J. Comp. Path. 2014, Vol. -, 1e6
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NEOPLASTIC DISEASE
Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma M. Romanucci*, D. Malatesta*, I. Berardi†, G. Pugliese†, D. Fusco† and L. Della Salda* * Faculty of Veterinary Medicine, University of Teramo, Teramo and † Veterinary Practitioner, Montesilvano, Pescara, Italy
Summary A 7-year-old female Shih-tzu dog was presented with severe dyspnoea. A large mass was palpated in the left cranial neck. Cytological examination of an aspirate sample revealed cells with marked anisokaryosis, giant elements and many bare nuclei. Scattered intact giant cells showed scant, granular cytoplasm and intranuclear inclusions. Histologically, neoplastic cells were subdivided into lobules by fine collagenous trabeculae. Numerous pleomorphic giant, or ‘monster’, cells were observed, showing a highly indented nuclear envelope, intranuclear cytoplasmic pseudoinclusions (ICPs) and ‘ground-glass’ nuclear appearance. Neoplastic emboli were present, but no distant metastases were detected grossly. Immunohistochemically, the neoplastic cells expressed synaptophysin and had variable expression of neuron-specific enolase and vimentin. The cells were negative for pan-cytokeratin, CAM 5.2, glial fibrillary acidic protein and S100. Nuclear abnormalities and cytoplasmic neurosecretory granules were noted ultrastructurally. These features were consistent with a diagnosis of carotid body carcinoma (chemodectoma). Monster cells with ICPs have not been documented previously in canine chemodectoma. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: carotid body carcinoma; dog; intranuclear cytoplasmic pseudoinclusions; monster cells
Chemodectoma, or non-chromaffin paraganglioma, is a tumour arising in chemoreceptor organs that are responsible for detecting changes in blood carbon dioxide content, oxygen tension and pH, and aid in the regulation of respiration and circulation. The aortic and carotid bodies are the most common sites for the development of chemodectomas in dogs (Capen, 2002). A genetic predisposition, aggravated by chronic hypoxia, may account for the higher prevalence of these tumours in brachycephalic dogs (Hayes, 1975). The carotid body is located at the bifurcation of the common carotid artery. Carotid body tumours appear less frequently, but tend to be more malignant than aortic body tumours (Hayes and Sass, 1988; Obradovich et al., 1992) and metastases have been found in regional lymph nodes, lung, liver, pancreas, bone and
Correspondence to: L. Della Salda (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.03.001
kidney (Capen, 2002; Deim et al., 2007). These tumours are not functional in animals and cause clinical signs late in the course of the disease, as they exert a ‘space occupying’ effect on adjacent structures. Carotid body tumours tend to spread the carotid bifurcation as they enlarge and surgical treatment is difficult (Deim et al., 2007; Kromhout et al., 2012). These tumours usually express neuroendocrine markers that can be detected by immunohistochemistry (IHC), such as synaptophysin, chromogranin A and, to a lesser extent, neuronspecific enolase (NSE) (Brown et al., 2003). Ultrastructurally, the cytoplasm of the tumour cells contains neurosecretory granules (Capen, 2002). Intranuclear cytoplasmic pseudoinclusions (ICPs) are found commonly in human phaeochromocytomas and paragangliomas (De Lellis et al., 1980). ICPs have also been reported in some animal tumours, including canine and feline meningiomas (Vernau Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
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et al., 2001; Harms et al., 2009), canine laryngopharyngeal rhabdomyoma (Liggett et al., 1985), interstitial cell tumour (Sanford et al., 1987), clear-cell apocrine ductular carcinoma (Mikaelian and Wong, 2003; Gross et al., 2005), oncocytic adrenocortical carcinoma of the putty-nosed monkey (Gruber-Dujardin et al., 2013), mouse hepatoma (Leduc and Wilson, 1959) and pulmonary tumour of cockatiels (Garner et al., 2009). Pleomorphic giant cells with significantly enlarged nuclei, typically visible using low-power microscopy, are usually referred to as ‘monster’ cells in the human literature (Hawryluk et al., 2013) and have been described in several human neoplasms (Boyd et al., 2005; Defty et al., 2011; Hawryluk et al., 2013). To our knowledge, monster cells with ICPs have not been reported in canine carotid body tumours. A 7-year-old, female Shih-tzu dog was presented with severe dyspnoea of one week’s duration. An extensive, firm mass was palpable in the left anterolateral cervical region near the angle of the mandible (Supplementary Fig. 1). Fine-needle aspiration cytology revealed quite bloody, highly cellular smears with poor cellular preservation and many bare nuclei in the background. Nuclei were round to oval and there was marked anisokaryosis, with presence of numerous giant nuclei. Scattered intact giant cells had sparse eosinophilic, granular cytoplasm and round to oval eosinophilic ICPs (Fig. 1), sometimes appearing as one or more empty vacuoles. The tumour mass was not surgically resectable and the dog was humanely destroyed. Necropsy examination revealed a large (9.0 5.0 cm), firm, multinodular grey to pink mass, incorporating the carotid bifurcation, external
Fig. 1. Fine-needle aspirate of the canine carotid body carcinoma. Many bare nuclei and an intact tumour cell showing a small amount of eosinophilic, granular cytoplasm (arrow) and an oval, eosinophilic ICP. Diff-QuikÔ. Bar, 15 mm.
