Path. Res. Pract. 188, 964-972 (1992)
.
Progressive Multifocal Leukoencephalopathy and Gliomas In a HIV-Negative Patient F. Gullotta, T. Masini1, G. Scarlato2 and K. Kuchelmeister Institut fOr Neuropathologie der Universitat Munster (Direktor Prof. Dr. F. Gullotta), Munster, FRG; I/stituto di Anatomia Patologica de//'Universita, Milano (Direttore Prof. Dr. L. Matturri); 2Clinica Neurologica de//'Universita, Milano (Direttore Prof. Dr. G. Scarlato), Milano, Italy
SUMMARY A case of progressive multifocal leukoencephalopathy (PML) is reported, detected at autopsy of a 30-year-old patient. The clinical picture was characterized by a progressive course of mental deterioration and ingravescent neurological symptoms. The patient was HI V-negative. He died of bronchopneumonia, after a clinical course of 13 months. iutopsy disclosed pulmonary tuberculosis with involvement of regional lymph nodes. In the brain, besides numerous PML-foci of varying age and structure, a pleomorphic astrocytoma was found in the white matter of the right parietal lobe. In the brain stem glial proliferation resembling diffuse gliomatosis was also present. In situ hybridization revealed Pap ova-virus (feV) in oligoglial nuclei, but not in neoplastic astrocytes. This is the third report on the concomitant occurrence of PML and glioma in man.
Progressive multifocal leukoencephalopathy (PML) is an opportunistic demyelinating disease of the central nervous system, caused by virally induced changes of glial cells in an immunological deficient host 1, 8, 14,25. Before the appearance of AIDS this infection was usually observed in patients affected by lympho-myeloproliferative disorders or by chronic diseases which required immunotherapy. In a few cases, however, no chronic pre-existing disease could be found. Since the appearance of AIDS the number of PML cases has tremendously increased. The agents responsible for PML (]C-, SV 40-, and BK-virus) are papova/polyoma viruses,]CV being the most frequent one, as also confirmed by our investigations on HIV-related encephalopathies lO , 16, 18. In a few cases some other viruses have also been identified 7 . Papova/polyoma viruses are oncogenic when inoculated into hamsters, and all are capable of producing cerebral tumors (ependymoma, plexus papilloma, glioma, medulloblastoma, etc. 14 ). However, no definitive evidence has linked any of such viruses to a cerebral tumor in man. 0344-0338/9210 18 8-0964$3.50/0
Here a case of PML and glioma observed in a HIVnegative patient, who had miliary tuberculosis, is reported. Case Report A 30-year-old right-handed man was admitted to the Neurological Clinic of the University of Milan in June 1989 because of a progressive deterioration of cerebral function. He was well until December 1988, when he began to experience difficulties in handline r'..lffs and in performing simple sequences of movements during his work as a sound technician. During the following three months he manifested a progressive left-sided weakness and speech disturbances. A CT scan was normal and he was given prednisone (60 mg/day for 20 days) without improvement. Neurological examination on admission revealed temporal and spatial disorientation, non-fluent aphasia with phonemic paraphasias, anosmias and impairment of com© 1992 by Gustav Fischer Verlag, Stungart
Leukoencephalopathy and Gliomas in a HIV-ncgative Patient· 965
prehension; there was buccofacial and ideomotor apraxia; the left limbs were weak with a Babinski sign on the left, but the patient was able to walk independently. During the following months there was a progressive deterioration of higher cortical functions (memory, attention, writing), with a worsening of the left hemiparesis, with gradual onset of right limb weakness, impairment in all directions of saccadic and pursuit ocular movements, and left hemianopia. By October 1989 the patient was bed-ridden, tetra paretic, severely demented, with global aphasia. CT brain scan was normal with and without contrast material in June 1989. One month later small hypodense areas in the white matter were observed in the left parietal lobe; similar lesions were also present in the right parietal and occipital lobes at a control CT scan in August. NMR, performed in July and again in August, confirmed the presence of small foci of increased intensity on T2weighted images in both hemispheres which, at a new examination in October 1989, had become larger with a confluent appearance.
