==== Front J Toxicol PatholJ Toxicol PatholTOXJournal of Toxicologic Pathology0914-91981881-915XJapanese Society of Toxicologic Pathology 2017-006710.1293/tox.2017-0067Case ReportSpontaneous pulmonary co-metastasis of hepatoblastoma arising within a hepatocellular carcinoma in an aged C57BL/6J mouse Castiglioni Vittoria 1 * Radaelli Enrico 2 3 1 IDEXX Laboratories Italia S.r.l., Via Guglielmo Silva 36, Milano 20149, Italy2 Comparative Pathology Core, Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3900 Delancey Street, Philadelphia, PA 19104-6051, U.S.A.3 VIB Center for the Biology of Disease and KU Leuven Center for Human Genetics, Herestraat 49, Leuven 3000, Belgium*Corresponging author: V Castiglioni (e-mail: vittoria-castiglioni@idexx.com)23 6 2018 7 2018 31 3 195 199 01 12 2017 20 4 2018 ©2018 The Japanese Society of Toxicologic Pathology2018This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/）. Murine hepatoblastoma (HB) is a rare spontaneous tumor with controversial histogenesis. It mainly occurs in aged males, frequently in close association with preexisting hepatocellular neoplasms. The present work describes a spontaneous HB arising within a hepatocellular carcinoma (HCC) in a 22-month-old male C57BL/6J mouse. The mouse also developed pulmonary co-metastases with either tumor components physically associated within the same metastatic foci. Microscopically, the HB consisted of a densely cellular neoplastic growth composed of palisades and perivascular pseudorosettes of poorly differentiated primitive cells, with a scant amount of cytoplasm, elongated hyperchromatic nuclei, and a high mitotic rate, whereas the hepatocellular carcinoma was composed of solid areas of neoplastic hepatocytes. Both in primary tumors and their metastases, β-catenin immunohistochemistry revealed a strong nucleocytoplasmic signal in HB cells, while neoplastic hepatocytes displayed a delicate membranous staining pattern. These findings suggest that the Wnt/β-catenin oncogenic pathway is upregulated in murine HB but not in the co-existing HCC, thus providing some insights into their divergent pathogenesis. Coexisting murine HB and HCC have been demonstrated to be completely distinct entities including origin, mutational landscape, and molecular profile. In this context, they might be regarded as collision tumors because of their intimate association, unique histologic features, and distinct immunohistochemical patterns. Nevertheless, the nature of their coevolution and progression to a co-metastatic phenotype reflects a close interdependence and support the overall idea that HB’s origin and progression might be promoted by not otherwise specified paracrine stimuli provided by the concurrent hepatocellular tumor (the so called “interaction theory”). hepatoblastomaimmunohistochemistrymousepathogenesispulmonary metastases ==== Body Hepatoblastoma (HB) is a primitive, poorly differentiated, embryonal hepatic neoplasm. In mice, HBs are rare spontaneous entities that mainly occur in aged male animals1 and are usually seen within or adjacent to preexisting hepatocellular adenomas (HCAs) or carcinomas (HCCs)2, 3. The cell of origin of HBs remains unknown, although liver blastemal cells, neoplastic hepatocytes, oval cells, and biliary epithelial cells have been proposed3. Previously considered to be the result of malignant progression of benign or low-grade hepatocellular tumors, it has been recently documented that murine hepatoblastoma most likely represents a distinct entity displaying a completely different mutational spectrum and transcriptome if compared with hepatocellular tumors4. In humans, HBs are mainly observed in young children below the age of 5 years and are exceptionally rare in adults5, 6. In contrast to their murine counterpart, human HBs are always solitary tumors unassociated with preexisting hepatocellular neoplasias and are thought to arise de novo from hepatoblast remnants of the embryonic liver2, 7, 8. On the other hand, HBs in both mice and humans share similarities, such as primitive morphologic features, predominance in the male gender, and a comparable metastatic rate with lungs as the most common metastatic site2, 9. Furthermore, genetic alterations have been associated with this malignancy, with human and mouse HBs both harboring oncogenic mutations in Ctnnb110, 11 leading to constitutive activation of the Wnt/β-catenin pathway. Considering nonhuman species, hepatoblastomas have been described in equine fetuses, neonates, and occasionally young adult horses 12, 13, 14, in young and adult sheep15, and in a cat16, llama17 and dog18. In all these species, HBs have been mainly reported in young animals, paralleling the human counterpart, with only a minority of cases occurring in adults. With reference to laboratory animals, there are rare reports of