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ORIGINAL ARTICLE
Natalizumab-Associated Progressive Multifocal Leukoencephalopathy in a Patient With Multiple Sclerosis: A Postmortem Study Christian Wu¨thrich, PhD, Bogdan F. Gh. Popescu, PhD, Sarah Gheuens, MD, Michael Marvi, MD, Ronald Ziman, MD, Stephen Pojen Denq, MD, Mylyne Tham, BS, Elizabeth Norton, BS, Joseph E. Parisi, MD, Xin Dang, PhD, Claudia F. Lucchinetti, MD, and Igor J. Koralnik, MD
Abstract Natalizumab, a monoclonal antibody directed against >4 integrins, has, to date, been associated with 399 cases of progressive multifocal leukoencephalopathy (PML) worldwide in patients receiving treatment for multiple sclerosis (MS). Because of the limited number of histologic studies, the possible interplay between MS and PML lesions has not been investigated. We report the clinical, radiologic, and histologic findings of an MS patient who developed PML after 32 months of natalizumab monotherapy. After withdrawal of natalizumab, she received plasma exchange, mefloquine, and mirtazapine but died soon thereafter. Postmortem examination was restricted to examination of the brain and spinal cord. Extensive PML lesions, characterized by the presence of JC virus DNA were found in the cerebral white matter and neocortex. Sharply demarcated areas of active PML lesions contained prominent inflammatory infiltrates composed of approximately equal numbers of CD4-positive and CD8-positive T cells, From the Division of Neurovirology, Department of Neurology (CW, SG, EN, XD, IJK), and Center for Virology and Vaccine Research, Department of Medicine (CW, SG, EN, XD, IJK), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; Department of Anatomy and Cell Biology (BFGhP, MT), and Cameco MS Neuroscience Research Center (BFGh.P, MT), University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Providence Saint Joseph Medical Center, The Hycy and Howard Hill Neuroscience Institute, Movement Disorder Center, Burbank (MM); David Geffen School of Medicine, University of California, Los Angeles, Los Angeles (RZ); Department of Medicine, Northridge Hospital Medical Center, Northridge (RZ); and Buddhist Tzu Chi Medical Center, Alhambra, California (SPD); and Department of Laboratory Medicine and Pathology (JEP), and Department of Neurology (CFL), Mayo Clinic, Rochester, Minnesota. Send correspondence and reprint requests to: Igor J. Koralnik, MD, Division of Neurovirology, Beth Israel Deaconess Medical Center, E/CLS-1005, 330 Brookline Ave, Boston, MA 02215; E-mail: [email protected] This study was supported in part by grants from the National Multiple Sclerosis Society and from Biogen Idec. This study was supported by NMSS grant RG 4523-A-1 and NIH grants R56 NS 041198, R01 NS 047029 and 074995, and K24 NS 060950 (to Igor Koralnik) and by grants from the Saskatchewan Health Research Foundation (2633; to Bogdan Popescu), the Canada Research Chairs program (to Bogdan Popescu), the National Multiple Sclerosis Society (NMSS RG3185-B-3; to Claudia Lucchinetti), and the National Institutes of Health (1R01NS049577; to Claudia Lucchinetti). The funding agencies had no input in the investigations or analysis of the results. Conflict of Interest: Sarah Gheuens and Elizabeth Norton are currently employees of Biogen Idec. The other authors declare that they have no competing interests.
consistent with an immune reconstitution inflammatory syndrome. Conversely, all MS lesions identified were hypocellular, long-standing inactive plaques characterized by myelin loss, relative axonal preservation, and gliosis and, importantly, were devoid of JC virus DNA and active inflammation. Chronic inactive MS lesions were separate and distinct from nearby PML lesions. This case demonstrates the coexistence and apparent lack of interplay between chronic inactive MS and PML lesions, and that immune reconstitution inflammatory syndrome seems to affect the shape and appearance of PML but not MS lesions. Key Words: Immune reconstitution inflammatory syndrome, JC virus, Multiple sclerosis, Natalizumab, Progressive multifocal leukoencephalopathy.