Hematological and biochemical findings were normal, except for mild to moderate eosinophilia (9-17 %, with a white blood cell count of 7,000/mm); serum ferritin was also normal, as were zinc and iron blood concentrations. Tests for T-cell subsets in the peripheral blood showed an inversion of the T4/T8 ratio, with increase of suppressor and natural killer cells and reduced response to mitogens. Cerebrospinal fluid on admission contained 2.6 mononuclear cells per cubic millimeter; the glucose was 55 mg per 100 ml, and the proteins 49 mg per 100 ml. Isoelectric focussing disclosed six oligoclonal bands, not detectable in serum. Findings were similar in September 1989. No antibodies to HIV, rubella virus, HTLV1, cytomegalovirus, Epstein Barr virus, toxoplasma gondii were detected in serum and cerebrospinal fluid, and serological tests for syphilis were negative. Repeated chest x-rays were normal and skin tests with tuberculin (up to 10 U) were negative. CT scan of the thorax revealed enlargement of a mediastinal lymph node, which was not considered to be accessible to biopsy by mediastinoscopy, and a bone marrow biopsy disclosed no evidence of lymphoma or other myeloproliferative disorders. EEG on admission showed intermittent polymorphous delta waves of the right hemisphere; subsequent recordings demonstrated progressive slowing which involved both hemispheres. The relatives refused permission to perform a brain biopsy, and the patient died in February 1990 of bulbar paralysis. Autopsy (S 31190) disclosed a miliary tuberculosis with involvement of paraesophageal lymph nodes and local secondary amyloidosis in both lungs. Foci of purulent pneumonia, bronchitis and bronchiolitis were also found. Further non-specific findings consisted of subacute myocarditis, interstitial nephritis, erosive gastritis and catarrhal enterocolitis. The brain was slightly atrophic, no signs of meningitis were evident. In the white matter of both hemispheres many greyish-yellow patches were seen, and a greyish "tumor" measuring about 1.5 cm in diameter was found in the right parietal lobe (Fig. 1). The consistence of this tumor was firmer than that of the surrounding tissue. A greyish discoloration of the white matter was also seen in the right cerebellar hemisphere and in the brainstem, whereas the left cerebellar hemisphere presented only some subcortical yellow patches.
Material and Methods
Fig. 1. Coronal slices of lUlfixed brain reveal many greyish demyelination patches in the white matter of both hemispheres, mainly subcortically located. In the right parietal lobe, a well demarcated tumor is evident (arrow).
For conventional histology organs were fixed in 10 % buffered formalin; paraffin sections were stained with H&E, van Gieson, Azan-Mallory, Congo red, PAS and Ziehl-Neelsen. Brain sections were also stained with Nissl, Kluver-Barrera and Bodian. Immunohistochemical investigations were carried out on selected deparaffinized brain slides using either peroxidase-anti peroxidase or avidin-biotin techniques. Reaction products were
966 . F. Gullotta ct al. visualized with diaminobenzidine. Antibodies to the following antigens (DAKO, Hamburg) were employed: GFAP (dilution 1: 100); Vimentin (1: 75); Leukocyte Common Antigen (1: 150); UCHL 1 (1: 150) and CD 3 (1: 125) as markers for T-Iymphocytes; and L26 (1:75) as marker for B-Iymphocytes. In situ hybridization of selected slides of brain and internal organs was carried out with ENZO-DNA Bioprobes for ]C-, SV 40- and BK-Virus on deparaffinized sections according to the procedure recommended by ORTHO Diagnostic Systems, Neckargemiind. Electron microscopic examination of the brain tumor was performed on formalin-fixed material. After washing, postfixation with 1.5 % osmium tetroxide and dehydration in graded ethanol, the material was embedded in Epon. Ultrathin sections were examined by a Philips EM 400.
Results The histological examination of internal organs confirmed the presence of miliary tuberculosis restricted to the lungs and the local lymph nodes.
