Inter-pathologist agreement on diagnosis, classification and grading of canine glioma.

Histopathological evaluation of tumours is a subjective process, but studies of inter-pathologist agreement are uncommon in veterinary medicine. The Comparative Brain Tumour Consortium (CBTC) recently published diagnostic criteria for canine gliomas. Our objective was to assess the degree of inter-pathologist agreement on intracranial canine gliomas, utilising the CBTC diagnostic criteria in a cohort of eighty-five samples from dogs with an archival diagnosis of intracranial glioma. Five pathologists independently reviewed H&E and immunohistochemistry sections and provided a diagnosis and grade. Percentage agreement and kappa statistics were calculated to measure inter-pathologist agreement between pairs and amongst the entire group. A consensus diagnosis of glioma subtype and grade was achieved for 71/85 (84%) cases. For these cases, percentage agreement on combined diagnosis (subtype and grade), subtype only and grade only were 66%, 80% and 82%, respectively. Kappa statistics for the same were 0.466, 0.542 and 0.516, respectively. Kappa statistics for oligodendroglioma, astrocytoma and undefined glioma were 0.585, 0.566 and 0.280 and were 0.516 for both low-grade and high-grade tumours. Kappa statistics amongst pairs of pathologists for combined diagnosis varied from 0.352 to 0.839. 8 % of archival oligodendrogliomas and 61% of archival astrocytomas were reclassified as another entity after review. Inter-pathologist agreement utilising CBTC guidelines for canine glioma was moderate overall but varied from fair to almost perfect between pairs of pathologists. Agreement was similar for oligodendrogliomas and astrocytomas but lower for undefined gliomas. These results are similar to pathologist agreement in human glioma studies and with other tumour entities in veterinary medicine.

Immunohistochemical evaluation of immune cell infiltration in canine gliomas.

Evasion of the immune response is an integral part of the pathogenesis of glioma. In humans, important mechanisms of immune evasion include recruitment of regulatory T cells (Tregs) and polarization of macrophages toward an M2 phenotype. Canine glioma has a robust immune cell infiltrate that has not been extensively characterized. The purpose of this study was to determine the distribution of immune cells infiltrating spontaneous intracranial canine gliomas. Seventy-three formalin-fixed, paraffin-embedded tumor samples were evaluated using immunohistochemistry for CD3, forkhead box 3 (FOXP3), CD20, Iba1, calprotectin (Mac387), CD163, and indoleamine 2,3-dioxygenase (IDO). Immune cell infiltration was present in all tumors. Low-grade and high-grade gliomas significantly differed in the numbers of FoxP3+ cells, Mac387+ cells, and CD163+ cells (P = .006, .01, and .01, respectively). Considering all tumors, there was a significant increase in tumor area fraction of CD163 compared to Mac387 (P < .0001), and this ratio was greater in high-grade tumors than in low-grade tumors (P = .005). These data warrant further exploration into the roles of macrophage repolarization or Treg interference therapy in canine glioma.

Chemical-Induced Oral Squamous Cell Neoplasms in Rodents: An Overview of NTP 2-Year Cancer Studies.

Oral cancer is the seventh most common malignancy worldwide, and lifestyle factors participate in its development. Rodent studies can help identify substances that contribute to its development and provide information on the early stages of carcinogenicity. The National Toxicology Program (NTP) has conducted more than 500 short-term and 2-year toxicology and carcinogenicity studies in rodents, and some of the tested compounds resulted in oral cancer. Our goal was to review the NTP carcinogenic studies to describe those chemicals that have oral carcinogenic outcome in rodents. For this project, we reviewed the results from all NTP carcinogenicity studies and a board-certified veterinary pathologist reviewed the slides from all neoplasms in the oral cavity that were considered treatment related. We have identified 26 chemicals with an adverse effect in the oral cavity. Fourteen chemicals demonstrated clear evidence of carcinogenicity in the oral cavity. We provide information on the carcinogenic findings in rodents together with a detailed description of the morphologic aspects of the oral cancers and speculate that the carcinogenic effects can be induced by different pathological modes of action. The findings reviewed here provide indicators for potential oral carcinogenesis processes in rodent models, which can be further investigated in future mechanistic studies.

Evaluation of 2-methoxy-4-nitroaniline (MNA) in hypersensitivity, 14-day subacute, reproductive, and genotoxicity studies.

