Multifocal Oligodendroglioma in Three Dogs
This report describes the clinical, histopathologic, and imaging findings of multifocal oligodendrogliomas from three canine patients. Clinical history varied but included seizure activity and behavior changes. Neurologic examination abnormalities included ataxia, proprioceptive deficits, cranial nerve deficits, and changes in mentation. MRI in one patient revealed multifocal brain lesions; however, the MRI was normal in another one of the patients. Histopathologic evaluation identified multifocal neoplastic infiltrates in all three patients involving the cerebral cortex, brainstem, and spinal cord, with leptomeningeal extension in two of the three patients. All three patients were euthanized due to progression of their neurologic condition and/or complications due to aspiration pneumonia. Oligodendrogliomas should be considered a differential diagnosis for patients with multifocal brain disease.
Introduction
To the authors' knowledge, this is the first report to describe the clinical, histopathologic, and imaging findings of multifocal oligodendrogliomas from multiple canine patients. This disease process is poorly documented in the veterinary literature, and, to the authors' knowledge, this report details a previously undocumented MRI description of multifocal oligodendrogliomas in dogs.
Case Report
Case 1
A 10.5 yr old sexually intact female mixed-breed dog was evaluated for seizures and ataxia. Fourteen days prior to presentation, the patient was treated with methocarbamola (16.7 mg/kg per os [PO] q 8 hr) and prednisoneb (0.4 mg/kg PO q 12 hr) for cervical pain. Five days prior to presentation, the patient had generalized cluster seizures and was treated with phenobarbitalc (2.8 mg/kg PO q 12 hr) and diazepamd (0.2 mg/kg PO q 12 hr). At the time of presentation to the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania, neurologic examination revealed an obtunded mentation and a diminished menace response bilaterally. Spinal reflexes were normal. The patient was ambulatory with decreased proprioception in all four limbs. Neuroanatomical localization was consistent with a prosencephalic lesion; however, involvement of the brainstem could not be ruled out on the basis of the spinal ataxia.
A normocytic, normochromic anemia (32.9%; reference range, 40.3–60.3%) was the only clinically significant abnormality noted on the complete blood count (CBC). Serum biochemical analysis showed increased alanine aminotransferase activity (317 U/L; reference range, 16–91 U/L), increased aspartate aminotransferase activity (88 U/L; reference range, 23–65 U/L), and increased alkaline phosphatase activity (694 U/L; reference range, 20–155 U/L). Phenobarbital serum level was 22.1 μg/ml (reference range, 15–40 μg/ml). IgM and IgG serology for Neospora caninum was negative (both titers <1:32), and Toxoplasma gondii IgG serology was also negative (titer <1:64). Thoracic radiographs were unremarkable, and abdominal ultrasound revealed hepatic nodular hyperplasia.
MRIe of the brain revealed three lesions. On T2-weighted transverse scans, the first lesion was a small area of increased signal intensity in the white matter of the left pyriform lobe, measuring approximately 4 mm in diameter. This first lesion was not visible on precontrast T1-weighted transverse scans. Following contrast administrationf, a small region of homogenous contrast enhancement in the left pyriform lobe was noted ventromedial to the most rostral part of the left lateral ventricle (Figure 1). The second lesion was approximately 1 cm in diameter, with a well-defined border. It was located on ventral midline and involved the hypothalamus and pituitary stalk. It extended rostrally and dorsally to the level of the internal capsule. The signal intensity of this lesion on T2-weighted scans was homogeneous and hyperintense relative to cerebrospinal fluid (CSF), and it appeared to be contiguous with the third ventricle. On T1-weighted scans, the second lesion appeared hypointense relative to surrounding brain tissue and had a rim of strong enhancement surrounding a nonenhancing center following gadolinium administration (Figures 2A, B). The third lesion was contiguous with the temporal and occipital horns of the left lateral ventricle and extended into the temporal and occipital lobes of the brain. On T2-weighted images, regions of this third lesion appeared hyperintense relative to CSF. Some of this hyperintensity extended into the ventricle. The third lesion was hypointense relative to surrounding tissue on T1-weighted scans, hyperintense relative to CSF, and extended into the left lateral ventricle. Following contrast administration, this lesion had a contrast-enhancing rim that merged with the lining of the left lateral ventricle. There was also evidence of contrast enhancement within the meninges surrounding the caudoventral aspect of the left occipital lobe (Figures 3A–C). Differential diagnoses for the lesions seen on MRI included neoplastic causes (including oligodendroglioma, metastatic tumors, or lymphosarcoma) and inflammatory causes (including cryptococcosis or other fungal infection).