INTRODUCTION Progressive multifocal leukoencephalopathy (PML) is a fulminant inflammatory demyelinating disease caused by the reactivation of the polyomavirus JC (JCV) in the setting of immunosuppression (1). As of September 3, 2013, 399 cases of PML have been diagnosed in multiple sclerosis (MS) patients who received immunomodulatory treatment with natalizumab (Tysabri) (2). Natalizumab is a humanized monoclonal antibody against >4 integrins that is administered monthly and prevents egress of all leukocytes from the bloodstream into brain parenchyma, thereby decreasing immune surveillance. Therefore, T lymphocytes are sequestered in the blood vessels and cannot prevent reactivation and spread of JCV in the CNS. Because the biologic activity of natalizumab can extend to up to 3 months, MS patients who develop PML usually are treated with plasma exchange. This decreases blood levels of natalizumab and hastens the return of lymphocytes into the CNS (3). This phenomenon usually is associated with an immune reconstitution inflammatory syndrome (IRIS), which by itself can be damaging to the brain. Immune reconstitution inflammatory syndrome is characterized clinically by paradoxic worsening of neurologic signs and symptoms at the time of immune recovery, and magnetic resonance imaging (MRI) may show enlargement of PML lesions, contrast enhancement, and swelling (3, 4). Of the 399 MS-PML patients, 23% have died (2), but histologic postmortem examinations of the brain have been limited. In the initial report of natalizumab-associated PML
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occurring in an MS patient, no residual MS lesions were identified in the CNS (5). More recently, a few reports have described limited results of stereotactic biopsies (6Y9). An additional detailed postmortem examination of a natalizumabassociated PML patient failed to demonstrate JCV infection in brain lesions (10). The coexistence of lesions of JCV and MS and their possible interactions have not been described. We present detailed clinical, radiologic, and pathologic findings in an MS patient who developed PML during natalizumab therapy and who, at autopsy, demonstrated lesions of both diseases.
slightly modified technique in which streptavidin-AP was replaced by streptavidin-HRP (DAB detection) and an additional tyramide amplification step (12). Polymerase chain reaction amplification of JCV DNA extracted from brain samples was performed using T AgYspecific primers, as previously described (13).
Characterization of the MS Lesions Demyelinating activities in MS lesions based on myelin degradation products in macrophages were classified according to previously published criteria (14).
Characterization of Inflammatory Infiltrates in the MS and PML Lesions
MATERIALS AND METHODS Histopathologic Methods Formalin-fixed, paraffin-embedded sections were studied using routine neuropathologic methods (i.e. hematoxylin and eosin, Luxol fast blueYperiodic acid-Schiff, and Bielschowsky impregnation), as well as single and double labeled immunohistochemistry (IHC) and immunofluorescence using antibodies listed in the Table, as previously described (11).
Characterization of PML Lesions Viral protein immunoreactivity for JCV T Antigen (T Ag) (v-300) or VP1 (PAB597) was correlated with the extent and pattern of demyelination using double labeling for myelin proteins, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase, myelinassociated glycoprotein, and myelin oligodendrocyte glycoprotein. Polyomavirus JCYinfected cells were identified by double labeling of specific cells (i.e. neurons and glia). In situ hybridization (ISH) for JCV DNA was performed using a
CD3-positive and CD8-positive T lymphocytes in both PML and MS lesions were examined relative to JCV VP1 (PAB597) expression. CD4-positive T cells were inferred based on double labeling for CD3 and CD8 (double positive consistent with CD8-positive T cells; CD3-positive/CD8-negative cells were considered CD4-positive T cells).
