Proceedings of the 2017 National Toxicology Program Satellite Symposium

Abstract

The 2017 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri," was held in Montreal, Quebec, Canada at the Society of Toxicologic Pathology's 36th annual meeting. The goal of this symposium was to present and discuss challenging diagnostic pathology and/or nomenclature issues. This article presents summaries of the speakers' talks along with select images that were used by the audience for voting and discussion. Various lesions and other topics covered during the symposium included renal papillary degeneration in perinatally exposed animals, an atriocaval mesothelioma, an unusual presentation of an alveolar-bronchiolar carcinoma, a paraganglioma of the organ of Zuckerkandl (also called an extra-adrenal pheochromocytoma), the use of human muscle samples to illustrate the challenges of manual scoring of fluorescent staining, intertubular spermatocytic seminomas, medical device pathology assessment and discussion of the approval process, collagen-induced arthritis, incisor denticles, ameloblast degeneration and poorly mineralized enamel matrix, connective tissue paragangliomas, microcystin-LR toxicity, perivascular mast cells in the forebrain thalamus unrelated to treatment, and 2 cases that provided a review of the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) bone nomenclature and recommended application of the terminology in routine nonclinical toxicity studies.

Keywords: INHAND; NTP Satellite Symposium; atriocaval mesothelioma; collagen-induced arthritis model; digital image analysis; incisor denticle; medical device evaluation; microcystin-LR; neural mast cells; paraganglioma; perinatal nephrotoxicosis; spermatocytic seminoma.

Figure 1

A-D. Renal tumor from 2-year-old Harlan Sprague Dawley male mid-dose rat presented in case 1. Figure A shows a low magnification image of the infiltrative unilateral renal tumor that distends the pelvis and extends into the ureter and peri-renal tissues. Higher magnifications of the neoplasm (B and C) show that in some areas the mass is composed of moderately to densely packed round to polygonal cells that resemble normal lipocytes, while in other areas, the cells are more spindloid to polygonal with rare intracytoplasmic vacuoles. There is marked proliferation of the overlying urothelium and frequent nests and islands of urothelium entrapped within the larger mesenchymal mass (D). H&E. E-H. Renal papillary changes from the high dose male HSD rat presented in case 2. The low magnification image shows that the papillae are bilaterally and diffusely expanded (E). A higher magnification demonstrates the expansion is due to marked dilation and/or distortion of the collecting ducts and increased amounts of pale eosinophilic to fibrillary amphophilic interstitial material (F and G). Figure 1H demonstrates that the affected ducts are lined by thin, attenuated epithelial cells and pyknotic nuclei are occasionally present (arrow). H&E.

Figure 1

A-D. Renal tumor from 2-year-old Harlan Sprague Dawley male mid-dose rat presented in case 1. Figure A shows a low magnification image of the infiltrative unilateral renal tumor that distends the pelvis and extends into the ureter and peri-renal tissues. Higher magnifications of the neoplasm (B and C) show that in some areas the mass is composed of moderately to densely packed round to polygonal cells that resemble normal lipocytes, while in other areas, the cells are more spindloid to polygonal with rare intracytoplasmic vacuoles. There is marked proliferation of the overlying urothelium and frequent nests and islands of urothelium entrapped within the larger mesenchymal mass (D). H&E. E-H. Renal papillary changes from the high dose male HSD rat presented in case 2. The low magnification image shows that the papillae are bilaterally and diffusely expanded (E). A higher magnification demonstrates the expansion is due to marked dilation and/or distortion of the collecting ducts and increased amounts of pale eosinophilic to fibrillary amphophilic interstitial material (F and G). Figure 1H demonstrates that the affected ducts are lined by thin, attenuated epithelial cells and pyknotic nuclei are occasionally present (arrow). H&E.

Figure 2

A-F. Atriocaval Mesothelioma in a male Sprague-Dawley (NCTR) rat. Low magnification H&E of the right atrial mass (A). Glandular structures are lined by nonciliated cuboidal to columnar epithelium and the lumen is filled with cellular debris admixed with inflammatory infiltrates, extravasated erythrocytes and hemoglobin crystals (B-D, H&E). Neoplastic cells exhibit moderate intracytoplasmic immunoreactivity for cytokeratin (E) and marked intracytoplasmic immunoreactivity of the stromal cells for vimentin (F).