Citation: Journal of the American Animal Hospital Association 47, 5; 10.5326/JAAHA-MS-5551
Citation: Journal of the American Animal Hospital Association 47, 5; 10.5326/JAAHA-MS-5551
Citation: Journal of the American Animal Hospital Association 47, 5; 10.5326/JAAHA-MS-5551
Cerebellomedullary cistern CSF analysis revealed a total protein of 138 mg/dL (reference range, <25 mg/dL), 64 red blood cells (RBC)/μL (reference range, <30 RBC/μL), and one nucleated cell/μL (reference range, <5 nucleated cells/μL). CSF cytospin evaluation revealed neither atypical cells nor infectious organisms. Aerobic and anaerobic bacterial cultures and fungal cultures on the CSF were negative. A Cryptococcus neoformans antigen latex agglutination test performed on the CSF was also negative.
Following MRI and spinal fluid procurement, the patient had a prolonged recovery from general anesthesia, displayed temporal muscle fasciculation, and remained severely obtunded. Due to the presumptive diagnosis of multifocal neoplasia and lack of clinical improvement, the owners elected euthanasia.
Grossly, a gray, soft, gelatinous mass was noted on necropsy. The mass was located adjacent to the left lateral ventricle and involved the thalamus. Histologically, the neoplasm extensively involved the ventricular system and the central canal of the spinal cord, with minimal involvement of the leptomeninges. Multiple noncontiguous foci of neoplastic cells were observed in the parenchyma of the cerebral cortex and brainstem, with multifocal effacement of the ependymal lining. Rostrally, the neoplastic cells occupied the lateral ventricle and infiltrated the caudate nucleus. Neoplastic cells effaced a large area of the optic chiasm bilaterally. Further caudally, neoplastic cells multifocally occupied the lateral and third ventricles and infiltrated the fornix of the hippocampus, dorsal thalamic nuclei, and the periventricular white matter, extending to the thalamus and hypothalamus, as well as the dorsal cerebral cortex. Neoplastic cells also invaded the pyriform lobe, extending to the adjacent cerebral cortex and hippocampus. Neoplastic cells were present within the mesencephalic aqueduct, fourth ventricle, and the central canal of the cervical, thoracic, and lumbar spinal cord, extending (occasionally) into the adjacent gray and white matter (Figures 4A–F).
Citation: Journal of the American Animal Hospital Association 47, 5; 10.5326/JAAHA-MS-5551
The neoplastic cells were arranged in either sheets or cords of round cells subtended by a fine fibrovascular stroma. Neoplastic cells were characterized by variably distinct cell borders, moderate amounts of eosinophilic or clear cytoplasm, and round or (occasionally) indented nuclei with finely stippled chromatin and inconspicuous nucleoli (Figure 5). Mitotic figures were infrequently observed, and there was mild anisokaryosis. Often, clusters of prominent branching vascular proliferations were interspersed with the neoplastic cells. Occasionally, small spaces filled with a basophilic mucinous material separated neoplastic cells. Neoplastic cells were morphologically similar in all examined locations along the central nervous system (CNS).
Citation: Journal of the American Animal Hospital Association 47, 5; 10.5326/JAAHA-MS-5551
Case 2
A 3.5 yr old spayed female boxer presented with a 3 wk history of behavioral changes, cervical hyperesthesia, left thoracic limb knuckling, and generalized seizures. The patient was initially treated with prednisone (0.5 mg/kg PO q 12 hr) and diazepamg (0.2 mg/kg IV) by the primary care veterinarian. On presentation to the Matthew J. Ryan Veterinary Hospital of the University of Pennsylvania, the patient experienced a generalized seizure, and the patient was treated with diazepam (0.55 mg/kg IV) and phenobarbitalh (4 mg/kg IV q 6 hr to a maximum of 16 mg/kg). Neurologic examination revealed an absent right-sided menace response and a decreased right-sided facial nociception. The patient was ambulatory; however, gait, postural reactions, and segmental reflexes were difficult to accurately assess due to the prior administration of diazepam. Neuroanatomic localization was most consistent with a multifocal lesion involving either the left and right sides of the prosencephalon or the left prosencephalon together with the left brainstem. The patient was prescribed ampicillini (20 mg/kg IV q 8 hr), enrofloxacinj (15 mg/kg IV q 24 hr), ranitidinek (2 mg/kg IV q 12 hr), and esomeprazolel (0.5 mg/kg IV q 12 hr).
CBC findings were unremarkable. Serum biochemistry abnormalities consisted of mildly increased alanine aminotransferase enzyme activity (124 U/L; reference range, 16–91 U/L). IgM and IgG serology for N. caninum was negative (both titers <1:32) and IgG T. gondii serology was negative (<1:64). A SNAP-4Dxm test was negative for Dirofilaria immitis antigen, and negative for serum antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia canis. A serum C. neoformans antigen latex agglutination test was also negative. Thoracic radiographs revealed a patchy alveolar pattern in the right cranial and right middle lung lobes, consistent with aspiration pneumonia. Arterial blood gas evaluation revealed a partial pressure of oxygen of 66 mm Hg and an alveolar-arterial gradient of 49.5 mm Hg. Due to progressive respiratory embarrassment and poor prognosis, the owner elected humane euthanasia.
On postmortem examination, a gray, gelatinous mass was located in the left cerebral cortex at the level of the cranial mesencephalon. Histologically, neoplastic cells were extensively scattered within the ventricular system and leptomeninges, including the spinal cord (Figures 6A, B). Foci of parenchymal invasion included the caudate nucleus, optic chiasm, hippocampus, parahippocampal gyrus, and medulla. Rostrally, the neoplastic cells infiltrated the periventricular white matter of the lateral and third ventricles, extending to the caudate nucleus. In addition, neoplastic cells invaded the dorsal hippocampus and parahippocampal gyrus from the level of the caudal thalamus to the mesencephalon. Neoplastic cells were present within the leptomeninges at the level of the cerebellum and medulla, with focal extension into the cochlear nucleus, and in the leptomeninges of the cervical, thoracic, and sacral spinal cord, occasionally separating adjacent nerve roots. Neoplastic cells were similar morphologically to those described in case 1.
Citation: Journal of the American Animal Hospital Association 47, 5; 10.5326/JAAHA-MS-5551
Case 3
A 7 yr old sexually intact male French bulldog presented with a 5 day history of lethargy, inappetence, and pelvic limb weakness and ataxia. Neurologic examination revealed: a quiet mentation; an occasional long-strided pelvic limb gait; decreased left-sided menace; decreased left-sided palpebral reflex; and decreased left pelvic limb proprioception. Neuroanatomic localization was consistent with either a right-sided prosencephalic lesion or a multifocal process involving both the right prosencephalon and the spinal cord from the third thoracic vertebra to the third lumbar vertebra.
Results of a CBC, serum biochemical analysis, and urinalysis were normal. Thoracic radiographs and abdominal ultrasound were within normal limits. MRI of the brain was performed. T2-weighted images (sagittal, transverse, and dorsal planes), precontrast T1-weighted images (transverse and sagittal planes), postcontrast T1-weighted images (transverse, dorsal, and sagittal planes), and proton-density images (transverse plane) were acquired. No obvious abnormalities were observed. CSF obtained from the cerebellomedullary cistern had a total protein of 46 mg/dL (reference range, <25 mg/dL), 25 RBC/ μL (reference range, <30 RBC/μL), and three nucleated cells/μL (reference range, <5 nucleated cells/μL). CSF cytospin evaluation revealed a nucleated cell population composed of 1% neutrophils, 32% lymphocytes, and 66% large mononuclear cells.
The patient re-presented 5 days later with continued pelvic limb weakness and ataxia, and appeared nonvisual in the left eye. The patient was discharged with clindamycinn (10 mg/kg PO q 8 hr) and doxycyclineo (10 mg/kg PO q 24 hr) pending infectious disease testing. IgM and IgG titers were <1:32 for N. caninum, and IgG serology for T. gondii was negative (<1:64). Serology for Rickettsia rickettsii (<1:40) and a serum C. neoformans antigen latex agglutination test were also negative. A SNAP-4Dx test was negative for D. immitis antigen and negative for serum antibodies to A. phagocytophilum and E. canis. Serum was positive for antibodies to B. burgdorferi, with a quantitative C6 antibody levelp of 25 U/mL (reference range, <30 U/mL).
The patient presented a third time 4 days later and was obtunded and laterally recumbent and tetraparetic on presentation. The patient lacked a menace response bilaterally and had fixed, mydriatic pupils. The patient was bradycardic (heart rate was 60 beats/min) and hypertensive, with an initial Doppler blood pressure of 240 mm Hg. A CBC and serum chemistry revealed no clinically significant abnormalities. Mannitolq (1 g/kg IV) and dexamethasone sodium phosphater (0.3 mg/kg IV q 8 hr) were administered. After an initial improvement in mentation, the patient remained obtunded over the following 12 hr and had multiple episodes of vomiting and suspected aspiration. Therapy with enrofloxacin (15 mg/kg IV q 24 hr), dolasetrons(0.5 mg/kg IV q 12 hr), and esomeprazole (0.5 mg/kg IV q 24 hr) was added. Due to deteriorating clinical status and poor prognosis, the owners elected humane euthanasia.
Grossly, the fourth ventricle was mildly dilated in this patient. No masses were observed grossly. Histologically, neoplastic cells were present within the brain and sacral spinal cord, involving the lateral and fourth ventricles, as well as the leptomeninges. At the level of the optic chiasm, neoplastic cells infiltrated the right septal nuclei. Caudally, at the level of the medulla, neoplastic cells within the fourth ventricle focally extended into the parenchyma of the right dorsolateral midmedulla. Neoplastic cells focally expanded the leptomeninges of the sacral spinal cord. Neoplastic cells were similar morphologically to those observed in previous cases described in this report.
Discussion
This report describes three dogs in which oligodendrogliomas occurred in multiple noncontiguous sites throughout the CNS. Although previous reports have referred to the ability of oligodendrogliomas to spread throughout the CNS, to the authors' knowledge, this report describes the first occurrence of multifocal oligodendrogliomas in multiple cases, and is the only report in dogs since the 1970s.1 Oligodendrogliomas comprise up to 14% of all primary canine intracranial neoplasms.2 The mean age of dogs with oligodendrogliomas is 8 yr; however, oligodendrogliomas have been reported in dogs as young as 15 mo.2,3 Brachycephalic breeds such as boxers, Boston terriers, and bulldogs are overrepresented.1,2,4
Oligodendrogliomas in dogs often occur in cerebral white or gray matter and commonly border on a ventricle and/or extend through the ependymal surface. Frequency of occurrence is generally higher in the olfactory bulb and frontal lobe.5–8 Grossly, these tumors are usually well-demarcated, gelatinous, and soft, with multifocal hemorrhages.1,3,5,6,9 Clinical signs described have largely reflected their location within the CNS. In one study, seizures (18/25 cases) and mentation change (10/25) were the most common presenting clinical signs.2
It is interesting to note that with all patients in this series the presenting clinical signs were predominantly prosencephalic in nature, with minor evidence of multifocal disease, despite the widespread infiltration of tumor cells. It would be interesting to speculate whether this was a function of mass size, with the largest masses being present in the region of most severe clinical signs, or if other factors (such as vascular compromise or neuronal necrosis secondary to the tumors) were involved. Other studies have reported that mass size alone has relatively less effect on clinical signs compared with features such as the speed of tumor growth, and it is likely the same holds true for the cases described herein.10,11
Primary oligodendrogliomas have also been reported in the spinal cord, with patients presenting with ataxia and proprioceptive deficits.8,12 All three patients in this study either presented with or developed signs referable to a spinal cord lesion. The first patient had neoplastic cells present in the central canal and adjacent white and gray matter of the spinal cord. In the second patient, neoplastic foci were present in the leptomeninges of the cervical, thoracic, and sacral spinal cord. In the third patient, a focus of neoplastic cells expanded the leptomeninges of the sacral spinal cord. Although none of these patients underwent imaging of their spinal cords, it is possible that the clinical signs of ataxia, weakness, and proprioceptive deficits were the result of neoplastic infiltrates in either the spinal cord parenchyma or leptomeninges.
In case 1, MRI revealed multifocal brain lesions that all appeared contiguous with the ventricular system. The presence of multifocal brain lesions on MRI is usually suggestive of inflammation, vascular disease, fungal infection, lymphosarcoma, and other metastatic neoplasia.13 Multifocal primary brain neoplasms have rarely been reported in dogs, with those reported either representing two distinct neoplasms, ependymomas, or choroid plexus tumors that have spread within the ventricular system and through CSF pathways.4,14,15 An MRI description of a multifocal oligodendroglioma in a cat has been documented; however, this report details a previously undocumented MRI description of multifocal oligodendrogliomas in the dog.16 The hypointensity of these lesions on T1-weighted images and hyperintensity on T2-weighted images, as well as the variability in homogeneity and postcontrast enhancement, were all consistent with previous reports for canine oligodendrogliomas.17,18 In particular, the hypointensity of the lesions seen on T1-weighted images together with relative hyperintensity seen on T2-weighted images are suggestive of increased intracellular fluid occurrence, relative to normal brain tissue, and a gelatinous consistency. This MRI appearance is also well described in humans.19
Unfortunately, fluid-attenuated inversion recovery (FLAIR) sequences were unavailable at the imaging facility where the cases were imaged. FLAIR sequences provide useful information regarding fluid within the brain that is not CSF (i.e., cerebral edema, regions of necrosis) and may have been useful in delineating the tumor from the ventricles and establishing the extent of cerebral edema.20 Nonetheless, the findings on T1- and T2-weighted scans were consistent with a diagnosis of oligodendrogliomas, with signal intensity of tumor tissue being sufficiently different from both CSF and brain parenchyma to be able to distinguish the tumor tissue without FLAIR scans. It is also possible that FLAIR sequences would have identified some abnormalities in the third case that were not visible on the T2-weighted scans.20 Other sequences that may have yielded more information about the tumors include diffusion-weighted and apparent diffusion coefficient (ADC) map sequences, which provide information on water movement within and between areas of the brain and are useful in ruling out vascular lesions.21,22 Other imaging findings associated with human oligodendrogliomas include areas of calcification, hemorrhage, and cyst formation.23 It is interesting that no lesion was seen on MRI in case 3, either pre- or postcontrast injection. This is most likely because of the small size of the tumor foci; however, a lack of contrast enhancement is a relatively common finding in human oligodendrogliomas, and it has been suggested that robust contrast-enhancement may correlate with more aggressive behavior.24
In all three cases described herein, it is likely that the lesion had spread along CSF pathways and that the neoplastic cells were in contact with either the ventricular or subarachnoid spaces. A single report in dogs of oligodendroglioma spread via the central canal has been described, but, to the authors' knowledge, no additional reports regarding the spread of oligodendrogliomas in dogs through the CNS have been published since then.1 In contrast, the spread of a primary tumor to the cerebellum or spinal cord along CSF pathways in humans is a well known phenomenon (known as “drop metastasis”), which has been recognized since the 1940s.19,25 A separate, but related, phenomenon known as “diffuse leptomeningeal oligodendrogliomatosis” is a rare disorder characterized by widespread invasion of the CSF-containing spaces without evidence of a primary intraparenchymal focus.26,27 This rare tumor has only been reported in a small number of human patients, and diagnosis is made challenging by the lack of a discrete mass visible on imaging studies.27 It has been documented that oligodendrogliomas can invade into, and grow along, the leptomeninges in dogs, although diffuse oligodendroglial tumor spread has not been reported.1,5 Despite the presumptive CSF spread in all of the patients described here, CSF cytologic evaluation in two of the patients was only compatible with nonspecific intracranial disease and offered no definitive criteria to specifically suggest neoplasia. In people with oligodendrogliomas, neoplastic cells can be found in CSF in up to 14% of patients following diagnosis.19 A case report of intracranial oligodendrogliomas in two cats describes the appearance of presumptive oligodendroglioma cells in samples of CSF; however, these tumor cells were only identified retrospectively after the tumor was identified on postmortem examination.16
True metastasis of primary intracranial tumors (to extracranial locations) in humans occurs rarely. Since 1951, only 32 cases of oligodendrogliomas metastasizing outside the CNS have been reported worldwide.28 Several reasons have been postulated for this finding, including the presence of a relatively impermeable barrier between the brain and vascular spaces in the form of the blood brain barrier, a lack of lymphatic drainage from the brain, and the rapid mortality associated with these tumors. The mechanism of tumor spread through CSF pathways requires a different biologic process compared with true metastasis via hematogenous spread. In the latter situation, neoplastic cells first need to evade local tissue boundaries, infiltrate into and survive in the vasculature, and then extravasate from the vasculature before invading a second tissue site and proliferating.29 Therefore, it seems likely that the spread of primary brain tumors throughout the CNS along CSF pathways does not represent true metastasis.