Case History A 52-year-old woman diagnosed as having relapsingremitting MS of 14 years’ duration had been treated with several immunomodulatory and immunosuppressive drugs, including interferon-A, glatiramer acetate, methotrexate, and cyclophosphamide. In November 2006, she began intravenous natalizumab monotherapy consisting of 300 mg every 4 weeks for refractory exacerbations. One year later, MRI showed stable MS lesions in the left periventricular area and the splenium of the corpus callosum (Fig. 1A, E). By June 2008, she was tetraplegic because of accumulating disability
TABLE. Antibodies Used for Immunohistochemistry and Immunofluorescence Staining Antigen; Product Name PAB597 SV40 T Ag (v-300); sc-20800 MAP-2; HM-2; M4403 MAP-2; ab5622 MAP-2; LS-B290 GFAP; M0761; 6F2 GFAP; Z0334 GFAP; ab4674 CNPase; C5922; 11-5B CNP; HPA023266 MOG; EP4281; ab109746 MAG; ab89780 CD8; 1A5; VP-C325 CD3; CD3-12; OASA11552 PLP NF KiM1P
Host
Isotype
Target
Source
m r m r ch m r ch m r r m m rat m m m
NA IgG IgG1 IgG IgY IgG1 IgG IgY IgG1 IgG IgG IgG1 IgG1 IgG
JCV VP1 JCV T Ag Neurons Neurons Neurons Astrocytes Astrocytes Astrocytes Myelin/Oligo Myelin/Oligo Myelin/Oligo Myelin/Oligo CD8 CD3 PLP NF KiM1P
Santa Cruz Biotechnology, Santa Cruz, CA Sigma, St. Louis, MO Chemicon, Temecula, CA Lifespan Bioscience, Seattle, WA Dako, Carpinteria, CA Dako Abcam, Cambridge, MA Sigma Sigma Abcam Abcam Vector Laboratories, Burlingame, CA Aviva Systems Biology, San Diego, CA Serotec, USA Dako, Glostrup, Denmark Dr. Radzun, Go¨ttingen, Germany
For double staining, primary antibodies (Abs) from different species (mouse, rat, rabbit, or chicken) were used with appropriately matched (species and isotypes) secondary Abs. These Abs were conjugated to either alkaline phosphatase (AP) or horseradish peroxidase (HRP) for immunohistochemistry (IHC) or Alexa Fluor 350, 488, and 568 according to the manufacturer’s instructions for immunofluorescence (IF). Single and double IHC were performed with the Vectastain Elite ABC Kits (Vector, Burlingame, CA) according to the manufacturer’s instructions. Negative controls, for IHC and IFA, included omission of the primary Abs, use of isotype matched controls, and use of sections from individuals known to be free of JCV-infected cells. ch, chicken; CNPase, CNP, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase; GFAP, glial fibrillary acidic protein; KiM1P, macrophage marker; m, mouse; MAG, myelin-associated glycoprotein; MAP-2, microtubule-associated protein 2; MOG, myelin-oligodendrocyte glycoprotein; NF, neurofilament; oligo, oligodendrocytes; PLP, myelin proteolipid protein; r, rabbit; SV40 T Ag, Simian virus 40 T antigen; NA, not available.
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Autopsy Case Report of MS and PML
FIGURE 1. Magnetic resonance imaging evolution of multiple sclerosis (MS) and progressive multifocal leukoencephalopathy (PML) lesions. Axial (AYD) and corresponding sagittal (EYH) FLAIR images. After 1 year of natalizumab monotherapy, MS lesions are present in the left periventricular area and the splenium of the corpus callosum (A, E, arrows). There is a new left subcortical lesion (B, arrowhead) in the left frontal lobe, remote from the old MS lesions 20 months later (B, F, arrows); 2.5 months later, the left frontal lobe lesion has extended posteriorly and contralaterally through the genu of the corpus callosum to the right frontal lobe white matter (C, G, arrowheads), whereas the MS lesions (arrows) seem stable. One month later, the PML lesions (D, H, arrowheads) have spread further in the right frontal lobe and the left frontal and parietal lobes, close, but separate from the MS lesions (D, H, arrows).