Figure 3

A-C. Case 1. Lung neoplasm, NCTR Sprague Dawley (CD) rat. A) An expansile, unencapsulated neoplasm is adhered to the rib and lung pleura, invading and compressing the adjacent lung parenchyma, subgross. B) The neoplasm is composed of epithelial cells forming alveolar (glandular) and papillary structures supported by moderate fibrous connective tissue stroma C) Neoplastic cells are cuboidal to columnar with moderate amounts of eosinophilic cytoplasm and contain a single, centrally to basally located nucleus with stippled chromatin. Note the large influx of macrophages into tumor alveoli. H&E D-I. Alveolar-bronchiolar (AB) carcinoma from the NTP archives, Sprague Dawley rat (D-F). Neoplastic cells exhibit positive cytoplasmic staining with SPC (D, surfactant protein C) and CK18 (E, cytokeratin 18) antibodies, while the mesenchymal cells between epithelial cells stain positive with vimentin (F). Malignant mesothelioma from the NTP archives, Sprague Dawley rat (G-I). There is no immunoreactivity observed with SPC immunostaining (G). However, intense cytoplasmic staining is observed with CK18 (H) and vimentin (I) antibodies. J-L. Lung neoplasm, NCTR Sprague Dawley (CD) rat. IHC staining patterns of the tumor resemble those of the positive AB carcinoma control (D-F) with positive cytoplasmic staining of neoplastic cells with SPC (J) and CK18 (K), and staining of mesenchymal cells between epithelial components with vimentin (L). M-O. Paraganglioma of the Organ of Zuckerkandl was presented as case 2. Subgross image (M) of abdominal mass recorded at the time of necropsy. The mass resembles a pheochromocytoma (N), composed of finely vacuolated, polygonal cells arranged in discrete packets and supported by fine, fibrovascular stroma. However, the adrenal glands from this animal (O) were unremarkable indicating that this tumor was most likely an “extra-adrenal pheochromocytoma” or, based on location, a “paraganglioma of the Organ of Zuckerkandl”, subgross. H&E.

Figure 3

A-C. Case 1. Lung neoplasm, NCTR Sprague Dawley (CD) rat. A) An expansile, unencapsulated neoplasm is adhered to the rib and lung pleura, invading and compressing the adjacent lung parenchyma, subgross. B) The neoplasm is composed of epithelial cells forming alveolar (glandular) and papillary structures supported by moderate fibrous connective tissue stroma C) Neoplastic cells are cuboidal to columnar with moderate amounts of eosinophilic cytoplasm and contain a single, centrally to basally located nucleus with stippled chromatin. Note the large influx of macrophages into tumor alveoli. H&E D-I. Alveolar-bronchiolar (AB) carcinoma from the NTP archives, Sprague Dawley rat (D-F). Neoplastic cells exhibit positive cytoplasmic staining with SPC (D, surfactant protein C) and CK18 (E, cytokeratin 18) antibodies, while the mesenchymal cells between epithelial cells stain positive with vimentin (F). Malignant mesothelioma from the NTP archives, Sprague Dawley rat (G-I). There is no immunoreactivity observed with SPC immunostaining (G). However, intense cytoplasmic staining is observed with CK18 (H) and vimentin (I) antibodies. J-L. Lung neoplasm, NCTR Sprague Dawley (CD) rat. IHC staining patterns of the tumor resemble those of the positive AB carcinoma control (D-F) with positive cytoplasmic staining of neoplastic cells with SPC (J) and CK18 (K), and staining of mesenchymal cells between epithelial components with vimentin (L). M-O. Paraganglioma of the Organ of Zuckerkandl was presented as case 2. Subgross image (M) of abdominal mass recorded at the time of necropsy. The mass resembles a pheochromocytoma (N), composed of finely vacuolated, polygonal cells arranged in discrete packets and supported by fine, fibrovascular stroma. However, the adrenal glands from this animal (O) were unremarkable indicating that this tumor was most likely an “extra-adrenal pheochromocytoma” or, based on location, a “paraganglioma of the Organ of Zuckerkandl”, subgross. H&E.

