View as PDF
Ute Bach, James R. Hailey, Georgette D. Hill, Wolfgang Kaufmann, Kenneth S. Latimer, David E. Malarkey, Robert M. Maronpot, Rodney A. Miller, Rebecca R. Moore, James P. Morrison, Thomas Nolte, Matthias Rinke, Susanne Rittinghausen, Andrew W. Suttie, Gregory S. Travlos, John L. Vahle, Gabrielle A. Willson, and Susan A. Elmore
View as PDF

The National Toxicology Program (NTP) Satellite Symposium is a one-day meeting that is held in conjunction with the annual Society of Toxicologic Pathology (STP) meeting. The topic of the 2009 Symposium was ‘‘Tumor Pathology and INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Nomenclature.’’ The goal of this article is to provide summaries of each speaker’s presentation, including the diagnostic or nomenclature issues that were presented, along with a few select images that were used for voting. The results of the voting process and interesting points of discussion that were raised during the presentation are also provided. A supplemental file with voting choices and voting results for each case presented at the symposium is available at http://tpx.sagepub.com/supplemental.

Keywords

NTP, satellite, symposium, INHAND, nomenclature.

Introduction

The NTP Satellite Symposium was originally conceived and implemented by Dr. Robert Maronpot as a way to present current diagnostic pathology issues to the toxicological pathology community. The first Symposium was held in 2000, and the topic was ‘‘Establishing an NTP Database for NonNeoplastic Lesions of Kidney and Urinary Bladder.’’ There was no Symposium the following year, but in 2002, the NTP Satellite Symposium returned by popular demand and has been a consistent and highly regarded component of the annual STP meeting since that time. The history of meeting topics, dates, and locations is presented in Table 1. The Symposium has served as a forum to present and discuss lesions that are rare and interesting, present a diagnostic challenge, are controversial, or have nomenclature dilemmas. It was decided to hold the Symposium on the Saturday before the annual Society of Toxicologic Pathology (STP) meeting, as doing so would conveniently allow any interested STP registrants to attend. The Symposium has always been held at no cost to the participants, and attendance has been around 200 for the past few years.

The objective of the NTP Satellite Symposium has always been to provide continuing education on the interpretation of pathology slides, in terms of diagnostic or nomenclature dilemmas, that the bench pathologist may see from time to time. An equally important component of the Symposium has been to incorporate audience participation through anonymous voting and the generation of lively and productive conversation after the voting. The theme for the 2009 Symposium was ‘‘Tumor Pathology,’’ corresponding with the STP meeting theme of cancer. We also included examples and discussion of International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) nomenclature as it pertains to neoplastic lesions. During each presentation, the speakers projected a series of lesion images on one screen with a choice of potential diagnostic choices listed on a separate screen. The members of the audience then voted anonymously with wireless keypads, and the voting results were displayed as bar graphs with percentages on the screen. After each voting session, the speakers sometimes presented additional data that may aid in the decision of diagnosis or choice of terminology, and then time was allowed for discussion. Occasionally, revotes were taken after the discussion period. In this article, the speakers have provided synopses of their presentations at the 2009 Symposium, including the diagnostic or nomenclature issues, a selection of images presented during the Symposium, voting choices, voting results, and discussion points.

2009 National Toxicology Program Satellite Symposium-t1

Proliferative Adrenal Medullary Lesions in Rats

The first presentation, given by Dr. Georgette Hill, focused on proliferative adrenal medullary lesions in rats. An initial overview of the various lesions and primary diagnostic criteria was presented. Three diagnostic dilemmas were shown to Satellite Symposium participants. For case 1, a medullary mass (Figure 1A) with pleomorphic neoplastic cells (Figure 1B) was shown. Similar neoplastic cells were present in the periadrenal vasculature (Figure 1C). The diagnostic voting choices for case 1 were (1) adrenal medullary hyperplasia, (2) benign pheochromocytoma, (3) malignant pheochromocytoma, (4) complex pheochromocytoma, and (5) ganglioneuroma. Issues raised during the participant discussion of case 1 included the difficulty of identifying vascular invasion in a low-power image as depicted in Figure 1A, as well as whether vascular invasion is considered a feature of malignancy. Dr. Hill mentioned that, depending on the reference book used, the primary features of adrenal medullary malignancy are adrenal capsule invasion and the presence of distant metastasis (Hamlin and Banas 1990). Although neither of these was a feature of case 1, the majority of the participants (63% after a revote) agreed that vascular invasion and pleomorphic neoplastic cells classified this mass as a malignant pheochromocytoma, which was also the NTP choice.