and required admission to a skilled nursing facility. She missed 2 injections of natalizumab in January and February 2009. In June 2009, she presented with cognitive decline. An MRI performed in July 2009, 32 months after onset of natalizumab therapy, showed a new left frontal subcortical nonenhancing white matter lesion in addition to stable chronic MS lesions (Fig. 1B, F). A few weeks later, she developed a speech deficit and worsening cognitive difficulties. She was treated with a short course of dexamethasone and received her last injection of natalizumab in early September 2009. Repeat MRI performed in September 2009 showed marked progression of the left frontal lesion, extending across the genu of the corpus callosum into the right frontal lobe, associated with a mild mass effect on the left lateral ventricle (Fig. 1C, G) and enhancement. A lumbar puncture performed in October 2009 showed 36 red blood cells/KL, 7 white blood cells/KL, glucose level of 67 mg/dL, and protein level of 77 mg/dL. Polyomavirus JC cerebrospinal fluid polymerase chain reaction was positive, showing 9900 JCV DNA copies/mL, thereby confirming the diagnosis of PML. She had a detectable cellular immune response against JCV, mediated by both CD4-positive and CD8-positive T cells, and was treated with 5 alternate day courses of plasma exchange, mirtazapine 30 mg/day, and mefloquine 250 mg/week. Later that month, cerebrospinal fluid showed undetectable JCV DNA, and MRI showed progression of the PML lesions in the right frontal, left frontal, and
parietal lobes (Fig. 1D, H) with minimal associated enhancement. Despite these interventions, there was no clinical improvement. She developed aspiration pneumonia and died in November 2009, 4 months after PML onset, 2 months after the last natalizumab injection, 3.5 weeks after onset of plasma exchange, and 12 days after the last MRI. Postmortem examination was restricted to the brain and spinal cord.
RESULTS Macroscopic Neuropathology The formalin-fixed brain showed mild generalized swelling and was sectioned in the axial plane to correlate with the recent MRI. Corresponding to the axial FLAIR images, the brain slices showed that the MS lesions were well demarcated and somewhat retracted firm areas of brown discoloration, whereas the PML lesions were softened, granular, and ‘‘motheaten’’ in appearance. Several MS lesions were identified in the white matter, including 5 periventricular lesions (3 around lateral ventricles, 1 in the midbrain tectum around the cerebral aqueduct, and 1 in the medulla around the fourth ventricle), 2 lesions in the cerebellar peduncles, 1 in subpial in the basis pontis, 1 in the medullary pyramid, 2 in the white matter of the spinal cord, and 2 in the optic tract. Also identified were 5 lesions in deep
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gray matter structures: 1 each in the amygdala, the tail of the caudate, putamen, claustrum, and inferior olivary nucleus.
Histology, IHC, and ISH Microscopically, all white and deep gray matter MS lesions were chronic, inactive, demyelinated, and hypocellular plaques, characterized by mild axonal loss, gliosis, and largely devoid of inflammatory T-cell infiltrates. All lesions showed mild microglial activation without macrophages, except for 1 spinal cord lesion that contained periodic acid-SchiffYpositive macrophages, consistent with more recent demyelination. Cortical MS lesions included 16 intracortical and subpial chronic lesions. Extensive PML lesions were present in the subcortical and deep white matter of both cerebral hemispheres, the corpus callosum, and the brainstem; they were characterized at the active edge of the lesion by relative myelin preservation and a high density of infected oligodendrocytes. Myelin loss was more apparent toward the centers of these lesions, which contained few infected cells. In situ hybridization showed abundance of JCV DNA localized mostly to sharply demarcated areas at the periphery of large lesions and involving the gray-white matter junction and the gray matter (Fig. 2A, C, E). Immunohistochemistry staining of sequential sections with an antibody against SV40 (which cross-reacts against JCV major capsid protein VP1) showed a similar staining pattern at the gray-white junction, indicating the presence of a productive lytic infection of glial cells (Fig. 2B, D). The extent of PML-associated demyelination in the white matter and the cortex is illustrated in Figure 2B inset. Only a few infected cells were present in gray matter (Fig. 2F). A particular feature of these lesions was the well-demarcated aspect of JCV-infected areas. CD3-positive T cells were