Figure 3

A-C. Case 1. Lung neoplasm, NCTR Sprague Dawley (CD) rat. A) An expansile, unencapsulated neoplasm is adhered to the rib and lung pleura, invading and compressing the adjacent lung parenchyma, subgross. B) The neoplasm is composed of epithelial cells forming alveolar (glandular) and papillary structures supported by moderate fibrous connective tissue stroma C) Neoplastic cells are cuboidal to columnar with moderate amounts of eosinophilic cytoplasm and contain a single, centrally to basally located nucleus with stippled chromatin. Note the large influx of macrophages into tumor alveoli. H&E D-I. Alveolar-bronchiolar (AB) carcinoma from the NTP archives, Sprague Dawley rat (D-F). Neoplastic cells exhibit positive cytoplasmic staining with SPC (D, surfactant protein C) and CK18 (E, cytokeratin 18) antibodies, while the mesenchymal cells between epithelial cells stain positive with vimentin (F). Malignant mesothelioma from the NTP archives, Sprague Dawley rat (G-I). There is no immunoreactivity observed with SPC immunostaining (G). However, intense cytoplasmic staining is observed with CK18 (H) and vimentin (I) antibodies. J-L. Lung neoplasm, NCTR Sprague Dawley (CD) rat. IHC staining patterns of the tumor resemble those of the positive AB carcinoma control (D-F) with positive cytoplasmic staining of neoplastic cells with SPC (J) and CK18 (K), and staining of mesenchymal cells between epithelial components with vimentin (L). M-O. Paraganglioma of the Organ of Zuckerkandl was presented as case 2. Subgross image (M) of abdominal mass recorded at the time of necropsy. The mass resembles a pheochromocytoma (N), composed of finely vacuolated, polygonal cells arranged in discrete packets and supported by fine, fibrovascular stroma. However, the adrenal glands from this animal (O) were unremarkable indicating that this tumor was most likely an “extra-adrenal pheochromocytoma” or, based on location, a “paraganglioma of the Organ of Zuckerkandl”, subgross. H&E.

Figure 3

A-C. Case 1. Lung neoplasm, NCTR Sprague Dawley (CD) rat. A) An expansile, unencapsulated neoplasm is adhered to the rib and lung pleura, invading and compressing the adjacent lung parenchyma, subgross. B) The neoplasm is composed of epithelial cells forming alveolar (glandular) and papillary structures supported by moderate fibrous connective tissue stroma C) Neoplastic cells are cuboidal to columnar with moderate amounts of eosinophilic cytoplasm and contain a single, centrally to basally located nucleus with stippled chromatin. Note the large influx of macrophages into tumor alveoli. H&E D-I. Alveolar-bronchiolar (AB) carcinoma from the NTP archives, Sprague Dawley rat (D-F). Neoplastic cells exhibit positive cytoplasmic staining with SPC (D, surfactant protein C) and CK18 (E, cytokeratin 18) antibodies, while the mesenchymal cells between epithelial cells stain positive with vimentin (F). Malignant mesothelioma from the NTP archives, Sprague Dawley rat (G-I). There is no immunoreactivity observed with SPC immunostaining (G). However, intense cytoplasmic staining is observed with CK18 (H) and vimentin (I) antibodies. J-L. Lung neoplasm, NCTR Sprague Dawley (CD) rat. IHC staining patterns of the tumor resemble those of the positive AB carcinoma control (D-F) with positive cytoplasmic staining of neoplastic cells with SPC (J) and CK18 (K), and staining of mesenchymal cells between epithelial components with vimentin (L). M-O. Paraganglioma of the Organ of Zuckerkandl was presented as case 2. Subgross image (M) of abdominal mass recorded at the time of necropsy. The mass resembles a pheochromocytoma (N), composed of finely vacuolated, polygonal cells arranged in discrete packets and supported by fine, fibrovascular stroma. However, the adrenal glands from this animal (O) were unremarkable indicating that this tumor was most likely an “extra-adrenal pheochromocytoma” or, based on location, a “paraganglioma of the Organ of Zuckerkandl”, subgross. H&E.