A compressive medullary mass was shown for case 2 (Figure 1D). The diagnostic voting choices for case 2 were (1) adrenal medullary hyperplasia, (2) benign pheochromocytoma, (3) malignant pheochromocytoma, (4) neuroblastoma, and (5) hemangiosarcoma. The NTP choice, from a pathology working group (PWG) vote, was malignant pheochromocytoma because of the necrosis and presence of neoplastic cells in vessels within the tumor; however, the majority of the Satellite Symposium participants (49%) voted for neuroblastoma. Issues raised during audience discussion for case 2 included neoplastic cells appearing neuroblastoma-like owing to poor resolution on the screen and that neuroblastomas are rare neoplasms in the adrenal medulla of rats. For comparative purposes, a neuroblastoma (Figure 1E) is shown at a similar magnification (40X) to the adrenal medulla mass from case 2 (Figure 1F). The closely packed neuroblast-like cells with scant cytoplasm in Figure 1E were compared to the sheet of neoplastic chromaffin cells with abundant cytoplasm along the area of compression in Figure 1F.

A focal medullary lesion that did not compress the surrounding parenchyma was shown for case 3 (Figure 1G). The diagnostic voting choices for case 3 were (1) adrenal medullary hyperplasia, (2) benign pheochromocytoma, (3) malignant pheochromocytoma, (4) angiectasis, and (5) hemangiosarcoma. The majority of the Satellite Symposium participants (67%) voted for adrenal medullary hyperplasia, which was also considered the NTP choice via PWG vote.

Image_002

Fig 1. (A) Malignant pheochromocytoma with neoplastic cells in the periadrenal vasculature. (B) Pleomorphic neoplastic cells from the malignant pheochromocytoma shown in (A). (C) Neoplastic cells in the lumen of a periadrenal blood vessel from the malignant pheochromocy-toma shown in (A). (D) Compressive adrenal medullary mass with necrosis. (E) Cellular features of an adrenal medullary neuroblastoma. Note the closely packed neoplastic neuroblasts with scant cytoplasm. (F) Cellular features of the malignant pheochromocytoma shown in (D). Note the sheet of neoplastic chromaffin cells with abundant cytoplasm. (G) Focal hyperplasia of the adrenal medulla.

Fig 1. (A) Malignant pheochromocytoma with neoplastic cells in the periadrenal vasculature. (B) Pleomorphic neoplastic cells from the malignant pheochromocytoma shown in (A). (C) Neoplastic cells in the lumen of a periadrenal blood vessel from the malignant pheochromocy-toma shown in (A). (D) Compressive adrenal medullary mass with necrosis. (E) Cellular features of an adrenal medullary neuroblastoma. Note the closely packed neoplastic neuroblasts with scant cytoplasm. (F) Cellular features of the malignant pheochromocytoma shown in (D). Note the sheet of neoplastic chromaffin cells with abundant cytoplasm. (G) Focal hyperplasia of the adrenal medulla.

Hepatocholangiocarcinoma: The Great Masquerader

The second presentation, by Dr. Rodney Miller, was based on the collaborative work of Drs. Rodney Miller, Rebecca Moore, and Gabrielle Willson and was titled “The Great Masquerader.” Dr. Miller’s discussion emphasized the various histomorphology patterns of hepatocholangiocarcinoma (HCCC) and thus the potential for diagnostic challenges.

