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Pulmonary alveolar microlithiasis with concurrent pleural mesothelioma in a dog
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Simone de Brot,1 Monika Hilbe
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From the Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zürich,
Zürich, Switzerland.
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Corresponding Author: Simone de Brot, Institute of Veterinary Pathology, Vetsuisse
Faculty, University of Zürich, Winterthurerstrasse 268, CH-8057 Zürich, Switzerland.
simone.debrot@uzh.ch
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Running head: Pulmonary microlithiasis and pleural mesothelioma in a dog
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Abstract. Pulmonary alveolar microlithiasis (PAM) is a rare pulmonary disorder characterized
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by the accumulation of calcium phosphate microliths within the alveoli, with only a few cases
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described in animals. A 10-year old female Bulldog was euthanized due to history of dyspnea
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and recurrent pleural and pericardial effusions. At necropsy, numerous multifocal to coalescent
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protruding nodules of 1–5 mm in diameter were scattered throughout the thoracic serosal
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surfaces. Moreover, lungs showed a diffuse pale gray color and had a generalized fine grainy
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consistency. Histological investigations revealed abundant intra-alveolar laminated microliths
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that stained positive with periodic acid–Schiff and von Kossa stains. The pulmonary interstitium
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showed multifocal, mild to moderate thickening, due to collagen deposition and mild hyperplasia
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of type 2 pneumocytes. The pulmonary lesion was not associated with any inflammatory
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response, and mineral deposition was not observed in any other organ or tissue. In addition,
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pulmonary, pericardial, and pleural surfaces were extensively infiltrated by an epithelioid
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mesothelioma. Immunohistochemical staining revealed neoplastic cells that strongly coexpressed
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vimentin and cytokeratin, supporting the diagnosis of mesothelioma. An overview of PAM,
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including pathogenesis and histopathological characteristics, are discussed in relation to the
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concurrent pleural mesothelioma. The potential cause and effect relationship between the 2
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conditions could neither be established nor ruled out.
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Keywords: Dogs; intra-alveolar filling disorder; lung; mesothelioma; microliths.
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Pulmonary alveolar microlithiasis (PAM) is a rare disorder characterized by the
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deposition of calcium phosphate within the alveoli of the lungs. The deposition usually occurs in
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the absence of any known disorder of calcium metabolism and quite often is not associated with
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an inflammatory response.6 The progression of the disease is generally very slow, and patients
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can be asymptomatic for years or even decades. Therefore, its diagnosis is usually incidental in
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both human and veterinary species.12,21 Pulmonary alveolar microlithiasis shows both sporadic
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and familial occurrence.9 In human beings, approximately one-third of cases are hereditary, with
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an autosomal recessive inheritance and complete penetrance.6,20 Mutations of the SLC34A2
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gene, which encodes a type IIb sodium phosphate co-transporter (NaPi-IIb), are considered to be
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the cause of the disease, leading to intra-alveolar accumulation of phosphate that favors the
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formation of microliths.10 However, the disease in animals is still of unknown cause, probably
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because of the low number of diagnosed cases. The condition was first described in human
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beings in 1918,13 whereas the first report in veterinary species was described 50 years later in a
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dog.18 To date, documented reports of PAM can be found only in 5 dogs,2,5,18,21 a cat,3 a
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binturong (Arctictis binturong),4 a breeding colony of Afghan pikas (Ochotona rufescens
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rufescens),19 an orangutan (Pongo pygmaeus),16 an alpaca (Vicugna pacos),17 and in mice.23
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Herein, a case of PAM associated with a pleural and pericardial mesothelioma in a Bulldog is
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described.
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A 10-year-old spayed female Bulldog weighing 30 kg was presented to the Veterinary
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Hospital Zurich (Vetsuisse Faculty of the University of Zürich, Switzerland) with a history of
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recurrent pleural and pericardial effusion and dyspnea. The dog had been treated with repeated
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pericardiocentesis and finally by subtotal pericardiectomy 2 years before the time of euthanasia.
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No gross lesions were observed in specimens of the parietal pericardium, and no histological
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examination of the pericardial tissue was performed. Following pericardiectomy, the dog had
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subsequent recurrent pleural hemorrhagic effusions. Due to poor prognosis, the dog was
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euthanized; postmortem examination was performed, and tissue specimens were obtained for
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detailed histological and immunohistochemical investigations.
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Specimens of various thoracic and abdominal organs including the pleural and epicardial
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masses and lung were fixed in 10% buffered formalin, routinely processed, paraffin-embedded,
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sectioned at 3 µm, and stained with hematoxylin and eosin (HE) for light microscopic
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examination. Selected sections of lung tissue were stained with periodic acid–Schiff (PAS)
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(0.5% periodic acid solution, Schiff reagent, counterstained with Mayer hematoxylin), von Kossa
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(3% aqueous silver nitrate solution, 5% sodium thiosulfate, counterstained with 0.1% nuclear
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fast red solution), and Prussian blue (2% aqueous solution of hydrochloric acid, 2% aqueous
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solution of potassium ferrocyanide, counterstained with nuclear fast red solution). For
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immunohistochemical evaluation of pleural and epicardial masses, sections of the tumor masses
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were incubated with commercially available antibodies to pan-cytokeratina (anti-mouse, 1:50)
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and vimentinb (anti-mouse, 1:100). The standard avidin–biotin–peroxidase technique was used to
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demonstrate the antigen.
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At necropsy, the pleural cavity contained 2–3 liters of serosanguinous fluids. Numerous
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multifocal to coalescent, tan to yellowish-red, firm, solid protruding nodules of 1–5 mm in
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diameter were scattered throughout the epicardial membrane, the pulmonary pleura, and,
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multifocally but less extensively, on the costal pleural surfaces (Fig. 1). Moreover, the lungs
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showed a diffuse pale gray color, were diffusely mildly rigid with a reduced elasticity, and had a
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generalized fine grainy consistency on palpation and on cut section.
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Histologically, lungs showed abundant multifocal accumulations of circular to irregularly
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shaped, variably sized (30–100 µm), densely basophilic, non-birefringent, amorphous to
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laminated concretions (microliths) that were either attached to the alveolar walls or found free
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within alveoli, involving up to 80% of the alveolar spaces (Fig. 2A). Some microliths were
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fragmented, with the majority of fracture lines showing a radial pattern. Others resembled
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homogeneous structureless spheroids or irregularly shaped masses. In HE sections of the lung,
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the staining of the central cores and peripheral lamellae of the microliths varied from red to
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bluish-purple, with the central areas appearing either lighter or darker than the periphery.
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Multifocally and quite often, there was mild to moderate disruption of the alveolar septae and
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compression of the pulmonary tissue adjacent to the microliths. Nevertheless, no inflammatory
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response was observed within the pulmonary parenchyma. Multifocally, the pulmonary
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interstitium showed mild to moderate thickening due to collagen deposition, as was
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demonstrated by Gomori blue trichrome stain (Fig. 2B). Mild hyperplasia and hypertrophy of
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type 2 pneumocytes was also multifocally present, and mineral deposition was not observed in
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any other organs or tissues. The concentric lamellae and cores of the microlith intensely stained
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in shades of purple with PAS stain (Fig. 2C). Furthermore, the microliths stained brownish to
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black with von Kossa stain (Fig. 2D). Finally, microliths did not stain with Prussian blue. A
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diagnosis of PAM was made based on the size, location, and morphology of the concretions and
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the absence of an inflammatory response.
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Histologically, pleural and epicardial surfaces were markedly thickened due to 1 or
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multiple layers of epithelial-like neoplastic cells showing papillary outgrowths supported by thin
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to moderately thick fibrous connective tissue (Fig. 3A). Neoplastic cells measured 15–25µm in
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diameter, were cuboidal to polygonal in shape, and were characterized by small to moderate
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amounts of eosinophilic homogenous to finely granular cytoplasm, distinct cell borders, and
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occasionally showed microvillus borders. The nuclei were central or eccentric, round to oval, and
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characterized by finely stippled basophilic staining chromatin and 1–2 prominent eosinophilic
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nucleoli. The degree of anisocytosis and anisokaryosis was moderate, and few regular mitotic
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figures were observed (0–1 per high power field). The neoplasm had multifocal areas of necrosis
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and hemorrhage, as well as multifocal areas with moderate numbers of infiltrating
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lymphohistiocytic cells and few neutrophils. No evidence of metastasis to any parenchymal
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organs was seen.
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Immunohistochemical staining of the tumor cells revealed a diffuse intracytoplasmic
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labeling of moderate and high intensity for vimentin and pan-cytokeratin, respectively (Fig. 3B,
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3C). The diagnosis of an epithelioid pleural and pericardial mesothelioma with associated
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pulmonary alveolar microlithiasis was made based on the characteristic gross, histological, and
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immunohistochemical findings.
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In summary, concurrent diagnosis of PAM and pleural mesothelioma is made in an adult
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spayed female Bulldog. The staining and other histomorphological characteristics of the intra-
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alveolar concretions are characteristic of PAM12 and remarkably dissimilar from that associated
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with metastatic and dystrophic calcification, where mineral deposits are located in the interstitial
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compartment or associated to areas of necrosis, respectively.8 In agreement with previous
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reports,12,22 characterization of the intra-alveolar microliths showed strong positive histochemical
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reactions with PAS and von Kossa stains. The strong positive reaction obtained with von Kossa
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stain demonstrates a high content of calcium salts within the microliths. Additionally,
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concretions did not stain with Prussian blue staining suggesting absence of iron. On the other
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hand, gross, histomorphological features, and immunohistochemical staining characteristics of
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the thoracic and pleural nodules were diagnostic for mesothelioma. Neoplastic cells were
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immunoreactive for both cytokeratin and vimentin. The 2 immunohistochemical markers are
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useful for differentiating mesothelioma from other neoplasms, as the latter tumors consistently
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co-express both epithelial and mesenchymal antigens.26
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Mesotheliomas are tumors that arise from cells lining the coelomic cavities including the
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pleura, pericardium, peritoneum and, occasionally, the vaginal tunic of the testis. In the present
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case, the neoplastic process involved both the pleural and visceral pericardial surfaces making it
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rather uncertain as to the primary site of origin of the tumor. However, the extensive
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involvement of the visceral pleura in both lungs together with the fact that most mesotheliomas
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tend to occur in the pleural surface rather than in the pericardium,15 makes pleural origin more
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likely in the present case. Mesothelioma has 3 distinct histological patterns: epithelioid, fibrous
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or sarcomatoid, and mixed cell or biphasic.14 The present case showed an epithelioid pattern
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which, together with the biphasic type, is one of the most common mesothelioma variants
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reported in dogs.15 Factors implicated in the pathogenesis of mesothelioma are exposure to
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asbestos, iron, or silica dust in industrial settings,26 in addition to viral or genetic factors.7,25,27 In
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the current case, there was no known history of exposure to industrial dusts nor occurrence of
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cases of mesotheliomas in relative dogs. Finally, no lesions suggestive of any viral infections
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were observed in the studied organs and tissues.
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The respiratory clinical signs caused by pleural effusions observed in the present case
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were attributed to the mesothelioma. On the other hand, however, PAM was considered to be an
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incidental finding in this dog. Indeed, PAM is an uncommon alveolar filling disorder usually
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diagnosed incidentally. Human patients affected with PAM are often asymptomatic for years
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with a generally stable, though progressive course of the disease.12 In those cases where clinical
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signs are observed, human patients typically present with progressive dyspnea and reduced
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pulmonary function. Respiratory insufficiency may eventually progress to pulmonary fibrosis,
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end-stage lung disease, and chronic pulmonary heart disease. In severe cases, death is often due
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to a combination of pulmonary dysfunction, pulmonary hypertension, and subsequent cor
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pulmonale.6,12 Studies have confirmed that familial PAM cases are caused by autosomal
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recessive inheritance of a mutation in the gene SLC34A2.10 In addition, several reports have
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suggested that environmental factors may play a role in the etiology.22 Whether individual
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susceptibility plays a major role in the pathogenesis of PAM following exposure to sand, silica,
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or other extrinsic factors is still unclear. The occurrence of PAM in animals is very rare with
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only a limited number of cases reported in the literature. Reports of 5 dogs with PAM can be
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found that showed a varied clinical and pathological presentation including ruptured chordae
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tendineae and hard, incompressible lungs in 1 dog18; 2 dogs with chronic Strongyloides
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stercoralis infection5; a dog with hypothyroidism2; and a dog suffering from osteoarthtitis.21
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When comparing the pathological findings described in the previous cases, no common lesion
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other than PAM could be found. All 5 cases showed a multifocal and generalized deposition of
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microliths within alveolar lumen, with variable interstitial fibrosis. It is worth mentioning that
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interstitial fibrosis was also observed in the present case, which, together with the hyperplasia of
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pneumocytes type 2, might be due to the physical damage that microliths cause to the alveolar
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walls. The 5 canine cases previously reported all belong to different and unrelated breeds and the
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dogs ranged from 4 months to 11 years old. Similarly, human PAM cases are reported to occur at
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any age, ranging from a few months to advanced age, with the mean age of diagnosis being 35
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years.12 Regardless, a higher number of descriptions in veterinary species is necessary to contrast
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this and other epidemiological factors. While the etiology of PAM in veterinary species remains
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obscure, both genetic and environmental factors are suspected. In the present case, it is unknown
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if PAM occurred in other blood-related dogs. Moreover, as mentioned above, the owner reported
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no exposure to dust or pollutants other than the ones usually present in the city where he lived
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with his dog.
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A possible association between PAM and mesothelioma in the present case cannot be
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ruled out. There are 3 possibilities to explain the concurrent presentation of both lesions: 1)
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initial development of mesothelioma followed by PAM; 2) PAM developing first and then
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followed by the mesothelioma; or 3) independent formation of both PAM and mesothelioma,
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without any cause-effect connection between the 2 lesions. In favor of the first hypothesis, there
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is a report describing human PAM cases in a single area of the lung as a secondary localized
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lesion associated with adenocarcinoma or pleural mesothelioma.1 In the present case, both the
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mesothelioma and PAM extensively affected the pulmonary pleura and pulmonary parenchyma,
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respectively. As such, a focal initial relationship of both lesions cannot be ruled out. Supporting
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the second hypothesis, a similar association has been suggested in some testicular tumors and
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testicular microlithiasis (TM) in human beings.11,24 Testicular microlithiasis has been linked to
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an increased risk for developing germ cell tumors; it is proposed that TM may result from the
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same genetic abnormalities as germ cell tumors.11 Even though TM is not uncommon in human
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beings, this condition, to the authors’ knowledge, has not been described in dogs. Additionally,
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an association between prostate microlithiasis and mammary calcifications and malignancies
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have also been described in human beings.10 On the other hand, it is well known that
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mesotheliomas are associated with inhalation of asbestos fibers or other foreign body
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materials.26,27 Taking into account that, in the present case, the pulmonary pleura was more
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extensively affected than the parietal pleura, it may be speculated that the presence of microliths
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could have acted as a foreign material inducing damage to visceral pleura and leading to tumor
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development. Nevertheless, it might also be possible that the relationship between the 2 lesions
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was not of cause-effect nature. In this regard, both lesions are reported to be etiologically linked
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to inhalation of environmental substances,22,27 although the influence of such factors could not be
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evidenced in the present case. Finally, it cannot be ruled out that concurrence of both lesions was
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purely incidental. The study of potential future cases showing both conditions in the same
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individuals might elucidate whether such an etiological relationship exists between both lesions.
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Further studies of PAM might also lead to a better understanding of the epidemiology and
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pathogenesis of this disorder in veterinary species.
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Acknowledgements
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The authors would like to thank Dr. Karoline Forster (Clinic for Small Animal Medicine,
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University of Zürich) for presenting the dog for necropsy.
13
Sources and manufacturers
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a. Product no. M082101, Dako Sweden AB, Stockholm, Sweden.
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b. Product no. M7020, Dako Sweden AB, Stockholm, Sweden.
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Declaration of conflicting interests
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The author(s) declare no potential conflicts of interest with respect to the research, authorship,
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and/or publication of this article.
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Funding
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The author(s) received no financial support for the research, authorship, and/or publication of
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this article.
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Figure legends
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Figure 1. Parietal pleura; Bulldog. Numerous multifocal to coalescent, tan to yellowish-red,
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firm, solid protruding nodules scattered throughout the costal and diaphragmatic pleural surface.
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Figure 2. Lung; Bulldog. A, multifocal accumulations of circular to irregularly shaped, variably
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sized (30–100 µm), densely basophilic stained concretions (microliths) within alveoli and
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alveolar septa. Hematoxylin and eosin stain. Bar = 100 µm. B, alveolar septa show mild to
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moderate amount of collagen deposition, shown stained in blue. Gomori blue trichrome stain.
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Bar = 50 µm. C, multiple purple-stained concentric lamella are evident. Periodic acid–Schiff
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stain. Bar = 20 µm. D, microliths intensely stained brownish to black. Von Kossa stain. Bar = 50
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µm.
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Figure 3. Pleural mesothelioma; Bulldog. A, papillary outgrowths of neoplastic cubical to
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cylindrical neoplastic cells within a moderately thick collagenous stroma. Cells show distinct
13
borders and moderate degree of anisocytosis and anisokaryosis. Moderate number of
14
lymphocytes and neutrophils are also present. Hematoxylin and eosin. Bar = 20 µm. B,
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cytoplasm of neoplastic cells is diffusely and strongly brown-stained. Pan-cytokeratin
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immunohistochemistry. Bar = 20 µm. C, pleural mesothelioma; cytoplasm of neoplastic cells is
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diffusely brown-stained. Vimentin immunohistochemistry. Bar = 20 µm.
14