Figure 4

A-F. Human frozen skeletal muscle samples (3 cases presented). Anti-dystrophin fluorescent labeling (TRITC; A, C, E). The first case (A) was a control muscle sample, displaying the tetramethylrhodamine (TRITC) channel only (anti-dystrophin staining). Overlaid with a digital image analysis markup (B), it shows that 100% of fibers are positive for dystrophin staining. Duchenne Muscular Dystrophy (DMD) sample presented as case 2 (C), with digital image analysis markup highlighting 14 positive fibers (D). Becker Muscular Dystrophy (BMD) sample presented as case 3 (E), with digital image analysis markup displaying 42 positive fibers (F). Images originally captured at 10× magnification, bars = 70μm. Markup (B, D, F): light blue lines = fiber membrane; red center of fiber = positive (membrane dystrophin staining above intensity threshold); blue center of fiber = negative (membrane dystrophin staining above intensity threshold). G-H. Craik-O'Brien-Cornsweet illusion (G) demonstrates the human's perception of surfaces based on the optical characteristics of their edges (Fibonacci, 2007). In this illusion, the brightness change along the edge in the middle influences the perception of both surfaces to the left and right of the middle having different brightness, when in fact the majority is of the same brightness. To confirm, cover the middle of the image to see that the majority of the area is the same intensity or shade of gray (Kurki et al., 2009, Purves et al., 1999, Masuda et al., 2014, Aeffner et al., 2017). Herman grid illusion (H) is a display of lateral inhibition, which in a perceived increase of contrast and sharpness (Aaen-Stockdale, 2007). Specifically, false gray dots appear at the intersection of the white lines. These are caused by the lateral inhibition, preventing a lateral spread of the action potential across the retina (Spillmann, 1994, Kingdom, 2014, Aeffner et al., 2017).

Figure 4

A-F. Human frozen skeletal muscle samples (3 cases presented). Anti-dystrophin fluorescent labeling (TRITC; A, C, E). The first case (A) was a control muscle sample, displaying the tetramethylrhodamine (TRITC) channel only (anti-dystrophin staining). Overlaid with a digital image analysis markup (B), it shows that 100% of fibers are positive for dystrophin staining. Duchenne Muscular Dystrophy (DMD) sample presented as case 2 (C), with digital image analysis markup highlighting 14 positive fibers (D). Becker Muscular Dystrophy (BMD) sample presented as case 3 (E), with digital image analysis markup displaying 42 positive fibers (F). Images originally captured at 10× magnification, bars = 70μm. Markup (B, D, F): light blue lines = fiber membrane; red center of fiber = positive (membrane dystrophin staining above intensity threshold); blue center of fiber = negative (membrane dystrophin staining above intensity threshold). G-H. Craik-O'Brien-Cornsweet illusion (G) demonstrates the human's perception of surfaces based on the optical characteristics of their edges (Fibonacci, 2007). In this illusion, the brightness change along the edge in the middle influences the perception of both surfaces to the left and right of the middle having different brightness, when in fact the majority is of the same brightness. To confirm, cover the middle of the image to see that the majority of the area is the same intensity or shade of gray (Kurki et al., 2009, Purves et al., 1999, Masuda et al., 2014, Aeffner et al., 2017). Herman grid illusion (H) is a display of lateral inhibition, which in a perceived increase of contrast and sharpness (Aaen-Stockdale, 2007). Specifically, false gray dots appear at the intersection of the white lines. These are caused by the lateral inhibition, preventing a lateral spread of the action potential across the retina (Spillmann, 1994, Kingdom, 2014, Aeffner et al., 2017).