After presentation of numerous images demonstrating the highly variable morphological patterns seen in the primary and metastatic lesions, a vote was taken, given the following diagnostic choices: (1) epithelioid hemangioendothelioma, (2) mesothelioma, (3) HCCC, (4) hepatocellular carcinoma, (5) atriocaval mesothelioma, and (6) hepatoblastoma. Hepatocholangiocarcinoma was the correct response and recorded a 57% vote. The other votes recorded were: epithelioid hemangioendothelioma (7%), mesothelioma (18%), hepatocellular carcinoma (2%), atriocaval mesothelioma (4%), and hepatoblastoma (12%). After the vote, a detailed presentation of HCCCs in the NTP database was given.

Hepatocholangiocarcinoma is a rare tumor with an overall incidence of less than 1% in male and female mice in the current NTP database. Hepatocholangiocarcinoma is defined as a combination of neoplastic hepatocytes and neoplastic biliary epithelial cells. In addition to the proliferative hepatocellular and biliary epithelial components, there may be a poorly differentiated cellular component that has a sarcomatous, anaplastic, and undifferentiated appearance. The variable histological morphology of the primary HCCC and the metastatic lesions may pose a diagnostic challenge. The purpose of this research and presentation was to describe the morphological features, incidence, and behavior of HCCC in B6C3F1 mice. The NTP historical database from 1982 to 2008 was reviewed for a diagnosis of HCCC in B6C3F1 mice (retrieved August 27, 2009, from the National Toxicology Program Web site: http://ntp-server.niehs.nih.gov). A total of 164 HCCCs (ninety-five male and sixty-nine female mice) from 74 two-year carcinogenicity studies were identified and reviewed.

Hepatocholangiocarcinoma occurred more commonly in males (58%) than in females (42%). Metastases were evident in 138 animals (84%) and often occurred in multiple sites. The sites most frequently observed to have metastatic lesions included lung (125/138, or 91%), mediastinum (104/138, or 75%), mesentery (80/138, or 58%), lymph nodes (71/138, or 51%), skeletal muscle (55/138, or 40%), kidney (47/138, or 34%), heart (46/ 138, or 33%), and pancreas (10/138, or 7%). Hepatocholangiocarcinomas were not considered to be treatment related in any study; however, in twenty-four of the seventy-four studies (32%), there was a concomitant treatment-related increase in hepatocellular tumors in one or both sexes. Although most of the NTP mouse control groups from this period did not have a diagnosis of HCCC, the highest incidence of spontaneous HCCC in any control group was as high as two of forty-nine for female controls and five of fifty for male controls.

All HCCCs reviewed in this study had a malignant hepatocellular component, as required by definition. The vast majority of HCCCs had small to large areas of necrosis and/or small to large areas of cystic spaces. The cystic areas had ill-defined epithelial linings or were partially lined by a flattened to cuboidal epithelium. Foci of proliferating epithelial cells forming ductal structures, representing the malignant biliary component, were often evident in close proximity to these areas of necrosis, or the cystic areas. Not infrequently, these foci were small and difficult to appreciate upon cursory examination (Figure 2A). Occasionally (about 16%), there were focal areas of proliferation of undifferentiated round to spindle cells in a loose fibrillar matrix within or adjacent to the malignant hepatocellular component (Figure 2B). It was not unusual to have transformation of neoplastic hepatocytes in the same proliferating hepatic cord into varieties of columnar epithelial cells to form ducts (Figure 2C).

The diagnostic challenge often began with the metastatic lesions. The metastatic lesions were often grossly visible, multifocal, and tan and occurred within the parenchyma of the tissue bearing the metastasis. The metastatic lesions also commonly extended to and occurred on serosal surfaces, where they stimulated serosal lining cells to react. If classical malignant hepatocellular components were present in the metastatic lesions along with the neoplastic duct-forming cells or the undifferentiated or spindle cells (Figures 2D and 2E), it stimulated the pathologist to return to the liver to confirm the presence of HCCC.