present at the edge of actively infected areas (Fig. 2B, D, F) and were often in close contact with JCV-infected cells (Fig. 2D, F). Topographic relationships between PML and MS lesions were studied by comparing ISH and IHC for JCV DNA. Progressive multifocal leukoencephalopathy lesions were more obvious and extensive, as demonstrated by ISH than by IHC and Luxol fast blue stain. Infected cells within PML lesions contained JCV T Ag (which is typically expressed early in the viral replication cycle), as well as JCV VP1 capsid protein (indicating the presence of mature viral particles). Chronic inactive MS lesions showed no evidence of JCV DNA or JCV proteins. The juxtaposition of PML and MS lesions is illustrated in an area of the left occipital lobe containing PML lesions adjacent to a cortical MS plaque (Fig. 3). A chronic subpial MS plaque is adjacent to PML lesions that are in the subcortical white matter and the lower layers of the cortex (Fig. 3A). The boxed areas (d and e in Fig. 3B and C, respectively) magnified in (Fig. 3D and E, respectively) show JCV-infected cells located immediately adjacent to the MS lesion (Fig. 3D) and a readily identified inflammatory infiltrate of CD3-positive T cells (Fig. 3E) in the PML lesions but not in the MS lesion. Higher-magnification images show that the MS lesion is entirely devoid of JCV infection or CD3-positive T-cell infiltrates, whereas CD3-positive T cells (Fig. 3G, I) are located in close proximity to JCV-infected cells (Fig. 3FYI).
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To determine the nature of the inflammatory T-cell infiltrate, double immunofluorescence staining for CD3 (green) and CD8 (red) in the PML lesions was performed (Fig. 4). As shown in Figure 4A, an approximately equal number of cells are CD3 positive/CD8 negative (green) or CD3 positive/CD8 positive (yellow). No CD3-negative/CD8-positive (red) cells were present. Higher-magnification shows a CD3-positive/ CD8-positive T-cell (B) and a CD3-positive/CD8-negative T cell (C), the latter corresponding to a CD4-positive T cell. The magnitude of the CD4-positive and CD8-positive T-cell infiltrates in the PML lesions is consistent with IRIS. In addition, occasional JCV-infected neurons expressing JCV T Ag, but not JCV VP1, were identified in lesions at the gray-white junction and in gray matter (Fig. 5A), findings consistent with nonproductive infection of cortical neurons by JCV, as in other PML cases (11). Extensive PML lesions were found in the corpus callosum, some extending into the septum pellucidum (Fig. 5B). A small PML focus also involved hippocampal white matter (not shown).
DISCUSSION Coexistence of Active PML and Chronic Inactive MS Lesions To our knowledge, this is the first histopathologically confirmed report showing coexistent MS and PML lesions in a natalizumab-treated patient. It provides a unique opportunity for comparing radiologic and histologic findings in natalizumabassociated PML as well as examining the early manifestations of superimposed IRIS. Progressive multifocal leukoencephalopathy lesions were extensive, corresponding to the MRI findings, and harbored ongoing productive JCV infection. Conversely, MS lesions were chronic inactive plaques devoid of active inflammatory infiltrates. Although PML lesions displayed a large number of cells containing JCV DNA or proteins, MS lesions were entirely devoid of JCV infection, even when they abutted PML lesions. This may be explained by the relative absence of remaining glial cells that could potentially become infected by JCV in chronic MS lesions. As such, the MS plaques and PML lesions were distinct and pathologically independent.
IRIS Affected PML but Spared MS Lesions Immune reconstitution inflammatory syndrome was originally described in HIV-infected patients treated with combination antiretroviral therapy, in which the recovering immune system was able to mount a cellular response. Progressive multifocal leukoencephalopathyYIRIS is a common consequence of plasma exchange immunomodulatory treatment and is thought to be caused by the return of lymphocytes to the brain parenchyma. The influence of IRIS may be heightened when natalizumab is withdrawn and the action of the drug is diminished, leading to catastrophic inflammation, with swelling, mass effect, and herniation, often requiring high doses of corticosteroids. In this case, the clinical manifestations of IRIS were limited to mild enhancement of PML lesions on MRI. Histologic examination of PML lesions revealed prominent T-cell infiltrates, characterized by equal numbers of CD4-positive Ó 2013 American Association of Neuropathologists, Inc.