2-Methoxy-4-nitroaniline (MNA), an intermediate in the synthesis of azo dyes used in textiles and paints, is structurally similar to carcinogenic anilines. Human exposure occurs primarily in the occupational setting through handling of dye dust, and through use and disposal of MNA-containing products. MNA has been reported to induce contact hypersensitivity in a human, myocardial necrosis in rats, and bacterial mutagenicity. This study assessed the subacute toxicity, genotoxicity, contact hypersensitivity, and reproductive toxicity of MNA in rodents in an effort to more fully characterize its toxicological profile. B6C3F1/N mice were exposed to 0, 650, 1250, 2500, 5000, or 10,000 ppm MNA by dosed feed for 14-days to evaluate subacute toxicity and histopathological endpoints. In female mice, decreased body weight (13.5 %) and absolute kidney weight (14.8 %), compared to control, were observed at 10,000 ppm MNA; increased relative liver weight (10-12 %), compared to control, occurred at 5,000-10,000 ppm MNA. In male mice, absolute (15 %) and relative liver weights (9-13 %) were increased at 2,500-5,000 ppm and 1250-10,000 ppm MNA, compared to control, respectively. In both sexes of mice, minimal elevations of hemosiderin pigmentation (a breakdown product of erythrocytes), relative to control, were observed in the liver (10,000 ppm); minimal to moderate elevations of hemosiderin pigmentation (5,000-10,000 ppm) and minimal increases in hematopoietic cell proliferation occurred in the spleen (≥ 1250 ppm). In a reproductive toxicity study, timed-mated female Harlan Sprague Dawley rats were exposed to 0-10,000 ppm MNA by dosed feed from gestation day 6 through postnatal day (PND) 21. Decreases in mean litter weights were observed at 5000 ppm MNA, compared to control, beginning at PND1. To evaluate potential contact hypersensitivity, MNA (2.5-50 %, in dimethylformamide) was applied to the dorsa of both ears of female Balb/c mice once daily for three days. The increase observed in lymph node cell proliferation (10-50 % increase in thymidine uptake compared to control) did not reproducibly achieve the Sensitization Index (SI) 3 level, and there was no ear swelling evident following sensitization with 10-50 % MNA and challenge with 25 % MNA in the mouse ear swelling test. In bacterial mutagenicity assays, MNA (250-1000 µg/plate) induced significant increases, compared to control, in mutant colonies with and without metabolic activation enzymes in Salmonella typhimurium strains TA100 and TA98. These data indicate that MNA is genotoxic, and may induce erythrocyte damage and reactive phagocytosis by macrophages in the liver and spleen.

Comparative Incidences and Biological Outcomes for Thymoma in Various Rat Strains in National Toxicology Program Studies.

Thymomas from 277 Fischer 344/N (F344/N), 10 Sprague Dawley (HSD:Sprague Dawley SD) (SD), 129 Wistar Han [Crl:WI(Han)] (WH), and 4 Wistar Outbred (WO) rats were reviewed from long-term studies in the National Toxicology Program (NTP) database. The incidence of thymomas in F344/N rats was slightly higher in males than in females, while the incidences in SD and WH rats were higher in females than in males. Only male WO rats were used in NTP studies. Of the 277 thymomas in F344/N rats, 235 (84.8%) were benign and 42 (15.2%) malignant, 14 of which exhibited metastasis. Of the 10 thymomas in SD rats, 5 (50%) were benign and 5 (50%) were malignant, one of which exhibited metastasis. Of the 129 thymomas in WH rats, 126 (98%) were benign and 3 (2%) were malignant, 1 with metastasis. Of the 4 thymomas in WO rats, 3 (75%) were benign and 1 (25%) was malignant, with no metastases. Malignant thymomas in F344/N and WH rats showed a propensity to be the cause of death and to result in early mortality, whereas the benign thymomas were associated less often with decreased survival. No occurrences of this neoplasm were reported to be related to exposure to any test articles.

Do GISTs Occur in Rats and Mice? Immunohistochemical Characterization of Gastrointestinal Tumors Diagnosed as Smooth Muscle Tumors in The National Toxicology Program.