Figure 5

A-E. Microscopic features of spermatocytic seminomas in NCTR Sprague Dawley (CD) rats. Low- (A), mid- (B) and high-magnification (C) images from a spermatocytic seminoma (H&E). The lesion is focal (A), does not invade the seminiferious tubules (B) and is present in dense sheets within the interstitium with cells often adhered to the capsular surfaces of the seminiferous tubules (C). Cellular features include small, medium and large, round to polygonal cells with marked pleomorphism, hyperchromatic nuclei, prominent nucleoli and a large nuclear to cytoplasmic ratio (C). Using H&E, the spermatocytic seminoma (C) can easily be distinguished from a classic intratubular seminoma (D), which consists of tubular structures filled with round to polyhedral cells (resembling primitive germ cells), that are mostly uniform in size, with eosinophilic cytoplasm, indistinct cell borders and numerous mitoses (H&E). A PAS stain (E) can help to better identify the tumor cells within a spermatocytic seminomoa that have a coarse and clumped chromatin pattern, resembling developing spermatocytes (arrows).

Figure 6

A-D. Skeletal muscle tissue an adult male New Zealand white rabbit implanted with articles. Figure A is a low magnification of a site implanted with a section of high density polyethylene (HDPE, open white space) that has minimal cellular and soft tissue responses to the implant. A higher magnification of the area (B) shows that the reaction is primarily fibroblasts and minimal collagen deposition. Figure C shows a low magnification of a site implanted with a section of xenogeneic collagen (black arrows) along site a location marker (HDPE, *); the cellular and soft tissue responses are much more severe than for HDPE alone (A). A higher magnification of the area (D) shows the article (black arrows) and inflammation is composed of numerous giant cells, heterophils, and lymphocytes. H&E.

Figure 7

A-D. Longitudinal sections of a hind foot from treated female Lewis rats on study for up to 21 days as presented in case 1. Figure A is a low magnification H&E section showing relationship of bones to each other and normal joint spaces (one outlined). Figure B shows a higher magnification of a joint space outline in (A), lined by plump synovial cells with strongly positive cytoplasmic immunoreactivity for CD68 on IHC. Figures C-D show a more advanced lesion from another animal on the study (H&E). The low magnification image (C) shows the irregularity of the surface of the smaller bones and expansion of the soft tissue by fibrosis and infiltrates of inflammatory cells. On high power of a joint space (D), the markedly thickened, inflamed synovial tissue and has irregularly eroded the periosteal surface of underlying bone (pannus formation). E-H. Figure 7E is an H&E example of a severe lesion with marked soft tissue swelling associated with fibrosis and inflammatory cell infiltrates, and extensive bone remodeling (area outlined) with distortion of joint spaces. Figure F is a high-power view of an area of bone remodeling. Trabeculae of bone are lined by large multinucleate osteoclasts with strongly positive cytoplasmic immunoreactivity for CD68. Figure 7G shows a low power of the hind foot with areas of new bone formation (outlined). Higher magnification of one area (H) shows irregular trabeculae of new bone radiating from the periosteal surface of the calcaneous bone. H&E. I-J. H&E stained longitudinal sections of incisor teeth from SD rats from a one month toxicity study. Treated rats on this study were noted to have white discolouration of the incisor teeth. Figure I is a high power view of the ameloblast layer in a control animal showing the normal regularly arranged single layer of columnar ameloblasts. Ameloblasts overlay an artefactual space caused by removal of the enamel layer in the process of decalcification of the section. Figure J is the same view in a treated animal showing disorganization, vacuolar degeneration and loss of ameloblasts, with formation of fluid-filled irregular spaces. The underlying enamel layer had not been removed by decalcification, but the cross-hatch appearance of the un-calcified enamel matrix was visible.