Conclusions
This report describes the spread of oligodendrogliomas along CSF pathways to multiple sites within the brain and spinal cord in three dogs. Findings presented suggest that oligodendroglioma should be a differential diagnosis for multifocal lesions, especially if they are either near or contiguous with ventricular spaces. This report demonstrates that MRI alone may be insufficient for making a diagnosis of intracranial oligodendrogliomas; however, some imaging findings, in particular T2-weighted hyperintensity together with T1-weighted hypointensity, are strongly suggestive of these tumors. Furthermore, the presence of multifocal CNS signs in patients does not exclude a diagnosis of oligodendroglioma in dogs. Due to their advanced state of disease, all three patients presented in this report suffered progressive clinical deterioration, suggesting that multifocal oligodendrogliomas carry a poor prognosis. It is hoped that the information in this report may make earlier antemortem diagnosis possible in some cases, thereby improving outcome in the future.
Postcontrast T1-weighted transverse magnetic resonance (MR) image of the dog presented in case 1 showing a small region of homogenous contrast enhancement (arrow) in the left pyriform lobe ventromedial to the most rostral part of the left lateral ventricle.
A: A T2-weighted transverse MR image of the dog presented in case 1 showing a second neoplastic lesion (arrow) on the ventral midline involving the hypothalamus and pituitary stalk. The signal intensity of this lesion is homogeneous and hyperintense relative to cerebrospinal fluid (CSF). B: A postcontrast T1-weighted transverse MR image showing the center of the same lesion (arrow) as hypointense relative to surrounding brain tissue, with a rim of strong contrast enhancement.
A: A T2-weighted transverse MR image of the dog presented in case 1 showing a third neoplastic lesion (arrow), which is contiguous with the temporal and occipital horns of the left lateral ventricle. Some regions of this lesion appear hyperintense relative to CSF. B: The same neoplastic lesion (arrow) is hypointense relative to the surrounding tissue, hyperintense relative to CSF, and extends into the left lateral ventricle, as seen on the transverse T1-weighted MR image. C: Postcontrast T1-weighted transverse MR image showing the neoplasm (arrow) with a contrast-enhancing rim that merges with the lining of the left lateral ventricle (closed arrow head). There is contrast enhancement within the meninges (open arrow head) surrounding the caudoventral aspect of the left occipital lobe.
Histopathology of the multifocal oligodendrogliomas diagnosed in the dog presented in case 1. Neoplastic cells (arrows) are present within the optic chiasm (A), thalamus and hypothalamus (B), lateral ventricle (C), pyriform lobe (D), fourth ventricle (E), and spinal cord (F). Hematoxylin and eosin (H&E) staining, magnification ×1.
Histopathology of the multifocal oligodendrogliomas diagnosed in the dog presented in case 1. Monomorphic neoplastic cells with hyperchromatic nuclei and clear cytoplasm are present. Note the prominent, branching, vascular proliferations (arrow). H&E staining, original magnification ×20.
Histopathology of the multifocal oligodendrogliomas diagnosed in the dog presented in case 2. Neoplastic cells (arrows) extensively infiltrate the leptomeninges (A) and ventricular system (B). H&E staining, magnification ×1.
Contributor Notes
M. Koch's present affiliation is Internal Medicine Service, Veterinary Specialists of Rochester, Rochester, NY.
M. Koch's updated credentials since article acceptance are VMD, DACVIM.
M. Sánchez's updated credentials since article acceptance are VMD, PhD, DACVP.
S. Long's present affiliation is Veterinary Teaching Hospital, University of Melbourne, School of Veterinary Science, Victoria, Australia.
S. Long's updated credentials since article acceptance are BVSc, MVM, PhD, DECVN, MRCVS.