jugular vein and several cranial nerves (Supplementary Fig. 1). The tumour was not associated with the neighbouring salivary glands, the parotid and submandibular lymph nodes or the thyroid glands. No distant metastases were found and no other gross abnormalities were observed. Samples of the mass were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (5 mm) were stained with haematoxylin and eosin (HE). Additional sections were also subjected to IHC using primary antibodies specific for pan-cytokeratin (1 in 100 dilution, AE1/ AE3, mouse monoclonal; Dako, Glostrup, Denmark), CAM 5.2 (ready to use, mouse monoclonal; BD Biosciences, San Jose, California, USA), vimentin (1 in 100 dilution, V9, mouse monoclonal; Dako), synaptophysin (1 in 100 dilution, SY38, mouse monoclonal; Dako), NSE (1 in 600 dilution, BBS/NC/VI-H14, mouse monoclonal; Dako), glial fibrillary acidic protein (GFAP; 1 in 500 dilution, rabbit polyclonal; Chemicon International, Inc., Temecula, California, USA) and S100 protein (1 in 400 dilution, rabbit polyclonal; Dako). Labelling was identified by use of secondary biotinylated goat anti-mouse or antirabbit antibodies (1 in 200 dilution; Vector Laboratories Inc., Burlingame, California, USA) and subsequently ‘visualized’ using an avidinebiotin complex (ABC) method (VectastainÒ ABC Kit, Vector Laboratories) with 0.1% H2O2 in 3,30 -diaminobenzidine solution (SigmaeAldrich, St Louis, Missouri, USA) as chromogen. Sections were counterstained with Mayer’s haematoxylin (Merck, Darmstadt, Germany). For transmission electron microscopy (TEM), small pieces of formalin-fixed tissue were re-fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide and embedded in epoxy resin. Ultrathin sections were counterstained with uranyl acetate and lead citrate and were examined using an EM 900Zeiss transmission electron microscope (Zeiss, Oberkochen, Germany). Histological examination revealed a thinly encapsulated proliferation of markedly pleomorphic tumour cells, subdivided into lobules and packets by branching collagenous trabeculae and connective tissue septa with numerous small nerves and capillaries. Most of the neoplastic cells were round to polyhedral with indistinct cell borders, a variable amount of lightly eosinophilic cytoplasm and a central, round to oval nucleus with one or more prominent nucleoli. Intermingled with this population were numerous pleomorphic giant cells with significantly enlarged atypical nuclei showing the following nuclear features: highly indented nuclear envelope and deep nuclear grooves; optically clear, ‘ground-glass’ nuclear
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
Monster Cells in Canine Carotid Body Carcinoma
appearance with fine chromatin pattern; one or rarely two, round, lightly to deeply eosinophilic, clearly demarcated ICPs (Fig. 2 and Supplementary Fig. 2). Frequently, the intranuclear inclusions also appeared as a conspicuous, apparently empty vacuole (Supplementary Fig. 2). A minor number (5%) of tumour giant cells showed bizarrely-shaped, densely basophilic nuclei. Neoplastic cells had a high mitotic index (>5 mitoses per 40 objective field) with abnormal mitotic figures noted. There was evidence of invasion of blood and lymphatic vessels. Enclosed within the tumour mass were multiple small, presumably retropharyngeal and cervical, lymph nodes. These had neoplastic emboli in the subcapsular sinuses. Immunohistochemically, the tumour cells diffusely expressed synaptophysin and the ICPs were also labelled with this marker (Fig. 3). Some single cells or groups of tumour cells also expressed NSE and vimentin and stromal cells were labelled with the vimentin marker (Supplementary Fig. 3). Tumour cells were negative for pan-cytokeratin, CAM 5.2, GFAP (Supplementary Fig. 3) and S100; however, positivity for GFAP and S100 was detected in scattered intermingled cells that were most likely sustentacular cells (Supplementary Fig. 3), as well as in small nerves enclosed within the tumour mass. The main ultrastructural nuclear features were one or more, variably-sized, irregularly-shaped invaginations of the nuclear envelope with fractionation of the nucleus into multiple lobes (Supplementary Fig. 3) and abundant euchromatin and scattered ICPs (Fig. 4), occasionally containing neurosecretory granules. Numerous spherical mitochondria, parallel ar-
Fig. 2. Histological features of the canine carotid body carcinoma. Pleomorphic giant, or ‘monster’, cells showing optically clear, ‘ground-glass’ nuclei with deep indentations and grooves of the nuclear membrane, lightly eosinophilic ICPs (arrow) and prominent nucleoli. HE. Bar, 22 mm.