The majority of the tumors in the gastrointestinal (GI) tract of rats and mice, with spindle cell morphology, are diagnosed as smooth muscle tumors (SMTs). Similarly, several decades ago human GI tumors with spindle cell morphology were also diagnosed as SMTs. However, later investigations identified most of these tumors in humans as gastrointestinal stromal tumors (GISTs). The GISTs are considered to arise from the interstitial cells of Cajal located throughout the GI tract. Positive immunohistochemical staining with CKIT antibody is a well-accepted diagnostic marker for GISTs in humans. Since there is a considerable overlap between the histomorphology of SMTs and GISTs, it is not possible to distinguish them on hematoxylin and eosin stained sections. As a result, GISTs are not routinely diagnosed in toxicological studies. The current study was designed to evaluate the tumors diagnosed as leiomyoma or leiomyosarcoma in the National Toxicology Program's 2-year bioassays using CKIT, smooth muscle actin, and desmin immunohistochemistry. The results demonstrate that most of the mouse SMTs diagnosed as leiomyoma or leiomyosarcoma are likely GISTs, whereas in rats the tumors are likely SMTs and not GISTs.

Uterine Paramesonephric Cysts in Sprague-Dawley Rats from National Toxicology Program Studies.

Congenital uterine wall cysts arising from paramesonephric (Müllerian) and mesonephric (Wolffian) ducts are typically incidental findings in most species. We used immunohistochemistry to characterize and determine the origin of uterine cysts in Sprague-Dawley (SD) rats from multigeneration studies conducted by the National Toxicology Program. Subserosal uterine cysts were observed in 20 of the 2,400 SD rats evaluated in five studies, and 10 cysts were characterized for this study. Single cysts were unilocular, fluid-filled, and occurred throughout the uterus. Microscopically, all cysts had a well-developed smooth muscle wall, lined by flattened to cuboidal, sometimes ciliated, epithelium that stained intensely positive for cytokeratin 18 and paired box protein 8 (PAX8). Most cyst epithelia displayed weak to moderate positivity for progesterone receptor (PR) and/or estrogen receptor α (ER-α), as well as were negative for GATA binding protein 3 (GATA3). Cyst lumens contained basophilic flocculent material. The cysts appeared to be developmental anomalies arising from paramesonephric tissue based on positive PAX8 and ER-α and/or PR staining. Additionally, 70% of the cysts lacked GATA3 expression. Taken together, the subserosal uterine cysts observed in adult rats in these studies most likely arose from the paramesonephric duct.

NTP/NIEHS Global Contributions to Toxicologic Pathology.

National Toxicology Program (NTP) pathologists are engaged in important initiatives that have significant global impact. These initiatives build on its leadership in pathology peer review and publications in the areas of toxicologic pathology, clinical pathology, and laboratory animal medicine. Over the past decade, NTP/National Institute of Environmental Health Sciences research initiatives have focused on cancer and noncancer hazard identification, with the goal of understanding cellular and molecular mechanisms of disease. New initiatives of significant global impact include the web-based nonneoplastic lesion atlas and an NTP partnership with international scientists to investigate molecular mechanisms at the whole genome level, which will be used to inform potential mechanisms of environmental exposures in human cancers. Also, we are dedicated to contributing to pathology and toxicology organizations through service on executive committees and editorial boards, participating in international projects and symposiums, and providing training for future leaders in toxicologic pathology. Herein, we provide highlights of our global contributions.

Comparative inhalation toxicity of ethyltoluene isomers in rats and mice.

The C9 alkylbenzenes, composed mostly of ethyltoluenes and trimethylbenzenes, comprise 75-90% of the naphtha fraction of crude oil. Occupational and environmental exposure to C9 alkylbenzenes occur via inhalation. We conducted short-term inhalation studies on the ethyltoluene isomers (2-, 3- or 4-) to select one isomer for more comprehensive studies. Male Hsd:Sprague Dawley rats and female B6C3F1/N mice (n = 10) were exposed by nose-only inhalation to 2-, 3- or 4-ethyltoluene (0, 1000 or 2000 ppm) or cumene (a reference compound: 0, 500 or 1000 ppm) 3 h/day, 5 days/week, for 2 weeks. Clinical observations included abnormal gait and delayed righting reflex. Rats and mice exposed to 2000 ppm 2-ethyltoluene and mice exposed to 2000 ppm 4-ethyltoluene were euthanized early in moribund condition; no exposure-related deaths were observed with 3-ethyltoluene or cumene. Histopathology of selected tissues revealed that the nose and liver (rats and mice) and lung (mice only) to be toxicity targets. In the mouse lung, all compounds except 4-ethyltoluene produced bronchial and bronchiolar hyperplasia. In rats and mice, 2-ethyltoluene was the only compound to produce lesions in the nose and liver: in mice, squamous metaplasia and neutrophilic inflammation of the respiratory epithelium and atrophy and degeneration of the olfactory epithelium were observed in the nose and centrilobular hypertrophy and necrosis were observed in the liver. In rats, 2-ethyltoluene exposure produced atrophy of the olfactory epithelium in the nose and centrilobular necrosis in the liver. Based on mortality, body weight effects and histopathology, the 2-ethyltoluene isomer was the most potent isomer.