Histological examination of the brain (SN 68/90) disclosed many small and/or large demyelinated foci, distributed irregularly in both cerebral hemispheres, in the midbrain, pons and cerebellum. The lesions were mainly subcortically located, the cortex appeared intact. In parts these foci were well demarcated, but many regions (e.g. capsula interna, pons, cerebellum) showed very large demyelinated areas, due to the confluence of many small foci of different age. Older lesions were characterized by severe tissue changes with spongiosis and necrosis, fibrillary astrocytes and macrophages. In early lesions a strong proliferation of protoplasmic astrocytes was present, partly intermingled with abnormal, giant, and/or multinucleated cells with large, atypical, "anaplastic" nuclei (Fig. 2A-D). Within spongio-necrotic lesions axons were partly destroyed, whereas in early foci, they were still intact and compressed by proliferating astrocytes (Fig. 2B). In some of the early lesions, perivascular cuffs of lymphocytes were present. At the periphery of many early lesions, some oligoglia cells with swollen basophilic nuclei were found (Fig. 3A). The white matter of basal ganglia contained many small foci consisting mainly of swollen oligoglia cells (Fig. 3D). Such cells were also present in the right parietal region around the tumor (Fig. 3E). The parietal tumor was quite well demarcated from the surrounding tissue. It displayed pleomorphic, bizarre, multinucleated glial cells with mitoses (Fig.4A,B), was partly well vascularized, but displayed no endothelial proliferations or micronecroses with pseudopalisading of nuclei. Within the tumor, no swollen oligoglia cells or nuclei were found. Around the tumor, demyelination with "reactive" protoplasmic astrocytes and many swollen oligoglial nuclei were seen (Fig. 3E). In the pons and in--the left cerebellar peduncle, besides demyelination with bizarre astrocytes, lipid laden macrophages, some perivascular cuffs and swollen oligoglia cells,
we also detected a diffuse proliferation of protoplasmic astrocytes with main involvement of long fiber tracts, as usually seen in diffuse gliomatosis. Neurons of pontine nuclei had been engulfed by proliferating glia cells (Fig. 4C,D). In the white matter of some cerebellar folia, distant from demyelination areas, single scattered astrocytes with large hyperchromatic nuclei were present (Fig.4E). No purulent or tuberculous meningitis was found. In swollen oligoglial nuclei in situ hybridization disclosed a strong positivity for JC virus only (Fig. 3B-D). Within the glioma, no virus-bearing nuclei were found, but many peritumoral oligoglia cells were positive. No virus positivity was detected in extracerebral organs.
Electron microscopic investigation of the tumor revealed a poor preservation of the tissue (only formalinfixed material was available). Nuclei of tumor cells presented extensive membrane infoldings and invagination of cytoplasmic organelles, patchy condensation of chromatin granules and clusters of dense granular structures, at first sight suggestive of virus assemblages; however, no viral particles or crystalline arrays of viruses could be ascertained. Immunohistochemistry: "Reactive" and "anaplastic" astrocytes within and around demyelinated foci expressed GF AP and Vimentin. The same pattern was seen in bizarre giant cells of the parietal glioma. Perivascular lymphocytic cuffs expressed Leukocyte Common Antigen; they consisted mainly of T-cells and presented a strong positivity for UCHL 1 and, to a lesser degree, for CD 3. Few T-cells were also distributed irregularly within early demyelinated foci and among the tumor cells of the parietal glioma. B-cells were not detected, L 26 reaction was negative. Discussion PML is morphologically characterized by 1) mostly patchy but also diffuse demyelination, mainly subcortical and partly confluent to extensive necroses; 2) swollen, hyperchromatic oligo glial nuclei which harbour virus colonies, mainly JC-virus; and 3) hypertrophic, bizarre, "reactive" or even "blastomatous" astrocytic cells. Our morphological findings and in situ hybridization established the diagnosis of PML in our patient. The occurrence of small and large demyelinating foci and their somewhat different histological structure (older foci are characterized by severe spongiosis or necrosis with fibrillary astrocytes and macrophages; younger foci present extensive proliferation of astrocytic cells and swollen oligoglia, but no tissue necrosis) correlates very well with the chronic course of the disease and with the neuroradiological findings. Certain signs of demyelination were first seen in this patient by means of CT-scan and particularly by NMR in July 1989, i.e. eight months after the appearance of the first clinical signs. Subsequent neuroradiological examinations in August and October 1989 confirmed the progression of the demyelinating process.