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Fig. 3. Canine carotid body carcinoma showing diffuse labelling of tumour cells for synaptophysin; an ICP also appears intensely labelled (arrow). IHC. Bar, 22 mm.
rays of rough endoplasmic reticulum and a moderate number of neurosecretory granules (Fig. 4, inset) were observed in the cytoplasm of neoplastic cells. On the basis of these findings, a diagnosis of carotid body carcinoma was made. Abnormal intranuclear substances that can be observed by light microscopy are usually referred to as nuclear inclusions. Two distinct types of inclusions may be encountered, which have different morphologies, mechanisms of
Fig. 4. Ultrastructural features of the canine carotid body carcinoma. Multiple infoldings of the nuclear membrane and formation of an ICP in the nucleus of a tumour cell. Condensed chromatin is visible on the inner aspect of the nuclear membrane and on the outer aspect of the ICP. TEM. Bar, 2 mm. Inset: Neurosecretory granules in the cytoplasm adjacent to the nucleus of a tumour cell. TEM. Bar, 0.5 mm.
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
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formation and diagnostic significance: bona fide nuclear inclusions and nuclear pseudoinclusions. Bona fide nuclear inclusions result from accumulation in the nuclear matrix of substances that are not normally found in the nucleus and are not delimited by nuclear membranes. The accumulated substances may be viral particles, cytoplasmic materials (e.g. surfactant, immunoglobulin or glycogen), biotin, nuclear lamins or polyglutamine. In contrast, nuclear pseudoinclusions represent herniations of the cytoplasm into the nucleus and are also known as ‘intranuclear cytoplasmic invaginations’ or ‘intranuclear vacuoles’. Microscopically, they typically appear as discrete, membrane-bound structures ‘floating’ into the nucleus, which are usually filled with material showing staining characteristics similar to or darker than the cytoplasm outside (Ip et al., 2010). However, it was only after the introduction of the electron microscope that the cytoplasmic origin of ICPs was understood (Kleinfeld et al., 1956). Cytoplasmic organelles, as well as secretory granules, are found in ICPs, whose immunoreactivity is the same as that of the cytoplasm (Sunba et al., 1980; Yang et al., 2003). Nuclear pseudoinclusions have been seen in a variety of normal and neoplastic cells (Sanford et al., 1987; Arora and Dey, 2012) and are commonly found in human phaeochromocytomas and paragangliomas (De Lellis et al., 1980). The pathogenesis of the formation of ICPs is unknown, although several hypotheses have been put forward (Arora and Dey, 2012). The high frequency of ICPs in human pheochromocytomas and paragangliomas is thought to result from the high degree of nuclear angulation and lobulation seen in these neoplasms (De Lellis et al., 1980). In fact, the infolding of the nuclear membrane has been suggested to be the early stage of the deep cytoplasmic invaginations that are responsible for the formation of ICPs (Deligeorgi-Politi, 1987). Invaginations of the nuclear envelope leading to the formation of ICPs have been considered as a mechanism involved in maintenance of the normal ratio of nuclear surface to nuclear volume in greatly enlarged cells (Leduc and Wilson, 1959; Sanford et al., 1987). In contrast, Sobel et al. (1969) suggested that most ICPs can be explained by the swollen cytoplasm extruding into the nucleus. Tumour giant cells with bizarre multilobed nuclei are reported in canine chemodectomas (Capen, 2002) and in the human literature such cells are typically referred to as ‘monster’ cells (Hawryluk et al., 2013). Tumour giant cells in canine chemodectomas usually display densely basophilic nuclei (Capen, 2002). However, pleomorphic giant cells similar to the monster cells observed in the present case showed previously undocumented nuclear features repre-
sented by a combination of optically clear, ‘groundglass’ nuclei due to abundant euchromatin, nuclear indentations and grooves with formation of ICPs, and prominent nucleoli. This spectrum of nuclear anomalies is characteristic of human papillary thyroid carcinoma (Beaumont et al., 1981; Ryska et al., 2000; Lugli et al., 2004). Among human neck tumours, ICPs may also occur in medullary carcinoma, parathyroid adenoma (Yang, 2013) and paraganglioma. Microscopically, the delicate pink cytoplasm with indistinct borders, delicate chromatin, nuclear pleomorphism and architectural features of paraganglioma are considered to be useful in differentiating from papillary thyroid carcinoma (Jacobs and Waisman, 1987; Rana et al., 1997). In the present case, thyroid involvement was excluded, since the thyroid gland was grossly normal and the findings clearly supported a diagnosis of carotid body carcinoma. ICPs have been described in canine laryngopharyngeal rhabdomyoma (Liggett et al., 1985), interstitial cell tumour (Sanford et al., 1987), clear-cell apocrine ductular carcinoma (Mikaelian and Wong, 2003; Gross et al., 2005) and meningioma (Vernau et al., 2001; Harms et al., 2009). Sanford et al. (1987) performed a light and electron microscopical study of ICPs in canine Leydig cell tumours. As in the present case, the ICPs were present in enlarged nuclei and were represented either by round, eosinophilic, clearly demarcated structures or by apparently empty vacuoles. In this respect, intranuclear vacuoles resulting from ICPs must be distinguished from the so-called nuclear pseudo-pseudoinclusions, which appear as single or multiple, apparently empty intranuclear ‘holes’, not delimited by membranes and present in the majority of cells in a microscopical field. These ‘bubbly’ nuclei are believed to result from incomplete fixation or dehydration, excessive heating, or other faults occurring during tissue processing (Ip et al., 2010; Arora and Dey, 2012). Harms et al. (2009) described the occurrence of ICPs in cytological and histological specimens of canine meningioma. Papanicolau’s stain has been used on cytological samples in human medicine in order to highlight ICPs in tumour cells (Villari et al., 2000). Similar to the case described by Harms et al. (2009), use of this stain was not necessary in the present case, since ICPs were detectable on Diff-QuikÔstained samples. To the best of our knowledge, this is the first light and ultrastructural description of multiple nuclear anomalies with formation of ICPs in ‘monster’ cells in a canine chemodectoma. Although rare, the possible occurrence of these aberrant nuclear features in canine carotid body tumours should be taken into
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
Monster Cells in Canine Carotid Body Carcinoma
consideration in the differential diagnosis from other types of neoplasia of the neck region, particularly for tumours of thyroid origin.