Proceedings of the 2016 National Toxicology Program Satellite Symposium.

The 2016 annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in San Diego, CA, at the Society of Toxicologic Pathology's (STP) 35th 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. Some lesions and topics covered during the symposium included malignant glioma and histiocytic sarcoma in the rodent brain; a new statistical method designed for histopathology data evaluation; uterine stromal/glandular polyp in a rat; malignant plasma cell tumor in a mouse brain; Schwann cell proliferative lesions in rat hearts; axillary schwannoma in a cat; necrosis and granulomatous inflammation in a rat brain; adenoma/carcinoma in a rat adrenal gland; hepatocyte maturation defect and liver/spleen hematopoietic defects in an embryonic mouse; distinguishing malignant glioma, malignant mixed glioma, and malignant oligodendroglioma in the rat; comparison of mammary gland whole mounts and histopathology from mice; and discussion of the International Harmonization of Nomenclature and Diagnostic Criteria collaborations.

Proceedings of the 2015 National Toxicology Program Satellite Symposium.

The 2015 Annual National Toxicology Program Satellite Symposium, entitled "Pathology Potpourri" was held in Minneapolis, Minnesota, at the American College of Veterinary Pathologists/American Society for Veterinary Clinical Pathology/Society of Toxicologic Pathology combined meeting. The goal of this symposium is to present and discuss diagnostic pathology challenges or nomenclature issues. Because of the combined meeting, both laboratory and domestic animal cases were presented. This article presents summaries of the speakers' talks, including challenging diagnostic cases or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included hepatocellular lesions, a proposed harmonized diagnostic approach to rat cardiomyopathy, crop milk in a bird, avian feeding accoutrement, heat exchanger in a tuna, metastasis of a tobacco carcinogen-induced pulmonary carcinoma, neurocytoma in a rat, pituicytoma in a rat, rodent mammary gland whole mounts, dog and rat alveolar macrophage ultrastructure, dog and rat pulmonary phospholipidosis, alveolar macrophage aggregation in a dog, degenerating yeast in a cat liver aspirate, myeloid leukemia in lymph node aspirates from a dog, Trypanosoma cruzi in a dog, solanum toxicity in a cow, bovine astrovirus, malignant microglial tumor, and nomenclature challenges from the Special Senses International Harmonization of Nomenclature and Diagnostic Criteria Organ Working Group.

Utilizing Whole Slide Images for Pathology Peer Review and Working Groups.

This article describes the results of comparisons of digitally scanned whole slide images (WSIs) and glass microscope slides for diagnosis of tissues under peer review by the National Toxicology Program. Findings in this article were developed as a result of the data collected from 6 pathology working groups (PWGs), 1 pathology peer review, and survey comments from over 25 participating pathologists. For each PWG, 6-14 pathologists examined 10-143 tissues per study from 6- and 9-month perinatal studies and 2-year carcinogenicity studies. Overall it was found that evaluation of WSIs is generally equivalent to using glass slides. Concordance of PWG consensus diagnoses based upon review of WSIs versus glass slides ranged from 74% to 100% (median 86%). The intra- and interobserver diagnostic variation did not appear to influence the conclusions of any study. Based upon user opinions collected from surveys, WSIs may be less optimal than glass slides for evaluation of subtle lesions, large complex lesions, small lesions in a large section of tissue, and foci of altered hepatocytes. These results indicate that, although there may be some limitations, the use of WSIs can effectively accomplish the objectives of a conventional glass slide review and definitely serves as a useful adjunct to the conduct of PWGs.

Genomic Profiling Reveals Unique Molecular Alterations in Hepatoblastomas and Adjacent Hepatocellular Carcinomas in B6C3F1 Mice.

