolidate the many conflicting terminologies [4]. We have recently proposed that some mouse hepatic carcinogens induce hepatocellular tumors which are morphologically and biologically different from those in control mice [ 9,161. In order to differentiate ‘spontaneous’ from ‘induced’ liver tumors, this paper reports the types of hepatocellular tumors seen in control R6C3Fl mice; tumors which occur in 2030% of the male and 3-10% of the female mice used as controls in carcinogenesis tests.
320 MATERIALS
AND METHODS
Female C57RL/6N mice were mated to male C3H/HeN mice at the Charles River Rreeding Laboratories (Wilmington, MA). The Fl generation were sent to 1 of 6 National Cancer Institute contract or subcontract bioassay laboratories [18] at 4 weeks of age over the past 7 years. At the bioassay laboratory, the mice were maintained generally 5 per polycarbonate or polypropylene cage with hardwood bedding. Wayne Lab Rlox (Allied Mills, Chicago, IL) and water ad lib. The bioassay commenced when mice were 6 weeks of age. All surviving mice were sacrificed at 90-104 weeks after initiation of the bioassay. A complete necropsy was performed [lo]. The liver lesions were classified according to morphologic characteristics 141.
TABLE 1 A MORPHOLOGIC B6C3Fl MICE
CLASSIFICATION
OF HEPATOCELLULAR
Tumor type Hepatocellular adenoma Basophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Eosinophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Hepatocellular carcinoma Basophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Eosinophilic cytoplasm Foci of vacuolated cells Foci of intracytoplasmic inclusions Foci of prominent trabecular formations Pulmonary metastases Total tumors
Male
NEOPLASMS OF
Female
51
15
38 26 19 0 0 13 0 0 0 0
13 7 6 0 0 2 0 1 0 0
64
17
56 21 12 56 11 8 0 1 8 0
17 2 3 17 1 0 0 0 0 0
115
32
321 RESULTS
AND DISCUSSION
The hepatocellular tumors were classified according to cytologic features in Table 1. Small tumors, usually l-5 mm in diameter, were most commonly composed of a uniform population of basophilic hepatocytes growing in a solid pattern, with a cell size smaller than normal hepatocytes (Fig. 1). Other small nodules contained predominantly esoinophilic or vacuolated hepatocytes, or a mixture of all 3 cytoplasmic types. The eosinophilic and vacuolated cells were generally larger than normal hepatocytes. The uniform population of hepatocytes and the general difficulty in transplanting these tumors [ 5,8,19] led to their diagnoses as hepatocellular adenomas. Also, certain biologic features of these lesions such as responsiveness to peroxisomal stimulators [ 131 and lack of a-fetoprotein production [ 1,2] by neoplastic hepatocytes in the adenomas suggest their differences from carcinoma; however, they were considered to be neoplastic because of their progressive growth, resistance to iron accumulation [ 191, lack of reversibility and general morphologic features [ 14,151. Other synonyms in the literature include hyperplastic nodules [ 71, type A nodule, neoplastic nodule and liver tumor [ 111. The larger liver tumors [ 5-10 mm] frequently resembled the small tumors histologically, but also frequently had foci of vacuolated (glycogen or fat) cells, intracytoplasmic inclusions and areas of prominent trabecular formations (Fig. 2), the inclusions were
Fig. 1. Hepatocellular adenoma on right side, composed of basophilic hepatocytes solid pattern or forming single cell cords. H & E, x 130.
in a
322
Fig. 2. Hepatocellular neoplasm with prominent trabecular area on right and soli d pattern on left. H & E, X 55.
Fig. 3. Trabecular hepatocellular carcinoma with poorly differentiated and better differentiated cords. H & E, X 130.
323
of the Type 2 previously reported [6]. The morphology of hepatocytes in trabecular areas found in 53-55s of the mouse liver tumors were identical to those found in trabecular carcinomas. These trabecular foci in adenomas have been previously reported in mice [ 3,4,11,15] and may represent the early stages of trabecular carcinoma. The presence of these foci should lead to diagnosis as carcinomas. The largest tumors (greater than 1 cm in diameter) were generally composed of a variety of areas; some resembling adenomas and other larger areas of prominent trabecular formations (Fig. 3). The hepatocytes in the trabecular foci were usually basophilic and formed cords that varied from well-differentiated to poorly differentiated. Thrombosis of the sinusoids and vascular invasion by the tumor cells were sometimes seen. All 12 mice in this study (Table 1) and many other B6C3Fl mice not in this study with hepatocellular carcinomas metastatic to the lung (Fig. 4) had large areas of prominent trabecular formations in the primary liver tumors, a finding previously reported in other strains of mice [ 141. The primary tumors were usually greater than 1 cm in diameter. The naturally-occurring hepatocellular tumors in B6C3Fl mice resemble those in other strains of mice [ 5--8,111. It has been suggested that some chemicals induce hepatocellular tumors which are morphologically and biologically different from those in the controls [ 9,16,17]. Several chemicals were shown to induce only eosinophilic hepatocellular tumors [ 91 while
Fig. 4. Metastatic hepatocellular trabecular area. H & E, x 130.