Conflict of Interest Statement The authors declare that they have no conflict of interest.
Supplementary data Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.jcpa. 2014.03.001.
References Arora SK, Dey P (2012) Intranuclear pseudoinclusions: morphology, pathogenesis, and significance. Diagnostic Cytopathology, 40, 741e744. Beaumont A, Ben Othman S, Fragu P (1981) The fine structure of papillary carcinoma of the thyroid. Histopathology, 5, 377e388. Boyd AS, Wu H, Shyr Y (2005) Monster cells in malignant melanoma. American Journal of Dermatopathology, 27, 208e210. Brown PJ, Rema A, Gartner F (2003) Immunohistochemical characteristics of canine aortic and carotid body tumours. Journal of Veterinary Medicine A, 50, 140e144. Capen CC (2002) Tumors of the endocrine glands. In: Tumors of Domestic Animals, 4th Edit., DJ Meuten, Ed., Iowa State Press, Ames, pp. 607e696. Defty CL, Segen J, Carter JJ, Ahmed I, Carr RA (2011) Basaloid squamous cell carcinoma with ‘monster’ cells: a mimic of pleomorphic basal cell carcinoma. Journal of Cutaneous Pathology, 38, 354e356. Deim Z, Szalay F, Glavitsm R, Bauer A, Cserni G (2007) Carotid body tumor in dog: a case report. Canadian Veterinary Journal, 48, 865e867. De Lellis RA, Suchow E, Wolfe HJ (1980) Ultrastructure of nuclear ‘inclusions’ in pheochromocytoma and paraganglioma. Human Pathology, 11, 205e207. Deligeorgi-Politi H (1987) Nuclear crease as a cytodiagnostic feature of papillary thyroid carcinoma in fine-needle aspiration biopsies. Diagnostic Cytopathology, 3, 307e310. Garner MM, Latimer KS, Mickley KA, Ritzman TK, Nordhausen RW (2009) Histologic, immunohistochemical, and electron microscopic features of a unique pulmonary tumor in cockatiels (Nymphicus hollandicus): six cases. Veterinary Pathology, 46, 1100e1108. Gross TL, Ihrke PJ, Walder EJ, Affolter VK (2005) Skin Diseases of the Dog and Cat: Clinical and Histopathologic Diagnosis, 2nd Edit., Blackwell Publishing, Ames, p. 686. Gruber-Dujardin E, Jurczynski K, Kaup F-J, M€ atzRensing K (2013) Oncocytic adrenocortical carcinoma in a putty-nosed monkey (Cercopithecus nictitans) with hyperadrenocorticism. Journal of Comparative Pathology, 149, 509e513.