Figure 7

A-D. Longitudinal sections of a hind foot from treated female Lewis rats on study for up to 21 days as presented in case 1. Figure A is a low magnification H&E section showing relationship of bones to each other and normal joint spaces (one outlined). Figure B shows a higher magnification of a joint space outline in (A), lined by plump synovial cells with strongly positive cytoplasmic immunoreactivity for CD68 on IHC. Figures C-D show a more advanced lesion from another animal on the study (H&E). The low magnification image (C) shows the irregularity of the surface of the smaller bones and expansion of the soft tissue by fibrosis and infiltrates of inflammatory cells. On high power of a joint space (D), the markedly thickened, inflamed synovial tissue and has irregularly eroded the periosteal surface of underlying bone (pannus formation). E-H. Figure 7E is an H&E example of a severe lesion with marked soft tissue swelling associated with fibrosis and inflammatory cell infiltrates, and extensive bone remodeling (area outlined) with distortion of joint spaces. Figure F is a high-power view of an area of bone remodeling. Trabeculae of bone are lined by large multinucleate osteoclasts with strongly positive cytoplasmic immunoreactivity for CD68. Figure 7G shows a low power of the hind foot with areas of new bone formation (outlined). Higher magnification of one area (H) shows irregular trabeculae of new bone radiating from the periosteal surface of the calcaneous bone. H&E. I-J. H&E stained longitudinal sections of incisor teeth from SD rats from a one month toxicity study. Treated rats on this study were noted to have white discolouration of the incisor teeth. Figure I is a high power view of the ameloblast layer in a control animal showing the normal regularly arranged single layer of columnar ameloblasts. Ameloblasts overlay an artefactual space caused by removal of the enamel layer in the process of decalcification of the section. Figure J is the same view in a treated animal showing disorganization, vacuolar degeneration and loss of ameloblasts, with formation of fluid-filled irregular spaces. The underlying enamel layer had not been removed by decalcification, but the cross-hatch appearance of the un-calcified enamel matrix was visible.

Figure 7

A-D. Longitudinal sections of a hind foot from treated female Lewis rats on study for up to 21 days as presented in case 1. Figure A is a low magnification H&E section showing relationship of bones to each other and normal joint spaces (one outlined). Figure B shows a higher magnification of a joint space outline in (A), lined by plump synovial cells with strongly positive cytoplasmic immunoreactivity for CD68 on IHC. Figures C-D show a more advanced lesion from another animal on the study (H&E). The low magnification image (C) shows the irregularity of the surface of the smaller bones and expansion of the soft tissue by fibrosis and infiltrates of inflammatory cells. On high power of a joint space (D), the markedly thickened, inflamed synovial tissue and has irregularly eroded the periosteal surface of underlying bone (pannus formation). E-H. Figure 7E is an H&E example of a severe lesion with marked soft tissue swelling associated with fibrosis and inflammatory cell infiltrates, and extensive bone remodeling (area outlined) with distortion of joint spaces. Figure F is a high-power view of an area of bone remodeling. Trabeculae of bone are lined by large multinucleate osteoclasts with strongly positive cytoplasmic immunoreactivity for CD68. Figure 7G shows a low power of the hind foot with areas of new bone formation (outlined). Higher magnification of one area (H) shows irregular trabeculae of new bone radiating from the periosteal surface of the calcaneous bone. H&E. I-J. H&E stained longitudinal sections of incisor teeth from SD rats from a one month toxicity study. Treated rats on this study were noted to have white discolouration of the incisor teeth. Figure I is a high power view of the ameloblast layer in a control animal showing the normal regularly arranged single layer of columnar ameloblasts. Ameloblasts overlay an artefactual space caused by removal of the enamel layer in the process of decalcification of the section. Figure J is the same view in a treated animal showing disorganization, vacuolar degeneration and loss of ameloblasts, with formation of fluid-filled irregular spaces. The underlying enamel layer had not been removed by decalcification, but the cross-hatch appearance of the un-calcified enamel matrix was visible.

Figure 8

A-F.Tumors within the cervical (Case 1; A, C, E) and thoracic mediastinum (Case 2; B, D, F) of a male and female Harlan Sprague-Dawley (Hsd: SD) rat, respectively, from National Toxicology Program (NTP) 2-year chronic whole body exposure carcinogenicity bioassays. The tumors are shown at low- (A and B, arrows), mid- (C and D), and high- (E and F) magnifications. Both tumors are well-demarcated from the adjacent adipose connective tissue but non-encapsulated. Both tumors are composed of small clusters or nests of neoplastic polygonal chief cells (“Zellballen”) separated by delicate fibrovascular stroma containing spindleoid sustentacular cells (E and F, arrows). The neoplastic chief cells have round to ovoid centrally located nuclei, typically with finely stippled chromatin and 1-2 prominent nucleoli, and moderate amounts of eosinophilic cytoplasm. Mitotic figures are rare. H&E. G-J. Immunohistochemical evaluation of the tumor from the case 2 female rat, including synaptophysin (G), cytokeratin (H), tyrosine hydroxylase (I) along with tyrosine hydroxlase from the case 1 male rat (J). The polygonal Type I chief cells had diffuse strong cytoplasmic staining for synaptophysin (G) and diffuse weak cytoplasmic staining for cytokeratin (H). Tyrosine hydroxlase staining in both the female (I) and male (J) rats was diffuse and of variable intensity. K-L. Immunohistochemical evaluation of the tumor from the case 2 female rat, including S-100 (K) and GFAP (L). The spindleoid Type II sustentacular cells (arrows) were immunopositive for S-100 (K) and GFAP (L).