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Autopsy Case Report of MS and PML
FIGURE 2. Progressive multifocal leukoencephalopathy (PML) lesions are sharply demarcated and harbor CD3-positive T cells. (A, B) In situ hybridization for JC virus (JCV) DNA (cresyl violet counterstaining) (A) and immunohistochemistry for JCV VP1 and CD3 (B) show that JCV-infected cells (arrowheads) are mostly restricted to the junction between white matter (WM) and gray matter (GM) and the lower layers of the cortex. Conversely, the deep WM is almost free of JCV-infected cells and totally demyelinated as shown by myelin proteolipid protein (PLP) immunostaining (brown [B inset]), but not cavitary. (CYF) Magnifications of rectangles c and e from (A) show a sharp demarcation of the areas containing JCV-infected cells (arrowheads) at the gray-white junction (C). The edge of JCV infection spreads in the lower layer of the cortex (E, arrowheads). Magnification of rectangles d and f from (B) show that cells expressing JCV VP1 protein (arrowheads) are surrounded by CD3-positive T cells (arrows in D and F). Most of these T cells (D and F insets, arrow) are located in close proximity to the JCV-infected cells (D and F insets, arrowheads). Overall, the T cells are mostly in areas where the cell nuclei contain JCV DNA (C) and express JCV VP1 (D). Ó 2013 American Association of Neuropathologists, Inc.
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and CD8-positive T cells within and at the active edge of lesions. This differs from PML-IRIS lesions in AIDS patients, who typically have low CD4-positive T-cell counts in peripheral
blood and a predominance of CD8-positive T cells in brain parenchyma (15). Inasmuch as JCV-specific CD4-positive and CD8-positive T cells were detected in the blood of our patient
FIGURE 3. Proximity of multiple sclerosis (MS) and progressive multifocal leukoencephalopathy (PML) lesions in the left occipital lobe. (AYC) Immunohistochemistry (IHC) for myelin proteolipid protein (A) shows a subpial MS lesion (delineated in red) and demyelination lesions of PML involving the subcortical white matter (WM) and lower layers of the cortical gray matter (GM) that contain numerous cells harboring JC virus (JCV) DNA ([B] JCV in situ hybridization with cresyl violet counterstaining) and expressing JCV VP1 protein in (C) (Luxol fast blue counterstaining). (D, E) Higher magnifications of boxes d and e in (B) and (C) show that cells harboring JCV DNA (D, arrowheads), those expressing JCV VP1 and CD3-positive T cells, are not present in the MS lesion (E). The distribution of the CD3-positive T cells is shaded orange (arrows). (FYI) Higher magnifications of the MS-PML interface (boxes f, g) and the lower GM side of the PML lesion (boxes h, i) show that cells harboring JCV DNA (F, arrowhead), those expressing JCV VP1 (G, arrowheads) and CD3-positive T cells (G, arrows), are present at the border of the MS lesion. Polyomavirus JC infection of the GM is shown in (H, I, arrowheads). Insets in G and I at higher magnification show CD3-positive T cells in close proximity to cells expressing JCV VP1 protein.
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Autopsy Case Report of MS and PML
FIGURE 4. CD8-positive and CD4-positive T cells are present in progressive multifocal leukoencephalopathy (PML) lesions. Double immunofluorescence staining for CD3 (Alexa Fluor 488, green) and CD8 (Alexa Fluor 568, red) and autofluorescence (blue). (A) Similar numbers of CD8-positive T cells (CD3-positive/CD8-positive yellow-red, arrows) and CD4-positive T cells (CD3-positive/CD8-negative, green, arrowheads) are present at the border of this PML lesion. (B) Higher magnification with insets show CD8-positive T cells (arrows) in both red (CD8) and green (CD3) channel. (C) Conversely, a CD4-positive T cell (arrowhead) is characterized by absence of staining in the red channel (CD8) but staining in the green channel (CD3). Ó 2013 American Association of Neuropathologists, Inc.