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Harms NJ, Dickinson RM, Nibblett BM, Wobeser BK (2009) What is your diagnosis? Intracranial mass in a dog. Veterinary Clinical Pathology, 38, 537e540. Hawryluk EB, Baran JL, Gerami P, Sepehr A (2013) ‘Monster cell’ melanoma with pulmonary metastasis and cyclin D1 amplification. Journal of Cutaneous Pathology, 40, 61e65. Hayes HH Jr. (1975) An hypothesis for the aetiology of canine chemoreceptor system neoplasms, based upon an epidemiological study of 73 cases among hospital patients. Journal of Small Animal Practice, 16, 337e343. Hayes HM, Sass B (1988) Chemoreceptor neoplasia: a study of the epidemiological features of 357 canine cases. Zentralbl Veterinarmed A, 35, 401e408. Ip YT, Dias Filho MA, Chan JK (2010) Nuclear inclusions and pseudoinclusions: friends or foes of the surgical pathologist? International Journal of Surgical Pathology, 18, 465e481. Jacobs DM, Waisman J (1987) Cervical paraganglioma with intranuclear vacuoles in a fine needle aspirate. Acta Cytologica, 31, 29e32. Kleinfeld RG, Greider MH, Frajola WJ (1956) Electron microscopy of intranuclear inclusions found in human and rat liver parenchymal cells. Journal of Biophysical and Biochemical Cytology, 2, 435e438. Kromhout K, Gielen I, De Cock HE, Van Dyck K, van Bree H (2012) Magnetic resonance and computer tomography imaging of a carotid body tumor in a dog. Acta Veterinaria Scandinavica, 54, 24. Leduc EH, Wilson JW (1959) An electron microscope study of intranuclear inclusions in mouse liver and hepatoma. Journal of Biophysical and Biochemical Cytology, 6, 427e430. Liggett AD, Weiss R, Thomas KL (1985) Canine laryngopharyngeal rhabdomyoma resembling an oncocytoma: light microscopic, ultrastructural and comparative studies. Veterinary Pathology, 22, 526e532. Lugli A, Terracciano LM, Oberholzer M, Bubendorf L, Tornillo L (2004) Macrofollicular variant of papillary carcinoma of the thyroid. A histologic, cytologic, and immunohistochemical study of 3 cases and review of the literature. Archives of Pathology and Laboratory Medicine, 128, 54e58. Mikaelian I, Wong V (2003) Follicular stem cell carcinoma: histologic, immunohistochemical, ultrastructural, and clinical characterization in 30 dogs. Veterinary Pathology, 40, 433e444. Obradovich JE, Withrow SJ, Powers BE, Walshaw R (1992) Carotid body tumors in the dog. Eleven cases (1978e1988). Journal of Veterinary Internal Medicine, 6, 96e101. Rana RS, Dey P, Das A (1997) Fine needle aspiration (FNA) cytology of extra-adrenal paragangliomas. Cytopathology, 8, 108e113. Ryska A, Nozicka Z, Hovorkova E, Suchy T (2000) Sclerosing papillary carcinoma of the thyroid with tubular features. Pathology Research and Practice, 196, 339e343. Sanford SE, Miller RB, Hoover DM (1987) A light and electron microscopical study of intranuclear cytoplasmic
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invaginations in interstitial cell tumours of dogs. Journal of Comparative Pathology, 97, 629e635. Sobel HJ, Schwarz R, Parquet E (1969) Nonviral nuclear inclusions. I. Cytoplasmic invaginations. Archives of Pathology, 87, 179e192. Sunba MS, Rahi AH, Morgan G (1980) Tumours of the anterior uvea. II. Intranuclear cytoplasmic inclusions in malignant melanoma of the iris. British Journal of Ophthalmology, 64, 453e456. Vernau KM, Higgins RJ, Bollen AW, Jimenez DF, Anderson JV et al. (2001) Primary canine and feline nervous system tumors: intraoperative diagnosis using the smear technique. Veterinary Pathology, 38, 47e57. Villari D, Grosso M, Vitarelli E, Tuccari G, Barresi G (2000) Nuclear pseudoinclusions in fine-needle aspira-
tion cytology of hepatic angiomyolipoma. Diagnostic Cytopathology, 22, 390e393. Yang GCH (2013) Thyroid Fine Needle Aspiration. Cambridge University Press, New York, p. 156. Yang SW, Yang KM, Kang Hy, Kim TS (2003) Intranuclear cytoplasmic pseudoinclusions in pituitary adenomas. Yonsei Medical Journal, 44, 816e820.
November 11th, 2013 ½ Received, Accepted, March 4th, 2014
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
Available online at www.sciencedirect.com
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NEOPLASTIC DISEASE
Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma M. Romanucci*, D. Malatesta*, I. Berardi†, G. Pugliese†, D. Fusco† and L. Della Salda* * Faculty of Veterinary Medicine, University of Teramo, Teramo and † Veterinary Practitioner, Montesilvano, Pescara, Italy