Figure 8

A-F.Tumors within the cervical (Case 1; A, C, E) and thoracic mediastinum (Case 2; B, D, F) of a male and female Harlan Sprague-Dawley (Hsd: SD) rat, respectively, from National Toxicology Program (NTP) 2-year chronic whole body exposure carcinogenicity bioassays. The tumors are shown at low- (A and B, arrows), mid- (C and D), and high- (E and F) magnifications. Both tumors are well-demarcated from the adjacent adipose connective tissue but non-encapsulated. Both tumors are composed of small clusters or nests of neoplastic polygonal chief cells (“Zellballen”) separated by delicate fibrovascular stroma containing spindleoid sustentacular cells (E and F, arrows). The neoplastic chief cells have round to ovoid centrally located nuclei, typically with finely stippled chromatin and 1-2 prominent nucleoli, and moderate amounts of eosinophilic cytoplasm. Mitotic figures are rare. H&E. G-J. Immunohistochemical evaluation of the tumor from the case 2 female rat, including synaptophysin (G), cytokeratin (H), tyrosine hydroxylase (I) along with tyrosine hydroxlase from the case 1 male rat (J). The polygonal Type I chief cells had diffuse strong cytoplasmic staining for synaptophysin (G) and diffuse weak cytoplasmic staining for cytokeratin (H). Tyrosine hydroxlase staining in both the female (I) and male (J) rats was diffuse and of variable intensity. K-L. Immunohistochemical evaluation of the tumor from the case 2 female rat, including S-100 (K) and GFAP (L). The spindleoid Type II sustentacular cells (arrows) were immunopositive for S-100 (K) and GFAP (L).

Figure 8

A-F.Tumors within the cervical (Case 1; A, C, E) and thoracic mediastinum (Case 2; B, D, F) of a male and female Harlan Sprague-Dawley (Hsd: SD) rat, respectively, from National Toxicology Program (NTP) 2-year chronic whole body exposure carcinogenicity bioassays. The tumors are shown at low- (A and B, arrows), mid- (C and D), and high- (E and F) magnifications. Both tumors are well-demarcated from the adjacent adipose connective tissue but non-encapsulated. Both tumors are composed of small clusters or nests of neoplastic polygonal chief cells (“Zellballen”) separated by delicate fibrovascular stroma containing spindleoid sustentacular cells (E and F, arrows). The neoplastic chief cells have round to ovoid centrally located nuclei, typically with finely stippled chromatin and 1-2 prominent nucleoli, and moderate amounts of eosinophilic cytoplasm. Mitotic figures are rare. H&E. G-J. Immunohistochemical evaluation of the tumor from the case 2 female rat, including synaptophysin (G), cytokeratin (H), tyrosine hydroxylase (I) along with tyrosine hydroxlase from the case 1 male rat (J). The polygonal Type I chief cells had diffuse strong cytoplasmic staining for synaptophysin (G) and diffuse weak cytoplasmic staining for cytokeratin (H). Tyrosine hydroxlase staining in both the female (I) and male (J) rats was diffuse and of variable intensity. K-L. Immunohistochemical evaluation of the tumor from the case 2 female rat, including S-100 (K) and GFAP (L). The spindleoid Type II sustentacular cells (arrows) were immunopositive for S-100 (K) and GFAP (L).