The cell of origin of hepatoblastoma (HB) in humans and mice is unknown; it is hypothesized to be a transformed hepatocyte, oval cell, or hepatic progenitor cell. In mice, current dogma is that HBs arise from preexisting hepatocellular neoplasms as a result of further neoplastic transformation. However, there is little evidence supporting this direct relationship. To better understand the relationship between hepatocellular carcinoma (HCC) and HB and determine molecular similarities between mouse and human HB, global gene expression analysis and targeted mutation analysis were performed using HB, HCC, and adjacent liver from the same animals in a recent National Toxicology Program bioassay. There were significant differences in Hras and Ctnnb1 mutation spectra, and by microarray, HBs showed dysregulation of embryonic development, stem cell pluripotency, and genomic imprinting compared to HCC. Meta-analysis showed similarities between HB, early mouse embryonic liver, and hepatocyte-derived stem/progenitor cells compared to HCC. Our data show that there are striking differences between HB and HCC and suggest that HB is a significantly different entity that may arise from a hepatic precursor cell. Furthermore, mouse HB is similar to the human disease at the pathway level and therefore is likely a relevant model for evaluating human cancer hazard.

Species and gender differences in the carcinogenic activity of trimethylolpropane triacrylate in rats and mice.

Trimethylolpropane triacrylate (TMPTA) is a multifunctional monomer with industrial applications. To determine the carcinogenic potential, male and female F344/N rats and B6C3F1/N mice were administered TMPTA (0, 0.3, 1.0, or 3.0mg/kg) in acetone dermally for 2 years. There were no differences in the body weights and survival in the treated animals compared to controls. Nonneoplastic skin lesions at the site of application included epidermal hyperplasia and hyperkeratosis in both rats and mice. There were no incidences of tumors at the site of application in rats and mice. Rare malignant liver neoplasms were observed in female mice that included hepatoblastoma in the 0.3 and 3.0mg/kg groups, and hepatocholangiocarcinoma in the 1.0 and 3.0mg/kg groups. The incidences of uterine stromal polyp and stromal polyp or stromal sarcoma (combined) in female mice occurred with positive trends and the incidences were significantly increased in the 3.0mg/kg group. A marginal increase in the incidences of malignant mesothelioma in male rats may have been related to TMPTA treatment. In conclusion, our studies show that TMPTA is a dermal irritant in both rats and mice of either sex. Increased incidences of tumor formation were observed in female mice and male rats.

Mutation spectra of Kras and Tp53 in urethral and lung neoplasms in B6C3F1 mice treated with 3,3',4,4'-tetrachloroazobenzene.

3,3',4,4'-tetrachloroazobenzene (TCAB) is a contaminant formed during manufacture of various herbicide compounds. A recent National Toxicology Program study showed B6C3F1 mice exposed to TCAB developed a treatment-related increase in lung carcinomas in the high-dose group, and urethral carcinomas, an extremely rare lesion in rodents, in all dose groups. As the potential for environmental exposure to TCAB is widespread, and the mechanisms of urethral carcinogenesis are unknown, TCAB-induced urethral and pulmonary tumors were evaluated for alterations in critical human cancer genes, Kras and Tp53. Uroplakin III, CK20, and CK7 immunohistochemistry was performed to confirm the urothelial origin of urethral tumors. TCAB-induced urethral carcinomas harbored transforming point mutations in K-ras (38%) and Tp53 (63%), and 71% displayed nuclear TP53 expression, consistent with formation of mutant protein. Transition mutations accounted for 88% of Tp53 mutations in urethral carcinomas, suggesting that TCAB or its metabolites target guanine or cytosine bases and that these mutations are involved in urethral carcinogenesis. Pulmonary carcinomas in TCAB-exposed animals harbored similar rates of Tp53 (55%) and Kras (36%) mutations as urethral carcinomas, suggesting that TCAB may induce mutations at multiple sites by a common mechanism. In conclusion, TCAB is carcinogenic at multiple sites in male and female B6C3F1 mice through mechanisms involving Tp53 and Kras mutation.

Proceedings of the 2013 National Toxicology Program Satellite Symposium.

The 2013 annual National Toxicology Program (NTP) Satellite Symposium, entitled "Pathology Potpourri," was held in Portland, Oregon, in advance of the Society of Toxicologic Pathology's 32nd annual meeting. The goal of the NTP Symposium is to present current diagnostic pathology or nomenclature issues to the toxicologic pathology community. This article presents summaries of the speakers' presentations, including diagnostic or nomenclature issues that were presented, along with select images that were used for audience voting and discussion. Some lesions and topics covered during the symposium included a caudal tail vertebra duplication in mice; nephroblastematosis in rats; ectopic C cell tumor in a hamster; granular cell aggregates/tumor in the uterus of a hamster; Pneumocystis carinii in the lung of a rat; iatrogenic chronic inflammation in the lungs of control rats; hepatoblastoma arising within an adenoma in a mouse; humoral hypercalcemia of benignancy in a transgenic mouse; acetaminophen-induced hepatotoxicity in rats; electron microscopy images of iatrogenic intraerythrocytic inclusions in transgenic mice; questionable hepatocellular degeneration/cell death/artifact in rats; atypical endometrial hyperplasia in rats; malignant mixed Müllerian tumors/carcinosarcomas in rats; differential diagnoses of proliferative lesions of the intestine of rodents; and finally obstructive nephropathy caused by melamine poisoning in a rat.