Summary A 7-year-old female Shih-tzu dog was presented with severe dyspnoea. A large mass was palpated in the left cranial neck. Cytological examination of an aspirate sample revealed cells with marked anisokaryosis, giant elements and many bare nuclei. Scattered intact giant cells showed scant, granular cytoplasm and intranuclear inclusions. Histologically, neoplastic cells were subdivided into lobules by fine collagenous trabeculae. Numerous pleomorphic giant, or ‘monster’, cells were observed, showing a highly indented nuclear envelope, intranuclear cytoplasmic pseudoinclusions (ICPs) and ‘ground-glass’ nuclear appearance. Neoplastic emboli were present, but no distant metastases were detected grossly. Immunohistochemically, the neoplastic cells expressed synaptophysin and had variable expression of neuron-specific enolase and vimentin. The cells were negative for pan-cytokeratin, CAM 5.2, glial fibrillary acidic protein and S100. Nuclear abnormalities and cytoplasmic neurosecretory granules were noted ultrastructurally. These features were consistent with a diagnosis of carotid body carcinoma (chemodectoma). Monster cells with ICPs have not been documented previously in canine chemodectoma. Ó 2014 Elsevier Ltd. All rights reserved. Keywords: carotid body carcinoma; dog; intranuclear cytoplasmic pseudoinclusions; monster cells
Chemodectoma, or non-chromaffin paraganglioma, is a tumour arising in chemoreceptor organs that are responsible for detecting changes in blood carbon dioxide content, oxygen tension and pH, and aid in the regulation of respiration and circulation. The aortic and carotid bodies are the most common sites for the development of chemodectomas in dogs (Capen, 2002). A genetic predisposition, aggravated by chronic hypoxia, may account for the higher prevalence of these tumours in brachycephalic dogs (Hayes, 1975). The carotid body is located at the bifurcation of the common carotid artery. Carotid body tumours appear less frequently, but tend to be more malignant than aortic body tumours (Hayes and Sass, 1988; Obradovich et al., 1992) and metastases have been found in regional lymph nodes, lung, liver, pancreas, bone and
Correspondence to: L. Della Salda (e-mail: [email protected]). 0021-9975/$ - see front matter http://dx.doi.org/10.1016/j.jcpa.2014.03.001
kidney (Capen, 2002; Deim et al., 2007). These tumours are not functional in animals and cause clinical signs late in the course of the disease, as they exert a ‘space occupying’ effect on adjacent structures. Carotid body tumours tend to spread the carotid bifurcation as they enlarge and surgical treatment is difficult (Deim et al., 2007; Kromhout et al., 2012). These tumours usually express neuroendocrine markers that can be detected by immunohistochemistry (IHC), such as synaptophysin, chromogranin A and, to a lesser extent, neuronspecific enolase (NSE) (Brown et al., 2003). Ultrastructurally, the cytoplasm of the tumour cells contains neurosecretory granules (Capen, 2002). Intranuclear cytoplasmic pseudoinclusions (ICPs) are found commonly in human phaeochromocytomas and paragangliomas (De Lellis et al., 1980). ICPs have also been reported in some animal tumours, including canine and feline meningiomas (Vernau Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
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et al., 2001; Harms et al., 2009), canine laryngopharyngeal rhabdomyoma (Liggett et al., 1985), interstitial cell tumour (Sanford et al., 1987), clear-cell apocrine ductular carcinoma (Mikaelian and Wong, 2003; Gross et al., 2005), oncocytic adrenocortical carcinoma of the putty-nosed monkey (Gruber-Dujardin et al., 2013), mouse hepatoma (Leduc and Wilson, 1959) and pulmonary tumour of cockatiels (Garner et al., 2009). Pleomorphic giant cells with significantly enlarged nuclei, typically visible using low-power microscopy, are usually referred to as ‘monster’ cells in the human literature (Hawryluk et al., 2013) and have been described in several human neoplasms (Boyd et al., 2005; Defty et al., 2011; Hawryluk et al., 2013). To our knowledge, monster cells with ICPs have not been reported in canine carotid body tumours. A 7-year-old, female Shih-tzu dog was presented with severe dyspnoea of one week’s duration. An extensive, firm mass was palpable in the left anterolateral cervical region near the angle of the mandible (Supplementary Fig. 1). Fine-needle aspiration cytology revealed quite bloody, highly cellular smears with poor cellular preservation and many bare nuclei in the background. Nuclei were round to oval and there was marked anisokaryosis, with presence of numerous giant nuclei. Scattered intact giant cells had sparse eosinophilic, granular cytoplasm and round to oval eosinophilic ICPs (Fig. 1), sometimes appearing as one or more empty vacuoles. The tumour mass was not surgically resectable and the dog was humanely destroyed. Necropsy examination revealed a large (9.0 5.0 cm), firm, multinodular grey to pink mass, incorporating the carotid bifurcation, external
Fig. 1. Fine-needle aspirate of the canine carotid body carcinoma. Many bare nuclei and an intact tumour cell showing a small amount of eosinophilic, granular cytoplasm (arrow) and an oval, eosinophilic ICP. Diff-QuikÔ. Bar, 15 mm.