Commonly, the metastatic lesions contained only epithelial cells of the malignant biliary component (as was often the case in mesenteric metastases), an undifferentiated component, or sometimes a combination of both. Less commonly, the metastatic lesions contained the malignant hepatocellular component. The undifferentiated component was composed of nondescript round cells or spindle cells resembling a sarcoma. When this sometimes puzzling, but virtually pathognomonic, pattern of metastasis is seen, it often leaves the pathologist wondering whether the primary tumor is a carcinoma, a sarcoma, a mesothelioma, or a neoplasm of unknown histogenesis. The pathologist should then be stimulated to review the primary liver mass and evaluate it closely, looking for the diagnostic trilogy that leads to a diagnosis of HCCC: the malignant hepatocellular component, areas of necrosis or cystic degeneration, and the ducts of the malignant biliary component. The presence of a focal and undifferentiated or spindle cell proliferation in the liver mass adds even more strength to the argument and helps confirm that the primary tumor should be called an HCCC.

In summary, HCCC is a rare tumor in mice and is an aggressive tumor that metastasizes readily. The HCCC metastatic rate of 84% is much higher than that of hepatocellular carcinomas (about 23%) or hepatoblastomas (about 23%) (Turusov et al. 2002). Hepatocholangiocarcinoma was more commonly observed in male mice than in female mice in these NTP studies. Although it has been reported to be induced by chemicals such as benzidine dihydrochloride and N-2-acetoaminofluorene (Frith, Ward, and Turusov 1994), treatment-related HCCCs were not observed in the NTP dataset. Hepatocholangiocarcinoma can have a highly variable histological morphology, and careful evaluation of all features of the primary and metastatic lesions must be taken into account to render the correct diagnosis. Careful consideration must be given to hepatocellular proliferation, biliary epithelial proliferation, undifferentiated cell proliferation, cystic areas, and necrotic areas in the primary liver tumor and put into context with any combination of hepatocellular, biliary or undifferentiated cell proliferation in the metastatic lesions.

Fig 2. (A) Primary liver neoplasm demonstrating the classic presentation of a hepatocholangiocarcinoma (HCCC) as seen in the B6C3F1 mouse. There is an area of malignant hepatocellular proliferation (arrow) and areas of malignant ductal cell proliferation (arrowhead) adjacent to an area of necrosis and near an area of a cystlike formation (asterisk). (B) A portion of a primary HCCC demonstrating an area of malignant duct proliferation (arrow) and a focus of undifferentiated malignant cell proliferation (arrowhead) in a bed of hepatocellular carcinoma. (C) A primary HCCC demonstrating cords of hepatocellular carcinoma transitioning/blending into malignant cuboidal and columnar epithelium, forming ducts of varying morphological character. (D) A metastatic lesion of an HCCC as seen in the lung. Note the presence of malignant hepatocytes and duct-forming epithelial cells. (E) An HCCC that metastasized to the lung that demonstrates malignant hepatocyte proliferation and adjacent proliferation of an undifferentiated cell type.

Fig 2. (A) Primary liver neoplasm demonstrating the classic presentation of a hepatocholangiocarcinoma (HCCC) as seen in the B6C3F1 mouse. There is an area of malignant hepatocellular proliferation (arrow) and areas of malignant ductal cell proliferation (arrowhead) adjacent to an area of necrosis and near an area of a cystlike formation (asterisk). (B) A portion of a primary HCCC demonstrating an area of malignant duct proliferation (arrow) and a focus of undifferentiated malignant cell proliferation (arrowhead) in a bed of hepatocellular carcinoma. (C) A primary HCCC demonstrating cords of hepatocellular carcinoma transitioning/blending into malignant cuboidal and columnar epithelium, forming ducts of varying morphological character. (D) A metastatic lesion of an HCCC as seen in the lung. Note the presence of malignant hepatocytes and duct-forming epithelial cells. (E) An HCCC that metastasized to the lung that demonstrates malignant hepatocyte proliferation and adjacent proliferation of an undifferentiated cell type.

“Kneejerks” Welcomed: Diagnostic Value of Immunohistochemistry

Dr. Dave Malarkey of the NTP presented three cases that didn’t necessarily elicit a “kneejerk” diagnosis from participants, each one illustrating the diagnostic utility of various immunohistochemical markers. The first case was a brain mass from a male B6C3F1 mouse treated daily for ninety days with sodium dichromate dihydrate (Figures 3A and 3B). The voting choices were: (1) meningothelial meningioma, (2) transitional meningioma, (3) meningioma not otherwise specified (NOS), (4) malignant glioma, (5) histiocytic sarcoma,