Leukoencephalopathy and Gliomas in a HIV-negati ve Patient· 967
Fig. 2. [n early demyelin atioft, foci of protoplasmic (" reactive") astrocytes are evident (A, B); the demyelinated area (A: upper half) is quite well demarcated from myelinated tissue. Axolls are still intact, appearing compressed by hypertrophic astrocytes (B). In some other foci, large astrocytic cells are multinucleated, others have large "anaplastic" nuclei (C, D: arrows). A: Kluver-Barrera, orig. magnif. x 50; B: Bodian silver imp regnation, x 64; C and D: H&E, x 64, x 50.
968 . F. Gullotta et al.
Fig. 3. In the periphery of early demyelinated areas some swollen oligocytes with hyperchromatic nuclei are already evident by· H&E-stain (A: arrows). In situ hybridization for ]Cvirus shows a clear-cut positivity in these nuclei (B, C) and discloses the high number of affected cells (D: focus in the capsula external, also in the peri tumorous area of the right parietal region (E). A: H&E, x 130; B-E: in situ hybridization for leV. B, e, x 130, D X 64, E X 40.
Fig. 4. The parietal glioma consists of bizarre, multinucleated, anaplastic astrocytes (A); the tumor is partly well vascularized, endothelial proliferations, however, are lacking (B). In the pons (e, D) a diffuse proliferation of protoplasmic astrocytes is present; "anaplastic" cells are absent here. Single astrocytes with large "anaplastic" nuclei are seen in the white matter of some cerebellar folia, far away from demyelinated areas (E: arrows). A-E, H&E stain. A and e, X 64; B, D and E, x 40.
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Leukoencephalopathy and Gliomas in a HIV-negative Patient" 969
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Progressive Multifocal Leukoencephalopathy and Gliomas In a HIV-Negative Patient F. Gullotta, T. Masini1, G. Scarlato2 and K. Kuchelmeister Institut fOr Neuropathologie der Universitat Munster (Direktor Prof. Dr. F. Gullotta), Munster, FRG; I/stituto di Anatomia Patologica de//'Universita, Milano (Direttore Prof. Dr. L. Matturri); 2Clinica Neurologica de//'Universita, Milano (Direttore Prof. Dr. G. Scarlato), Milano, Italy
SUMMARY A case of progressive multifocal leukoencephalopathy (PML) is reported, detected at autopsy of a 30-year-old patient. The clinical picture was characterized by a progressive course of mental deterioration and ingravescent neurological symptoms. The patient was HI V-negative. He died of bronchopneumonia, after a clinical course of 13 months. iutopsy disclosed pulmonary tuberculosis with involvement of regional lymph nodes. In the brain, besides numerous PML-foci of varying age and structure, a pleomorphic astrocytoma was found in the white matter of the right parietal lobe. In the brain stem glial proliferation resembling diffuse gliomatosis was also present. In situ hybridization revealed Pap ova-virus (feV) in oligoglial nuclei, but not in neoplastic astrocytes. This is the third report on the concomitant occurrence of PML and glioma in man.