jugular vein and several cranial nerves (Supplementary Fig. 1). The tumour was not associated with the neighbouring salivary glands, the parotid and submandibular lymph nodes or the thyroid glands. No distant metastases were found and no other gross abnormalities were observed. Samples of the mass were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (5 mm) were stained with haematoxylin and eosin (HE). Additional sections were also subjected to IHC using primary antibodies specific for pan-cytokeratin (1 in 100 dilution, AE1/ AE3, mouse monoclonal; Dako, Glostrup, Denmark), CAM 5.2 (ready to use, mouse monoclonal; BD Biosciences, San Jose, California, USA), vimentin (1 in 100 dilution, V9, mouse monoclonal; Dako), synaptophysin (1 in 100 dilution, SY38, mouse monoclonal; Dako), NSE (1 in 600 dilution, BBS/NC/VI-H14, mouse monoclonal; Dako), glial fibrillary acidic protein (GFAP; 1 in 500 dilution, rabbit polyclonal; Chemicon International, Inc., Temecula, California, USA) and S100 protein (1 in 400 dilution, rabbit polyclonal; Dako). Labelling was identified by use of secondary biotinylated goat anti-mouse or antirabbit antibodies (1 in 200 dilution; Vector Laboratories Inc., Burlingame, California, USA) and subsequently ‘visualized’ using an avidinebiotin complex (ABC) method (VectastainÒ ABC Kit, Vector Laboratories) with 0.1% H2O2 in 3,30 -diaminobenzidine solution (SigmaeAldrich, St Louis, Missouri, USA) as chromogen. Sections were counterstained with Mayer’s haematoxylin (Merck, Darmstadt, Germany). For transmission electron microscopy (TEM), small pieces of formalin-fixed tissue were re-fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide and embedded in epoxy resin. Ultrathin sections were counterstained with uranyl acetate and lead citrate and were examined using an EM 900Zeiss transmission electron microscope (Zeiss, Oberkochen, Germany). Histological examination revealed a thinly encapsulated proliferation of markedly pleomorphic tumour cells, subdivided into lobules and packets by branching collagenous trabeculae and connective tissue septa with numerous small nerves and capillaries. Most of the neoplastic cells were round to polyhedral with indistinct cell borders, a variable amount of lightly eosinophilic cytoplasm and a central, round to oval nucleus with one or more prominent nucleoli. Intermingled with this population were numerous pleomorphic giant cells with significantly enlarged atypical nuclei showing the following nuclear features: highly indented nuclear envelope and deep nuclear grooves; optically clear, ‘ground-glass’ nuclear
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
Monster Cells in Canine Carotid Body Carcinoma
appearance with fine chromatin pattern; one or rarely two, round, lightly to deeply eosinophilic, clearly demarcated ICPs (Fig. 2 and Supplementary Fig. 2). Frequently, the intranuclear inclusions also appeared as a conspicuous, apparently empty vacuole (Supplementary Fig. 2). A minor number (5%) of tumour giant cells showed bizarrely-shaped, densely basophilic nuclei. Neoplastic cells had a high mitotic index (>5 mitoses per 40 objective field) with abnormal mitotic figures noted. There was evidence of invasion of blood and lymphatic vessels. Enclosed within the tumour mass were multiple small, presumably retropharyngeal and cervical, lymph nodes. These had neoplastic emboli in the subcapsular sinuses. Immunohistochemically, the tumour cells diffusely expressed synaptophysin and the ICPs were also labelled with this marker (Fig. 3). Some single cells or groups of tumour cells also expressed NSE and vimentin and stromal cells were labelled with the vimentin marker (Supplementary Fig. 3). Tumour cells were negative for pan-cytokeratin, CAM 5.2, GFAP (Supplementary Fig. 3) and S100; however, positivity for GFAP and S100 was detected in scattered intermingled cells that were most likely sustentacular cells (Supplementary Fig. 3), as well as in small nerves enclosed within the tumour mass. The main ultrastructural nuclear features were one or more, variably-sized, irregularly-shaped invaginations of the nuclear envelope with fractionation of the nucleus into multiple lobes (Supplementary Fig. 3) and abundant euchromatin and scattered ICPs (Fig. 4), occasionally containing neurosecretory granules. Numerous spherical mitochondria, parallel ar-
Fig. 2. Histological features of the canine carotid body carcinoma. Pleomorphic giant, or ‘monster’, cells showing optically clear, ‘ground-glass’ nuclei with deep indentations and grooves of the nuclear membrane, lightly eosinophilic ICPs (arrow) and prominent nucleoli. HE. Bar, 22 mm.
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Fig. 3. Canine carotid body carcinoma showing diffuse labelling of tumour cells for synaptophysin; an ICP also appears intensely labelled (arrow). IHC. Bar, 22 mm.
rays of rough endoplasmic reticulum and a moderate number of neurosecretory granules (Fig. 4, inset) were observed in the cytoplasm of neoplastic cells. On the basis of these findings, a diagnosis of carotid body carcinoma was made. Abnormal intranuclear substances that can be observed by light microscopy are usually referred to as nuclear inclusions. Two distinct types of inclusions may be encountered, which have different morphologies, mechanisms of
Fig. 4. Ultrastructural features of the canine carotid body carcinoma. Multiple infoldings of the nuclear membrane and formation of an ICP in the nucleus of a tumour cell. Condensed chromatin is visible on the inner aspect of the nuclear membrane and on the outer aspect of the ICP. TEM. Bar, 2 mm. Inset: Neurosecretory granules in the cytoplasm adjacent to the nucleus of a tumour cell. TEM. Bar, 0.5 mm.