this lesion in rats3. A single case was described in a 10-day-preterm fetus of a male cynomolgus macaque (Macaca fascicularis)19. This study aimed to describe pathological features of a spontaneous murine condition characterized by the co-occurrence of HB and HCC and development of pulmonary co-metastases consisting of closely associated neoplastic cells from both the hepatic cancers. The animal considered in this study was an experimentally naïve 22-month-old male C57BL/6J mouse included as control in an aging study. The mouse was sacrificed because of generalized deterioration of clinical conditions associated with marked abdominal enlargement and dyspnea. A complete necropsy and histopathological examination were performed following the same protocols and procedures as described by Castiglioni et al.20. For immunohistochemistry (IHC), 4 µm formalin-fixed paraffin-embedded sections were dewaxed and rehydrated. Endogenous peroxidase was blocked by incubating sections in 3% H2O2 for 15 min. For antigen retrieval, sections were immersed in citrate buffer (pH 6.0), heated, and cooled at room temperature. β-catenin (rabbit monoclonal, clone 6B3, dilution 1:150, Cell Signaling) specific antibody was applied for 1.5 hours at room temperature. The reaction was amplified with an avidin-biotin method (Vectastain Elite ABC Kit PK-6100, Vector Laboratories Inc., Burlingame, CA, USA) and visualized with 3,3′-diaminobenzidine. Sections were counterstained with hematoxylin. A negative immunohistochemical control was prepared by replacing the primary antibody with an irrelevant one, and a known positive control section was included in the immunolabeling assay. Procedures involving animals were performed in accordance with the guidelines of the Catholic University of Leuven (KU Leuven) Animal Care and Use Ethical Committee, which specifically approved this study (reference number 072/2015). At necropsy, ascites was detected, and the entire left hepatic lobe and part of the median were markedly enlarged by a large, mottled gray-brown multinodular mass measuring approximately 1.5 × 1.5 × 1 cm. The lungs were characterized by multiple small brownish nodules (measuring approximately 1 to 3 mm in diameter) that were particularly evident along the margins of the lobes. Histologically, the affected hepatic parenchyma was almost completely effaced by two distinct neoplastic growths (Fig. 1Fig. 1. Liver, 22-month-old male C57BL/6J mouse. The hepatic parenchyma is expanded and effaced by two different neoplastic growths that are in close proximity: a hepatocellular carcinoma (black asterisk) and a hepatoblastoma (white asterisk). Hematoxylin and eosin. Scale bar: 2 mm. ). The first one was characterized by multiple coalescing HCAs consisting of expansile masses that were moderately cellular, unencapsulated, relatively well demarcated and composed of well-differentiated neoplastic hepatocytes arranged in cords or thin trabeculae separated by a delicate sinusoidal vascular network. Scattered throughout the HCAs there were foci of increased atypia with higher N/C ratios, hyperchromatic nuclei, with neoplastic hepatocytes arranged in thicker trabeculae (more than 3 cells in thickness) separated by irregularly distended sinusoids often forming pseudocystic spaces, and with foci of coagulative necrosis. These were interpreted as foci of progression suggesting a morphological diagnosis of trabecular type HCC arising within an HCA (Fig. 2Fig. 2. Liver, 22-month-old male C57BL/6J mouse. Hepatocellular carcinoma arising within a hepatocellular adenoma. Note the carcinomatous focus (arrows) characterized by increased trabecular thickness and findings of atypia in tumor cells including a higher N/C ratio and hyperchromasia. Hematoxylin and eosin. Scale bar: 100 µm. ). The second neoplastic entity arose within the previously described hepatocellular tumor as a multicentric process. These growths were infiltrating, unencapsulated, and composed of dense palisades and perivascular pseudorosettes of poorly differentiated primitive cells, with a scant amount of cytoplasm, elongated hyperchromatic nuclei, and a high mitotic rate (Fig. 3Fig. 3. Liver, 22-month-old male C57BL/6J mouse. The hepatoblastoma consists of poorly differentiated primitive cells with elongated hyperchromatic nuclei, scant cytoplasm, and a high mitotic rate. Tumor cells are typically arranged in dense palisades and perivascular pseudorosettes. Small scattered foci of squamous differentiation (inset) are also evident. Hematoxylin and eosin. Scale bar: 100 µm. ). Scattered throughout there were foci of coagulative necrosis and hemorrhages and few foci of squamous differentiation (Fig. 3, inset). Morphologically, the tumor was consistent with a diagnosis of HB. Histopathology of the pulmonary lesions revealed disseminated neoplastic aggregates