Progressive multifocal leukoencephalopathy (PML) is an opportunistic demyelinating disease of the central nervous system, caused by virally induced changes of glial cells in an immunological deficient host 1, 8, 14,25. Before the appearance of AIDS this infection was usually observed in patients affected by lympho-myeloproliferative disorders or by chronic diseases which required immunotherapy. In a few cases, however, no chronic pre-existing disease could be found. Since the appearance of AIDS the number of PML cases has tremendously increased. The agents responsible for PML (]C-, SV 40-, and BK-virus) are papova/polyoma viruses,]CV being the most frequent one, as also confirmed by our investigations on HIV-related encephalopathies lO , 16, 18. In a few cases some other viruses have also been identified 7 . Papova/polyoma viruses are oncogenic when inoculated into hamsters, and all are capable of producing cerebral tumors (ependymoma, plexus papilloma, glioma, medulloblastoma, etc. 14 ). However, no definitive evidence has linked any of such viruses to a cerebral tumor in man. 0344-0338/9210 18 8-0964$3.50/0
Here a case of PML and glioma observed in a HIVnegative patient, who had miliary tuberculosis, is reported. Case Report A 30-year-old right-handed man was admitted to the Neurological Clinic of the University of Milan in June 1989 because of a progressive deterioration of cerebral function. He was well until December 1988, when he began to experience difficulties in handline r'..lffs and in performing simple sequences of movements during his work as a sound technician. During the following three months he manifested a progressive left-sided weakness and speech disturbances. A CT scan was normal and he was given prednisone (60 mg/day for 20 days) without improvement. Neurological examination on admission revealed temporal and spatial disorientation, non-fluent aphasia with phonemic paraphasias, anosmias and impairment of com© 1992 by Gustav Fischer Verlag, Stungart
Leukoencephalopathy and Gliomas in a HIV-ncgative Patient· 965
prehension; there was buccofacial and ideomotor apraxia; the left limbs were weak with a Babinski sign on the left, but the patient was able to walk independently. During the following months there was a progressive deterioration of higher cortical functions (memory, attention, writing), with a worsening of the left hemiparesis, with gradual onset of right limb weakness, impairment in all directions of saccadic and pursuit ocular movements, and left hemianopia. By October 1989 the patient was bed-ridden, tetra paretic, severely demented, with global aphasia. CT brain scan was normal with and without contrast material in June 1989. One month later small hypodense areas in the white matter were observed in the left parietal lobe; similar lesions were also present in the right parietal and occipital lobes at a control CT scan in August. NMR, performed in July and again in August, confirmed the presence of small foci of increased intensity on T2weighted images in both hemispheres which, at a new examination in October 1989, had become larger with a confluent appearance.
Hematological and biochemical findings were normal, except for mild to moderate eosinophilia (9-17 %, with a white blood cell count of 7,000/mm); serum ferritin was also normal, as were zinc and iron blood concentrations. Tests for T-cell subsets in the peripheral blood showed an inversion of the T4/T8 ratio, with increase of suppressor and natural killer cells and reduced response to mitogens. Cerebrospinal fluid on admission contained 2.6 mononuclear cells per cubic millimeter; the glucose was 55 mg per 100 ml, and the proteins 49 mg per 100 ml. Isoelectric focussing disclosed six oligoclonal bands, not detectable in serum. Findings were similar in September 1989. No antibodies to HIV, rubella virus, HTLV1, cytomegalovirus, Epstein Barr virus, toxoplasma gondii were detected in serum and cerebrospinal fluid, and serological tests for syphilis were negative. Repeated chest x-rays were normal and skin tests with tuberculin (up to 10 U) were negative. CT scan of the thorax revealed enlargement of a mediastinal lymph node, which was not considered to be accessible to biopsy by mediastinoscopy, and a bone marrow biopsy disclosed no evidence of lymphoma or other myeloproliferative disorders. EEG on admission showed intermittent polymorphous delta waves of the right hemisphere; subsequent recordings demonstrated progressive slowing which involved both hemispheres. The relatives refused permission to perform a brain biopsy, and the patient died in February 1990 of bulbar paralysis. Autopsy (S 31190) disclosed a miliary tuberculosis with involvement of paraesophageal lymph nodes and local secondary amyloidosis in both lungs. Foci of purulent pneumonia, bronchitis and bronchiolitis were also found. Further non-specific findings consisted of subacute myocarditis, interstitial nephritis, erosive gastritis and catarrhal enterocolitis. The brain was slightly atrophic, no signs of meningitis were evident. In the white matter of both hemispheres many greyish-yellow patches were seen, and a greyish "tumor" measuring about 1.5 cm in diameter was found in the right parietal lobe (Fig. 1). The consistence of this tumor was firmer than that of the surrounding tissue. A greyish discoloration of the white matter was also seen in the right cerebellar hemisphere and in the brainstem, whereas the left cerebellar hemisphere presented only some subcortical yellow patches.