Perinatal toxicity and carcinogenicity studies of styrene-acrylonitrile trimer, a ground water contaminant.

Styrene acrylonitrile (SAN) trimer is a by-product in the production of acrylonitrile styrene plastics. Following a report of a childhood cancer cluster in the Toms River section of Dover Township, New Jersey, SAN Trimer was identified as one of the groundwater contaminants at Reich Farm Superfund site in the township. The contaminants from the Reich Farm site's ground water plume impacted two wells at the Parkway well field. The National Toxicology Program (NTP) studied the toxicity and carcinogenicity of SAN Trimer in rats exposed during their perinatal developmental period and adulthood. The chronic toxicity and carcinogenicity studies in F344/N rats were preceded by 7- and 18-week perinatal toxicity studies to determine the exposure concentrations for the 2-year studies. Subsequently, Fisher 344 pregnant dams were exposed to SAN Trimer containing diet at 400, 800, or 1600ppm concentrations during gestation, nursing and weaning periods of offspring followed by two year of adult exposures to both male and female pups. There was no statistically significant evidence of carcinogenic activity following SAN-Trimer exposure; however, rare neoplasms in the brain and spinal cord were observed in males and to lesser extent in female rats. These incidences were considered within the range of historical background in the animal model used in the current studies. Therefore, the presence of a few rarely occurring CNS tumors in the treated groups were not judged to be associated with the SAN Trimer exposure. The major finding was a dose-related peripheral neuropathy associated with the sciatic nerves in females and spinal nerve roots in males and females thereby suggesting that SAN Trimer is potentially a nervous system toxicant.

Evaluation of expression profiles of hematopoietic stem cell, endothelial cell, and myeloid cell antigens in spontaneous and chemically induced hemangiosarcomas and hemangiomas in mice.

It is unclear whether the process of spontaneous and chemically induced hemangiosarcoma and hemangioma formation in mice involves the transformation of differentiated endothelial cells (ECs) or recruitment of multipotential bone marrow-derived hematopoietic stem cells or endothelial progenitor cells (EPCs), which show some degree of endothelial differentiation. In the present study, immunohistochemical staining for hematopoietic stem cell markers (CD45 and CD34), EC markers (vascular endothelial growth factor receptor 2 [VEGFR2], CD31, and factor VIII-related antigen), and a myeloid lineage marker (CD14) was employed to better define the origin of hemangiosarcomas and hemangiomas in mice. Staining was negative for CD45, factor VIII-related antigen, and CD14 and positive for CD34, VEGFR2, and CD31, indicating that mouse hemangiosarcomas and hemangiomas are composed of cells derived from EPCs expressing CD34, VEGFR2, and CD31 but not factor VIII-related antigen. The lack of CD45 expression suggests that mouse vascular tumors may arise from EPCs that are at a stage later than hematopoietic stem cells. Since factor VIII-related antigen expression is known to occur later than CD31 expression in EPCs, our observations may indicate that these tumor cells are arrested at a stage prior to complete differentiation.  In addition, myeloid lineage cells do not appear to contribute to hemangiosarcoma and hemangioma formation in mice.

Comparative histomorphological review of rat and human hepatocellular proliferative lesions.

In this comparative review, histomorphological features of common nonneoplastic and neoplastic hepatocyte lesions of rats and humans are examined using H&E-stained slides. The morphological similarities and differences of both neoplastic (hepatocellular carcinoma and hepatocellular adenoma) and presumptive preneoplastic lesions (large and small cell change in humans and foci of cellular alteration in rats) are presented and discussed. There are major similarities in the diagnostic features, growth patterns and behavior of both rat and human hepatocellular proliferative lesions and in the process of hepatocarcinogenesis. Further study of presumptive preneoplastic lesions in humans and rats should help to further define their role in progression to hepatocellular neoplasia in both species.

Testing an aflatoxin B1 gene signature in rat archival tissues.

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 µg of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ≤ 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and ß-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures.