Antemortem Diagnosis of Localized Central Nervous System Histiocytic Sarcoma in 2 Dogs

Abstract

A6-year-old male castrated Greyhound was presented to the William R. Pritchard Veterinary Medical Teaching Hospital, University of California, Davis, with a 4-week history of apparent spinal pain and paraparesis. There was no improvement despite treatment consisting of oral carprofen (unknown dose) for 10 days followed by a tapering course of oral prednisone for 6 days before referral (1.2–0.2 mg/kg/d). Abnormalities on physical examination included 5% dehydration, a large flaccid bladder that was easily expressed, moderate conjunctivitis, and mild generalized patchy alopecia. On neurological examination, the dog was ambulatory with marked paraparesis and absent conscious proprioception in both pelvic limbs. The patellar, gastrocnemius, and pelvic limb withdrawal reflexes were decreased bilaterally, the cutaneous trunci reflex was absent caudal to L4, and the perineal reflex and anal tone were absent. The tail was flaccid with intact superficial pain sensation. The dog was painful with dorsal palpation of the lumbar spine. The neurological abnormalities were consistent with an L4–Cd myelopathy. A CBC and serum biochemistry had mild abnormalities, interpreted as clinically insignificant. Urinalysis indicated a specific gravity of 1.048 and marked proteinuria (4+ on sulfosalicylic acid method). Thoracic radiographs and abdominal ultrasound examination indicated no clinically relevant findings. After vertebral column radiographs, where no abnormalities were seen, two 22 G 3.5-in. spinal needles were placed, 1 at the L4–5 and 1 at the L5–6 intervertebral disc spaces, but cerebrospinal fluid (CSF) could not be obtained. CSF was collected from the cerebellomedullary cistern. A diagnostic myelogram was not performed and, therefore, magnetic resonance (MR) images of the lumbar spinal cord followed by those of the brain were acquired with a 1.5-T scanner.a T1-weighted (T1W) images and T2-weighted (T2W) images included the L3 vertebra to the caudal vertebrae and the entire brain to the C3 vertebra. The spinal cord was enlarged and slightly elevated and dorsoventrally flattened from the L4 through the S2 vertebrae. On the T2W images, there was intramedullary hyperintensity of the lumbar and sacral segments of the spinal cord and the cauda equina, with partial loss of epidural fat signal and gray and white matter definition in the mid lumber region (Fig 1A). After IV contrast administration,b there was moderate parenchymal contrast enhancement and mild diffuse meningeal enhancement from the cranial lumbar region to the conus medullaris (Fig 1B and C). On the postcontrast T1W images of the brain, there was diffuse meningeal enhancement, most evident along the tentorium cerebelli and the falx cerebri (not shown). (A) T2-weighted sagittal magnetic resonance images (MRI) from dog 1. Note the intramedullary hyperintensity compared with spinal cord gray matter, loss of epidural fat signal, and gray/white matter definition. (B) T1-weighted sagittal MRI and (C) T1-weighted sagittal MRI postcontrast from dog 1. There is moderate parenchymal contrast enhancement and mild diffuse meningeal enhancement (arrow) from L4 to the conus medullaris. The CSF had a markedly increased nucleated cell count (660 cells/μL; reference range, ≤ 3 cells/μL) and a moderate increase in the protein concentration (72 mg/dL; reference range, ≤ 25 mg/dL). The differential cell count was 15% neutrophils, 12% lymphocytes, 68% large mononuclear cells, and 5% eosinophils. On cytologic evaluation, most of the large mononuclear cells were considered typical macrophages; however, some of the cells were extremely large and variably vacuolated with large immature nuclei (Fig 2). Within this small subpopulation of large immature cells, numerous mitoses were noted (2, 3). There were occasional large multinucleate tumor cells, with a marked anisokaryosis of the component nuclei and nuclear fragmentation (Fig 3). Rare bizarre multinucleate tumor cell variants also were present (Fig 4). The interpretation was marked, mixed, predominantly histiocytic inflammation with underling neoplasia, most likely histiocytic sarcoma. Cerebrospinal fluid cytospin slide, dog 1. There is a marked, predominantly large mononuclear pleocytosis. Note the scattered, extremely large, single round cells with large immature nuclei. Within this small subpopulation of large immature cells, there are numerous mitoses. Wright–Giemsa stain, scale bar = 50 μm. Cerebrospinal fluid cytospin slide, dog 1. Occasional large multinucleate tumor cells are present that have marked anisokaryosis of component nuclei and nuclear fragmentation. Wright–Giemsa stain, scale bar = 30 μm. Cerebrospinal fluid cytospin slide, dog 1. Rare bizarre multinucleate tumor cell variants are present that also have marked anisokaryosis of component nuclei and nuclear fragmentation. Wright–Giemsa stain, scale bar = 30 μm. Treatment with an anti-inflammatory dosage of dexamethasone sodium phosphate IV (0.22 mg/kg/d) was initiated immediately, but the dog deteriorated neurologically over the next 36 hours after anesthesia and became nonambulatory with marked apparent spinal pain. Immunophenotyping of the cells in the CSF was carried out by staining CSF cytospin slides using canine-specific monoclonal antibodies against CD1c, CD11b, CD11c, CD11d, CD3, CD79a, CD34, pancytokeratin, vimentin, and a negative control using previously described methods.1 The large immature cells strongly expressed CD1c (Fig 5) and CD11c. The neutrophils expressed CD11c. As expected, all leukocytes expressed vimentin. The diagnosis was histiocytic sarcoma with secondary marked, mixed inflammation. Based on the deteriorating neurologic status and the poor prognosis associated with histiocytic sarcoma, the dog was euthanized and submitted for necropsy. Cerebrospinal fluid cytospin slide, dog 1. The large immature single round tumor cells have a variable, predominantly cytoplasmic membrane, expression of CD1c. Immunocytochemistry stain, AEC substrate, hematoxylin counterstain, scale bar = 30 μm. At necropsy, clinically relevant findings on gross and histological examination were restricted to the central nervous system (CNS). The meninges of the brain and the spinal cord were diffusely opaque. On the cut surface of the spinal cord, the subarachnoid space was diffusely filled by an infiltrative, pale tan mass that entrapped the nerve rootlets from L1 to the cauda equina and invaded the spinal cord from L4 to the cauda equina. Tissues were immersed in 10% neutral buffered formalin and routinely processed. Five micrometer paraffin-embedded sections were stained with hematoxylin and eosin (H&E) for routine histologic assessment. Immunohistochemistry was performed on sections of the spinal cord using canine leukocyte-specific antibodies for which the sources, reactivity, and staining protocol have been documented previously.2, 3 Histologically, dense sheets of pleomorphic, neoplastic round cells were present within the subarachnoid space of the spinal cord and brain, often obscuring the leptomeninges and multifocally invading the dura mater and subpial neuropil. The most extensive invasion involved the L4–6 and caudal spinal cord segments, in which neoplastic cells extended to and within the central canal. The neoplasm tapered from the thoracic to the cervical spinal cord, markedly expanded again around the medulla oblongata, cerebellum, and thalamus, and then tapered toward the cerebral hemispheres. It invaded along the subarachnoid space around the blood vessels within the superficial neuropil, outlined the cerebellar folia, and occasionally invaded the subpial neuropil. The affected nerve rootlets and the neuropil of the spinal cord and brain contained extensive edema and Wallerian degeneration. Consistent with the histiocytic origin demonstrated by immunocytochemistry of the CSF, neoplastic cells were broadly immunoreactive for CD18, inconsistently immunoreactive for CD45, and not immunoreactive for all other leukocyte antibodies (CD3, CD11d, CD79a, and pancytokeratin). A 9-year-old male castrated Pembroke Welsh Corgi was presented to the William R. Pritchard Veterinary Medical Teaching Hospital, University of California, Davis, for an acute onset of obtundation, head tilt, and difficulty in walking. The dog had received nonsteroidal anti-inflammatory treatment (unknown drug and dose) before presentation. Neurologic examination indicated moderate obtundation, tetraparesis, and mild generalized ataxia with a tendency to walk against the wall on the left side. Inconsistent menace response in the right eye, decreased response to nasal stimulation in the left naris, a mild head tilt to the right, and miotic pupils were noted. Proprioceptive positioning was evaluated by paw replacement reaction, and was absent in the right thoracic and pelvic limbs, and intact on the left. Neuroanatomic localization was consistent with multifocal brain disease. A CBC, serum biochemistry, urinalysis, thoracic radiographs, and abdominal ultrasound examination were unremarkable except for a mildly enlarged and heterogeneously echogenic right pancreatic limb interpreted as most consistent with previous pancreatitis. The dog was anesthetized and MR imaging of the brain was performed, including pre- and postcontrast T1W, T2W, proton density, and fluid-attenuated inversion recovery (FLAIR) images. A mass was identified, which appeared to arise from the lateral margin of the hippocampus, with an extensive component within the lateral ventricles bilaterally. The mass was a hypointense to gray matter on T1W images (Fig 6A) and isointense to slightly hyperintense on T2W images. On T2W images and FLAIR sequences, bilateral periventricular and peritumoral edema was observed (Fig 6B). The mass enhanced homogeneously with contrast administration (Fig 6C and D). (A) T1-weighted transverse magnetic resonance imaging (MRI) at the level of the zygomatic process of the temporal bone of dog 2. Note the bilateral hypointense mass associated with the lateral margin of the hippocampus. (B) Transverse fluid-attenuated inversion recovery image. Note the bilateral periventricular and peritumoral edema. Transverse (C) and dorsal plane (D) T1-weighted postcontrast images. Note the uniform, bilateral contrast enhancement of the mass with asymmetrical bilateral extension into the ventricular lamina. Cisternal CSF had a protein concentration of 135mg/dL (reference range, ≤ 25 mg/dL), 220 RBC/μL (reference range, 0 RBC/μL), and a markedly increased nucleated cell count of 1,550/μL (reference range, ≤ 3 cells/μL), with a differential cell count of 1% lymphocytes, 19% neutrophils, and 80% macrophages. On cytological evaluation, macrophages predominated, with nondegenerate neutrophils in lower numbers. A population of individual giant cells with rounded cytoplasmic margins was also present. These cells had a moderate to high N : C ratio and abundant, homogeneous, medium blue cytoplasm. Rare cells were erythrophagic. Nuclei were oval to reniform with lacy, open chromatin and were somewhat eccentrically placed within the cytoplasm. Binucleated and multinucleated cells were seen occasionally and featured marked anisocytosis and anisokaryosis. Numerous mitotic figures were noted within this population of large, immature round cells. The diagnosis was malignant neoplasia with associated marked mixed inflammation and mild prior hemorrhage. Additional CSF cytospin slides were made and assessed with antibodies against CD1c, CD11b, CD11c, cytokeratin (pancytokeratin and AE1/AE3), and GFAP along with a negative control. The neoplastic cells were not immunoreactive for cytokeratin or GFAP, moderately immunoreactive for CD1c, weakly to moderately immunoreactive for CD11c, and variably and weakly immunoreactive for CD11b. The diagnosis was histiocytic sarcoma with associated marked mixed inflammation. The dog recovered from anesthesia and was treated with an anti-inflammatory dosage of dexamethasone sodium phosphate (0.2 mg/kg/d) IV and lactated ringers solution with 20 mEq KCl/L was added IV (6.7 mL/kg/h). Twelve hours later, the dog was markedly obtunded. Because of rapid deterioration in neurological status and the poor prognosis associated with histiocytic sarcoma, the dog was euthanized. Clinically relevant gross necropsy findings were limited to the CNS. After fixation in 10% neutral buffered formalin, the brain was sectioned frontally and a soft tan mass that was bilaterally within the lateral ventricles and third ventricle was observed. It extensively invaded and effaced the dentate gyri and corpus callosum. Tissues were fixed in formalin, embedded in paraffin, sectioned at 5 μm, and stained with H&E. A section of the brain that included the dentate gyrus was immunohistochemically stained using a panel of leukocyte markers, as previously described in this report. On histopathologic examination, densely cellular sheets of neoplastic round cells, supported by moderate fibrovascular connective tissue, extensively effaced the dentate gyri and the corpus callosum. Few to numerous neoplastic round cells were scattered throughout the subarachnoid space and there was multifocal invasion of the subpial neuropil. The neoplastic cells were strongly immunoreactive with antibodies directed against CD45 and CD18. A subset of the neoplastic cell population, most notably within the meninges, was CD11d immunopositive. The neoplastic cell population exhibited no immunoreactivity with antibodies directed against CD3, CD79a, or CD20. Based on the cellular morphology and results of the immunohistochemical staining of the paraffin-embedded brain tissue, the mass was diagnosed as a histiocytic sarcoma. These results confirmed the diagnosis made antemortem by cytological and immunocytochemical evaluation of the CSF. The neoplasm was considered a primary CNS histiocytic sarcoma because there was no gross or histological evidence of neoplasia outside of the CNS. Histiocytic disorders are common in dogs and can be divided into reactive histiocytoses, cutaneous histiocytoma, localized histiocytic sarcoma, and disseminated histiocytic sarcoma (dHS)/malignant histiocytosis (mHS). dHS and mHS are histiocytic cell malignancies that either arise in 1 location with secondary widespread dissemination (dHS) or in many locations simultaneously (mHS).2, 3 These 2 diseases may be difficult to differentiate and, therefore, the terms frequently are used interchangeably. Dogs with dHS/mHS often have clinical signs of systemic disease because the internal organs are affected.3 Predilection sites include spleen, liver, lymph nodes, lungs, and bone marrow.4, 5 Bernese Mountain Dogs, Rottweilers, and Golden, Labrador, and Flatcoat Retrievers are predisposed to this disease,3, 6-18 whereas other breeds are affected sporadically.5, 19-22 Although many reports describe dHS/mHS affecting the CNS,4, 16, 20, 21 localized CNS histiocytic sarcoma has been infrequently reported.19, 20, 22 Three reports describe localized histiocytic sarcoma confined to the CNS of dogs, sparing other visceral organs,19, 20, 22 but bone marrow was not evaluated in these dogs. All 3 dogs were diagnosed with localized HS postmortem, 2 dogs had the CSF evaluated.20, 22 In 1 dog with diffuse leptomeningeal histiocytic sarcoma, the CSF nucleated cell count was 152 cells/μL, with a differential cell count of 50% neutrophils and 50% mononuclear cells and a total protein of 95 mg/dL. No atypical cells were recognized and CSF immunphenotyping was not performed.20 The other dog had a parenchymal brain mass and the cisternal CSF had a total nucleated cell count of 520 cells/μL, with a differential cell count of 7% neutrophils, 17% lymphocytes, and 76% monocytoid cells. The total protein (pyrogallol red methodology) was 51.8 mg/dL. The cytological interpretation was severe monocytoid—predominant, mixed cell pleocytosis, with cellular atypia suggestive of neoplasia.22 Postmortem, archived, unstained, air-dried, cytocentrifuged slides were immunophenotyped. The monocytoid cells were immunoreactive for CD1c and CD11d and were not immunoreactive for T and B cell markers (CD3 and CD79a). The CSF immunocytochemistry results supported the postmortem diagnosis of HS.22 The MR imaging findings were similar in both dogs reported here. In the first dog, the primary tumor was located in the L4–Cd segments whereas in the second dog, the primary tumor arose from the anatomical margins of the hippocampus with an extensive component in both lateral ventricles. In both cases, the tumors were contrast enhancing on T1W images, with diffuse meningeal enhancement in the area of the tumor as well as in other locations within the CNS. In the second dog, peritumoral edema was present. To our knowledge, this is the first report of antemortem diagnosis of primary CNS HS using CSF analysis. Similar to the dogs in previous reports,20, 22 the dogs in this report had marked, mixed, predominantly mononuclear pleocytosis with a moderate increase in the CSF protein concentration. CSF cytology warranted immunophenotyping because the neoplastic cell population was highly pleomorphic, exhibited both round to stellate and spindloid morphology, and was accompanied by a high number of inflammatory leukocytes, which complicated definitive interpretation without concurrent immunophenotyping. The large immature cells in the CSF were immunoreactive for CD1c and CD11c, variably and weakly immunoreactive for CD11b, but not immunoreactive for CD11d, CD3, or CD79a. The cell morphology and immunophenotype were indicative of histiocytic sarcoma of a dendritic cell origin.2, 3 In a report assessing histiocytic sarcomas of a dendritic cell origin in 39 dogs, the neoplastic cells were consistently immunoreactive for CD1c, CD11c, CD18, and CD45. The cell of origin in these tumors is believed to be an interstitial myeloid dendritic antigen-presenting cell. Postmortem immunohistochemistry performed on the CNS tissue from the dogs reported here confirmed the antemortem diagnosis. Neoplastic cells were strongly and diffusely immunoreactive for CD18, strongly and variably immunoreactive for CD45, and not immunoreactive for CD3, CD79a, and CD20. The MR image characteristics and CSF analysis, including immunocytochemical immunophenotyping and postmortem findings, confirmed the presence of a primary HS with metastasis within the CNS, possibly tracking along the meninges. Dogs with localized CNS HS may have a marked CSF pleocytosis with moderately increased protein concentration. When large, discrete atypical cells are present, immunophenotyping of CSF cells may be used to definitively confirm the histiocytic lineage of the atypical cells and hence diagnose HS. In the 2 dogs reported here, the MR findings of a primary tumor with diffuse T1W meningeal enhancement, CSF analysis, and CSF immunophenotyping permitted an antemortem diagnosis of HS, which was confirmed on postmortem evaluation. aGeneral Electric Signa LX, Milwaukee, WI bMagnevist (Gadopentate dimeglumine 469.01 mg/mL), Berlex Laboratories, Wayne, NJ