Material and Methods
Fig. 1. Coronal slices of lUlfixed brain reveal many greyish demyelination patches in the white matter of both hemispheres, mainly subcortically located. In the right parietal lobe, a well demarcated tumor is evident (arrow).
For conventional histology organs were fixed in 10 % buffered formalin; paraffin sections were stained with H&E, van Gieson, Azan-Mallory, Congo red, PAS and Ziehl-Neelsen. Brain sections were also stained with Nissl, Kluver-Barrera and Bodian. Immunohistochemical investigations were carried out on selected deparaffinized brain slides using either peroxidase-anti peroxidase or avidin-biotin techniques. Reaction products were
966 . F. Gullotta ct al. visualized with diaminobenzidine. Antibodies to the following antigens (DAKO, Hamburg) were employed: GFAP (dilution 1: 100); Vimentin (1: 75); Leukocyte Common Antigen (1: 150); UCHL 1 (1: 150) and CD 3 (1: 125) as markers for T-Iymphocytes; and L26 (1:75) as marker for B-Iymphocytes. In situ hybridization of selected slides of brain and internal organs was carried out with ENZO-DNA Bioprobes for ]C-, SV 40- and BK-Virus on deparaffinized sections according to the procedure recommended by ORTHO Diagnostic Systems, Neckargemiind. Electron microscopic examination of the brain tumor was performed on formalin-fixed material. After washing, postfixation with 1.5 % osmium tetroxide and dehydration in graded ethanol, the material was embedded in Epon. Ultrathin sections were examined by a Philips EM 400.
Results The histological examination of internal organs confirmed the presence of miliary tuberculosis restricted to the lungs and the local lymph nodes.
Histological examination of the brain (SN 68/90) disclosed many small and/or large demyelinated foci, distributed irregularly in both cerebral hemispheres, in the midbrain, pons and cerebellum. The lesions were mainly subcortically located, the cortex appeared intact. In parts these foci were well demarcated, but many regions (e.g. capsula interna, pons, cerebellum) showed very large demyelinated areas, due to the confluence of many small foci of different age. Older lesions were characterized by severe tissue changes with spongiosis and necrosis, fibrillary astrocytes and macrophages. In early lesions a strong proliferation of protoplasmic astrocytes was present, partly intermingled with abnormal, giant, and/or multinucleated cells with large, atypical, "anaplastic" nuclei (Fig. 2A-D). Within spongio-necrotic lesions axons were partly destroyed, whereas in early foci, they were still intact and compressed by proliferating astrocytes (Fig. 2B). In some of the early lesions, perivascular cuffs of lymphocytes were present. At the periphery of many early lesions, some oligoglia cells with swollen basophilic nuclei were found (Fig. 3A). The white matter of basal ganglia contained many small foci consisting mainly of swollen oligoglia cells (Fig. 3D). Such cells were also present in the right parietal region around the tumor (Fig. 3E). The parietal tumor was quite well demarcated from the surrounding tissue. It displayed pleomorphic, bizarre, multinucleated glial cells with mitoses (Fig.4A,B), was partly well vascularized, but displayed no endothelial proliferations or micronecroses with pseudopalisading of nuclei. Within the tumor, no swollen oligoglia cells or nuclei were found. Around the tumor, demyelination with "reactive" protoplasmic astrocytes and many swollen oligoglial nuclei were seen (Fig. 3E). In the pons and in--the left cerebellar peduncle, besides demyelination with bizarre astrocytes, lipid laden macrophages, some perivascular cuffs and swollen oligoglia cells,
we also detected a diffuse proliferation of protoplasmic astrocytes with main involvement of long fiber tracts, as usually seen in diffuse gliomatosis. Neurons of pontine nuclei had been engulfed by proliferating glia cells (Fig. 4C,D). In the white matter of some cerebellar folia, distant from demyelination areas, single scattered astrocytes with large hyperchromatic nuclei were present (Fig.4E). No purulent or tuberculous meningitis was found. In swollen oligoglial nuclei in situ hybridization disclosed a strong positivity for JC virus only (Fig. 3B-D). Within the glioma, no virus-bearing nuclei were found, but many peritumoral oligoglia cells were positive. No virus positivity was detected in extracerebral organs.