multifocally plugging small and mid-sized pulmonary vessels (tumor emboli) and invading the surrounding parenchyma (tumor metastases). Pulmonary lesions recapitulated the two different neoplastic growths observed within the liver, with neoplastic hepatocytes closely associated with primitive, poorly differentiated neoplastic cells (interpreted as pulmonary metastases of HB arising within an HCC) (Fig. 4Fig. 4. Lung, 22-month-old male C57BL/6J mouse. Metastatic focus consisting of combined hepatoblastoma and hepatocellular carcinoma. Hematoxylin and eosin. Scale bar: 50 µm. ). Immunohistochemistry of β-catenin revealed that neoplastic hepatocytes in both hepatic and pulmonary locations showed mild to moderate membranous immunoreactivity, whereas a strong nucleocytoplasmic signal was evident in HB tumor cells (Fig. 5 Fig. 5. Liver, 22-month-old male C57BL/6J mouse. Hepatoblastoma (left) and adjacent hepatocellular adenoma (right). β-catenin immunohistochemistry reveals a strong nucleocytoplasmic signal in hepatoblastoma tumor cells, while neoplastic hepatocytes in the surrounding adenoma display a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 100 µm. and 6Fig. 6. Lung, 22-month-old male C57BL/6J mouse. The same metastatic focus is depicted as in Fig. 4. β-catenin immunohistochemistry reveals a strong nucleocytoplasmic signal in the hepatoblastoma fraction of the metastatic lesion, while the hepatocarcinomatous component maintains a delicate membranous staining pattern. β-catenin immunohistochemistry (IHC); hematoxylin counterstain. Scale bar: 50 µm. ). The complex hepatic changes observed in this aged C57BL/6J mouse recapitulate the well-characterized scenario in which HB is usually seen within or adjacent to preexisting hepatocellular tumors1. The presence of areas of HCC within HCA supports the overall idea that hepatocellular carcinogenesis in the mouse often follows the preneoplastic pathway, with preneoplastic lesions evolving into adenomas and later on into foci of carcinomatous progression arising within adenomas (hence the diagnostic designation of HCC arising within HCA)3. The hepatoblastoma here examined showed foci of squamous differentiation, in agreement with current literature that frequently reports small areas of bone and squamous differentiation along with foci of extramedullary hematopoiesis3. Both murine HCC and HB exhibit metastatic potential, with the lung being the most frequently involved distant organ2. To the authors’ knowledge, evidence of pulmonary co-metastases has been reported only once prior to this report2. The different patterns of β-catenin expression observed in both hepatic and pulmonary locations, with a strong nucleocytoplasmic signal in HB and delicate membranous immunoreactivity displayed by the hepatocellular proliferations, are in line with the well-established notion that the Wnt/β-catenin oncogenic pathway drives murine HB but not HCC tumorigenesis4. The divergent oncogenic mechanisms activated by these two tumors further support the hypothesis that HB does not progress from the concurrent hepatocellular tumor and that it should rather be regarded as a separate neoplasm. Comparative transcriptomic analysis demonstrated a high degree of concordance among murine HB, embryonic mouse hepatoblasts, and hepatic pluripotent stem cells, suggesting that these poorly differentiated and primitive tumors likely originate from a primordial hepatic precursor cell4, 21. Recent studies also showed that coexisting HCC and HB do not share the same mutational spectrum, thus providing compelling evidence that they are indeed distinct tumor entities4, 10. In light of these considerations, we propose to consider the entity here described as a collision tumor, that is defined as two distinct neoplasms with diverse origins and phenotypes coinciding at the same location22. Furthermore, as suggested by the frequent formation of HB within (or near) a well-developed hepatocellular neoplasm, the molecular events that lead to hepatic precursor cell transformation and HB formation are facilitated by not otherwise specified paracrine stimuli provided by the preexisting neoplastic hepatocytes. In this context, we support the idea that the microenvironment created by the hepatocellular tumors promotes the development of HB via a still unclear paracrine mechanism. Such a type of interaction, known as the “interaction theory”, has already been proposed for a series of co-developing neoplasms in humans23, 24. In this context, the evidence of co-metastases, in which both tumor cell types are physically associated within the same metastatic focus, possibly indicates that the close biological interdependence between HB and HCC also contributes to tumor progression and combined metastatic spread. 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