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
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formation and diagnostic significance: bona fide nuclear inclusions and nuclear pseudoinclusions. Bona fide nuclear inclusions result from accumulation in the nuclear matrix of substances that are not normally found in the nucleus and are not delimited by nuclear membranes. The accumulated substances may be viral particles, cytoplasmic materials (e.g. surfactant, immunoglobulin or glycogen), biotin, nuclear lamins or polyglutamine. In contrast, nuclear pseudoinclusions represent herniations of the cytoplasm into the nucleus and are also known as ‘intranuclear cytoplasmic invaginations’ or ‘intranuclear vacuoles’. Microscopically, they typically appear as discrete, membrane-bound structures ‘floating’ into the nucleus, which are usually filled with material showing staining characteristics similar to or darker than the cytoplasm outside (Ip et al., 2010). However, it was only after the introduction of the electron microscope that the cytoplasmic origin of ICPs was understood (Kleinfeld et al., 1956). Cytoplasmic organelles, as well as secretory granules, are found in ICPs, whose immunoreactivity is the same as that of the cytoplasm (Sunba et al., 1980; Yang et al., 2003). Nuclear pseudoinclusions have been seen in a variety of normal and neoplastic cells (Sanford et al., 1987; Arora and Dey, 2012) and are commonly found in human phaeochromocytomas and paragangliomas (De Lellis et al., 1980). The pathogenesis of the formation of ICPs is unknown, although several hypotheses have been put forward (Arora and Dey, 2012). The high frequency of ICPs in human pheochromocytomas and paragangliomas is thought to result from the high degree of nuclear angulation and lobulation seen in these neoplasms (De Lellis et al., 1980). In fact, the infolding of the nuclear membrane has been suggested to be the early stage of the deep cytoplasmic invaginations that are responsible for the formation of ICPs (Deligeorgi-Politi, 1987). Invaginations of the nuclear envelope leading to the formation of ICPs have been considered as a mechanism involved in maintenance of the normal ratio of nuclear surface to nuclear volume in greatly enlarged cells (Leduc and Wilson, 1959; Sanford et al., 1987). In contrast, Sobel et al. (1969) suggested that most ICPs can be explained by the swollen cytoplasm extruding into the nucleus. Tumour giant cells with bizarre multilobed nuclei are reported in canine chemodectomas (Capen, 2002) and in the human literature such cells are typically referred to as ‘monster’ cells (Hawryluk et al., 2013). Tumour giant cells in canine chemodectomas usually display densely basophilic nuclei (Capen, 2002). However, pleomorphic giant cells similar to the monster cells observed in the present case showed previously undocumented nuclear features repre-
sented by a combination of optically clear, ‘groundglass’ nuclei due to abundant euchromatin, nuclear indentations and grooves with formation of ICPs, and prominent nucleoli. This spectrum of nuclear anomalies is characteristic of human papillary thyroid carcinoma (Beaumont et al., 1981; Ryska et al., 2000; Lugli et al., 2004). Among human neck tumours, ICPs may also occur in medullary carcinoma, parathyroid adenoma (Yang, 2013) and paraganglioma. Microscopically, the delicate pink cytoplasm with indistinct borders, delicate chromatin, nuclear pleomorphism and architectural features of paraganglioma are considered to be useful in differentiating from papillary thyroid carcinoma (Jacobs and Waisman, 1987; Rana et al., 1997). In the present case, thyroid involvement was excluded, since the thyroid gland was grossly normal and the findings clearly supported a diagnosis of carotid body carcinoma. ICPs have been described in canine laryngopharyngeal rhabdomyoma (Liggett et al., 1985), interstitial cell tumour (Sanford et al., 1987), clear-cell apocrine ductular carcinoma (Mikaelian and Wong, 2003; Gross et al., 2005) and meningioma (Vernau et al., 2001; Harms et al., 2009). Sanford et al. (1987) performed a light and electron microscopical study of ICPs in canine Leydig cell tumours. As in the present case, the ICPs were present in enlarged nuclei and were represented either by round, eosinophilic, clearly demarcated structures or by apparently empty vacuoles. In this respect, intranuclear vacuoles resulting from ICPs must be distinguished from the so-called nuclear pseudo-pseudoinclusions, which appear as single or multiple, apparently empty intranuclear ‘holes’, not delimited by membranes and present in the majority of cells in a microscopical field. These ‘bubbly’ nuclei are believed to result from incomplete fixation or dehydration, excessive heating, or other faults occurring during tissue processing (Ip et al., 2010; Arora and Dey, 2012). Harms et al. (2009) described the occurrence of ICPs in cytological and histological specimens of canine meningioma. Papanicolau’s stain has been used on cytological samples in human medicine in order to highlight ICPs in tumour cells (Villari et al., 2000). Similar to the case described by Harms et al. (2009), use of this stain was not necessary in the present case, since ICPs were detectable on Diff-QuikÔstained samples. To the best of our knowledge, this is the first light and ultrastructural description of multiple nuclear anomalies with formation of ICPs in ‘monster’ cells in a canine chemodectoma. Although rare, the possible occurrence of these aberrant nuclear features in canine carotid body tumours should be taken into
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
Monster Cells in Canine Carotid Body Carcinoma
consideration in the differential diagnosis from other types of neoplasia of the neck region, particularly for tumours of thyroid origin.
Conflict of Interest Statement The authors declare that they have no conflict of interest.
Supplementary data Supplementary material related to this article can be found online at http://dx.doi.org/10.1016/j.jcpa. 2014.03.001.
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November 11th, 2013 ½ Received, Accepted, March 4th, 2014
Please cite this article in press as: Romanucci M, et al., Cytological, Histological and Ultrastructural Nuclear Features of Monster Cells in a Canine Carotid Body Carcinoma, Journal of Comparative Pathology (2014), http://dx.doi.org/10.1016/j.jcpa.2014.03.001