carcinoma in lung. Primary tumor had a large prominent
324
others were shown to induce primarily basophilic tumors [14,16]. The chemical carcinogen may either induce a specific genetic change which results in specific phenotypic expression or may affect directly the morphology of tumor cells [ 131. The present morphological study and review of mouse liver tumors induced by more than 50 carcinogens [ 171 lead to a simple classification scheme for diagnosis of liver tumors in mice [4]. The terms adenoma and carcinoma may be used and each may be subclassified by cytologic features, pattern of development, and differentiation [4]. The utilization of this scheme, hopefully, will allow us to better study and understand liver neoplasms in mice. Using this scheme, 81 of the 147 liver neoplasms in this study would be classified as trabecular carcinomas and 66 as adenomas. Of the adenomas, 51 were of basophilic hepatocytes and 15 of eosinophilic hepatocytes. ACKNOWLEDGEMENTS
This study was supported in part by PHS Contract NOl-CP-43350 to Tracer-Jitco, Inc. The aid of Robert Nye, Jack Romine, Jean Keller, Ann Johnson, Amelia Grant, and Joan O’Brien is gratefully acknowledged. REFERENCES 1 Becker, F.F., Stillman, D. and Sell, S. (1977) Serum a-fetoprotein in a mouse strain (CBH-AvyfB) with spontaneous hepatocellular carcinomas. Cancer Res., 37, 870-872. 2 Becker, F.F. and Sell, S. (1979) a-Fetoprotein levels and hepatic alterations during chemical carcinogenesis in C57BL/6N mice. Cancer Res., 39, 3491-3494. 3 Frith, C.H. and Dooley, K. (1976) Hepatic cytologic and neoplastic changed in mice given benzidine dihydrochloride. J. Natl. Cancer Inst., 56, 679-682. 4 Frith, C.H. and Ward, J.M. A morphologic classification of proliferative and endoplastic hepatic lesions in mice. J. Environ. Pathol. Toxicol., (in press). 5 Gellatly, J.B.M. (1975) The natural history of hepatic parenchymal nodule formation in a colony of C57BL mice with reference to the effect of diet. In: Mouse Hepatic Neoplasia, Chapter 5, pp. 229-248. Editors: W.H. Butler and P.M. Newberne. Elsevier, Amsterdam. 6 Helyer, B.J. and Petrelli, M. (1978) Histochemistry and spontaneous hepatomas of CBA/H-T6T6 mice. Histochemistry and electron microscopy. J. Natl. Cancer Inst., 60,861-869. 7 Jones, G. and Butler, W.H. (1975) Morphology of spontaneous and induced neoplasia. In: Mouse Hepatic Neoplasia, Chapter 3. Editors: W.H. Butler and P.M. Newberne. Elsevier, Amsterdam. 8 Reuber, M.D. (1971) Morphologic and biologic correlation of hyperplastic and neoplastic lesions occurring “spontaneously” in C3HXY hybrid mice. Br. J. Cancer, 25, 538-543. 9 Reznik, G. and Ward, J.M. (1979) Carcinogenicity of the hair dye component 2nitro-p-phenylenediamine: induction of eosinophilic hepatocellular neoplasms in female B6C3Fl mice. Food Cosmet. Toxicol., 17, 493-500. 10 Squire, R.A. (1976) Bioassay pathology procedures. In: Guidelines for Carcinogen Bioassay in Small Rodents, pp. 51-57. Editors: J.M. Sontag, N.P. Page and U. Saffiotti. National Cancer Institute Carcinogenesis Technical Report Series NO. 1, Washington, D.C., U.S. Govt. Printing Office.
325 11 Stewart, H.L. (1975) Comparative aspects of certain cancers. In: Cancer - A Comprehensive Treatise, Vol. 4, pp. 320-329. Editor: F.F. Becker, Plenum Press. 12 Tomatis, L., Partensky, C. and Montesano, R. (1973) The predictive value of mouse liver tumor induction in carcinogenicity testing - a literature survey. Int. J. Cancer, 12, l-20. 13 Tsukada, H., Gotoh, M., Mochizuki, Y. and Furutkawa, K. (1979) Changes in peroxisomes in preneoplastic liver and hepatoma of mice induced by a-benzene hexachloride. Cancer Res., 39, 1628-1634. 14 Vesselinovitch, S.D., Michailovich, N. and I&o, K.V.N. (1978) Morphology and metastatic nature of induced hepatic nodular lesions in C57BL XC3HFl mice. Cancer Res., 38, 2003-2010. 15 Ward, J.M. and Vlahakis, G. (1978) Evaluation of hepatocellular neoplasms in mice. J. Natl. Cancer Inst., 61, 807-811. 16 Ward, J.M., Bernal, E., Buratto, B., Goodman, D.G., Strandberg, J.D. and Schueler, R. (1979) Histopathology of neoplastic and non-neoplastic lesions in mice fed-diets containing tetrachlorvinphos. J. Natl. Cancer Inst., 63, 111-118. 17 Ward, J.M., Griesemer, R.A. and Weisburger, J.H. (1979) The mouse liver tumor as an endpoint in carcinogenesis tests. Toxicol. Appl. Pharmacol., 51, 389-397. 18 Ward, J.M., Goodman, D.G., Squire, R.A., Chu, K.C. and Linhart, M.S. (1979) Neoplastic and non-neoplastic lesions in aging (C57BL/6N X C3H/HeN) Fl (B6C3Fl) mice. J. Natl. Cancer Inst., 63, 849-854. 19 Williams, G.M., Hirota, N. and Rice, J.M. (1979) The resistance of spontaneous mouse hepatocellular neoplasms to iron accumulation during rapid iron loading by parenteral administration and their transplantability. Am. J. Pathol., 94, 65-74.