Electron microscopic investigation of the tumor revealed a poor preservation of the tissue (only formalinfixed material was available). Nuclei of tumor cells presented extensive membrane infoldings and invagination of cytoplasmic organelles, patchy condensation of chromatin granules and clusters of dense granular structures, at first sight suggestive of virus assemblages; however, no viral particles or crystalline arrays of viruses could be ascertained. Immunohistochemistry: "Reactive" and "anaplastic" astrocytes within and around demyelinated foci expressed GF AP and Vimentin. The same pattern was seen in bizarre giant cells of the parietal glioma. Perivascular lymphocytic cuffs expressed Leukocyte Common Antigen; they consisted mainly of T-cells and presented a strong positivity for UCHL 1 and, to a lesser degree, for CD 3. Few T-cells were also distributed irregularly within early demyelinated foci and among the tumor cells of the parietal glioma. B-cells were not detected, L 26 reaction was negative. Discussion PML is morphologically characterized by 1) mostly patchy but also diffuse demyelination, mainly subcortical and partly confluent to extensive necroses; 2) swollen, hyperchromatic oligo glial nuclei which harbour virus colonies, mainly JC-virus; and 3) hypertrophic, bizarre, "reactive" or even "blastomatous" astrocytic cells. Our morphological findings and in situ hybridization established the diagnosis of PML in our patient. The occurrence of small and large demyelinating foci and their somewhat different histological structure (older foci are characterized by severe spongiosis or necrosis with fibrillary astrocytes and macrophages; younger foci present extensive proliferation of astrocytic cells and swollen oligoglia, but no tissue necrosis) correlates very well with the chronic course of the disease and with the neuroradiological findings. Certain signs of demyelination were first seen in this patient by means of CT-scan and particularly by NMR in July 1989, i.e. eight months after the appearance of the first clinical signs. Subsequent neuroradiological examinations in August and October 1989 confirmed the progression of the demyelinating process.
Leukoencephalopathy and Gliomas in a HIV-negati ve Patient· 967
Fig. 2. [n early demyelin atioft, foci of protoplasmic (" reactive") astrocytes are evident (A, B); the demyelinated area (A: upper half) is quite well demarcated from myelinated tissue. Axolls are still intact, appearing compressed by hypertrophic astrocytes (B). In some other foci, large astrocytic cells are multinucleated, others have large "anaplastic" nuclei (C, D: arrows). A: Kluver-Barrera, orig. magnif. x 50; B: Bodian silver imp regnation, x 64; C and D: H&E, x 64, x 50.
968 . F. Gullotta et al.
Fig. 3. In the periphery of early demyelinated areas some swollen oligocytes with hyperchromatic nuclei are already evident by· H&E-stain (A: arrows). In situ hybridization for ]Cvirus shows a clear-cut positivity in these nuclei (B, C) and discloses the high number of affected cells (D: focus in the capsula external, also in the peri tumorous area of the right parietal region (E). A: H&E, x 130; B-E: in situ hybridization for leV. B, e, x 130, D X 64, E X 40.
Fig. 4. The parietal glioma consists of bizarre, multinucleated, anaplastic astrocytes (A); the tumor is partly well vascularized, endothelial proliferations, however, are lacking (B). In the pons (e, D) a diffuse proliferation of protoplasmic astrocytes is present; "anaplastic" cells are absent here. Single astrocytes with large "anaplastic" nuclei are seen in the white matter of some cerebellar folia, far away from demyelinated areas (E: arrows). A-E, H&E stain. A and e, X 64; B, D and E, x 40.
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Leukoencephalopathy and Gliomas in a HIV-negative Patient" 969
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