Figure 9

A-D. Liver from male C57BL/6j mice, control (A) or treated with microcystin-LR (MCLR, B-D). (B). After 4 weeks of MCLR treatment, there is hepatocyte dissociation, multinucleation and karyocytomegaly, as well as loss of hepatocytes. Cellular infiltration by mononuclear cells and a few neutrophils is also present. (C) After 8 weeks of MCLR treatment, hepatocyte apoptosis, apoptotic bodies and karyocytomegaly are prominent. (D) After 13 weeks of MCLR treatment, there is almost complete regeneration, however cell proliferation continues with some abnormal mitoses, mild cellular infiltrates, and hepatocellular cytoplasmic alteration.

Figure 10

A-D. Coronal brain sections of 8-10-week-old Sprague Dawley (SD) rats showing the thalamus region. (A) Vehicle treated male SD rat. (B) Antibody treated male SD rat, (C) Vehicle treated male SD rat. (D) Antibody treated female SD rat. Note large mononuclear cells (arrows) with abundant granular cytoplasm present around blood vessels or in the neuroparenchyma. H&E. E-H. Coronal brain sections of 8-10-week-old control rats (E-F) show a few perivascular mast cells (arrows) in the thalamus region and the dark purple metachromatic granules are highlighted with toluidine blue stain. Sagittal brain section (G-H) of a ∼8-9-month-old untreated Sprague Dawley rat. Low magnification view shows the location of the thalamus (Thal), hippocampus (Hipp) and choroid plexus (CP) (G inset). The high magnification H&E (G) shows perivascular mast cells in the thalamus region (arrows). Tryptase immunohistochemistry (IHC) on a serial section (H) reveals immunopositive perivascular mast cells (arrows) in the same region as in (G). Tryptase IHC with fast red chromagen and hematoxylin counterstain.

Figure 10

A-D. Coronal brain sections of 8-10-week-old Sprague Dawley (SD) rats showing the thalamus region. (A) Vehicle treated male SD rat. (B) Antibody treated male SD rat, (C) Vehicle treated male SD rat. (D) Antibody treated female SD rat. Note large mononuclear cells (arrows) with abundant granular cytoplasm present around blood vessels or in the neuroparenchyma. H&E. E-H. Coronal brain sections of 8-10-week-old control rats (E-F) show a few perivascular mast cells (arrows) in the thalamus region and the dark purple metachromatic granules are highlighted with toluidine blue stain. Sagittal brain section (G-H) of a ∼8-9-month-old untreated Sprague Dawley rat. Low magnification view shows the location of the thalamus (Thal), hippocampus (Hipp) and choroid plexus (CP) (G inset). The high magnification H&E (G) shows perivascular mast cells in the thalamus region (arrows). Tryptase immunohistochemistry (IHC) on a serial section (H) reveals immunopositive perivascular mast cells (arrows) in the same region as in (G). Tryptase IHC with fast red chromagen and hematoxylin counterstain.

Figure 11

A-D. Increased bone, trabeculae and cortex in the tibia from an adult rat that had been given an anti-sclerostin antibody presented in case 1 (A-B). Figure A shows decalcified tibia of an age-matched control rat of the same sex. Figure B shows a section of the tibia from a treated rat with increased bone that is characterized by expanded thickness of metaphyseal trabeculae and cortex compared to the untreated rat. H&E. Reproduced with permission of the Japanese Society of Toxicologic Pathology from Fossey S, et al. Nonproliferative and proliferative lesions of the rat and mouse skeletal tissues (Bones, joints, and teeth). J Toxicol Pathol 29: 49S-103S, 2016. Decreased physeal thickness in the femur of an adult rat with severe food restriction presented in case 2 (C-D). Figure C shows a stained section of decalcified bone in an age-matched control rat of the same sex. Figure D shows the physis of a rat with severe food restriction at the same magnification. The growth plate of the rat with food restriction is slightly narrowed compared to that of the control. This change was confirmed with quantitative methods. H&E. Reproduced with permission of the Japanese Society of Toxicologic Pathology from Fossey S, et al. Nonproliferative and proliferative lesions of the rat and mouse skeletal tissues (Bones, joints, and teeth). J Toxicol Pathol 29: 49S-103S, 2016.