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FIGURE 5. Nonproductive infection of hemispheric cortical neurons and productive infection of glial cells in the septum pellucidum. (A) Triple immunohistochemistry (IHC) for JC virus (JCV) T Antigen (T Ag) (blue), MAP-2 (orange), and 2¶,3¶-cyclicnucleotide 3¶-phosphodiesterase (CNPase) (brown). Rare JCV-infected cortical neurons expressing T Ag are present at the edge of a progressive multifocal leukoencephalopathy (PML) lesion in the gray matter (GM) (inset). (B) IHC for JCV VP1 (blue). Frequent glial cells expressing JCV VP1 can be seen in the corpus callosum (upper inset) and in the white matter (WM) of the septum pellucidum (lower inset). LFB, Luxol fast blue.
4 weeks before death, it is possible that T cells in the brain were actively involved in lysing JCV-infected cells. In contrast, inflammatory infiltrates spared chronic MS lesions. Because the patient did not develop an MS exacerbation during her brief course off natalizumab, it seems that the inflammatory reaction associated with IRIS did not lead to an MS-like autoimmunemediated response directed against myelin. These data suggest that, in MS patients with PML-IRIS, T cells are driven by JCV antigen specificity and do not cause an exuberant response as in HIV-infected patients (15).
PML Lesions Involve Both White and Gray Matter Progressive multifocal leukoencephalopathy lesions were extensive in white matter, including in the septum pellucidum, an area that is not classically regarded as a target of JCV infection. Such involvement might result in dissemination of infection to the fornix and hippocampus. There were numerous leukocortical and intracortical PML lesions that were occa-
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sionally in close proximity to subpial MS lesions, but no subpial lesions of PML were identified, consistent with previous reports (16, 17). This suggests that this location may be more characteristic of MS. In addition, some cortical neurons sustained nonproductive infection by JCV, as in other PML cases (11). This case exemplifies the risks of natalizumab therapy and provides further insights in PML pathogenesis in the context of MS. This patient’s aggressive relapsing form of MS required multiple immunomodulatory and immunosuppressive therapies before switching to natalizumab, which she received as monotherapy for 32 months. If, like 98% of natalizumab-associated PML patients (2), she had been JCV seropositive, the risk of her developing PML in the setting of prior immunosuppression use and natalizumab treatment for more than 24 months is estimated to be 1/90, according to a recent algorithm (18). This is significant and second only to the risk in patients with advanced AIDS not receiving antiretroviral treatment, which is 1/20 (19). The patient’s clinical presentation with progressive cognitive decline and speech difficulties superimposed on her Ó 2013 American Association of Neuropathologists, Inc.
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neurologic deficits attributed to MS was commensurate with the location of the PML lesions, as reported in other series (3, 4).
Lack of Concomitant Demonstration of JCV Infection and MS Lesions in Other Cases Our study expands previous biopsy and autopsy findings of natalizumab-associated PML. Earlier biopsy studies found no evidence of JCV infection (6, 7, 9) or only rare single-infected cells in 4 of 5 cases (8). In the original autopsy case report by Kleinschmidt-DeMasters and Tyler (5), no residual MS lesions were identified histologically among widespread PML lesions containing numerous cells harboring JCV DNA, suggesting the possibility that PML may have obscured entirely the MS lesions. This also may have occurred in some of the MS lesions in our case, given the extent of PML that was observed. In contrast to our patient, another reported case demonstrated no detectable JCV-infected cells by IHC for JCV T Ag within extensive demyelinated lesions attributed to PMLIRIS (10). Failure to demonstrate JCV T Ag by IHC may have been caused by technical factors related to the suboptimal sensitivity of PAb416 (DP02) for detection of JCV in formalinfixed, paraffin-embedded tissue (20, 21). Alternatively, it is possible that PML lesions were ‘‘burned out’’ in that case because the postmortem examination was performed 9 months after the clinical onset of PML (10).
CONCLUSIONS This case demonstrates the coexistence of chronic inactive MS and PML lesions harboring active JCV infection characterized by the presence of JCV DNA, T Ag, and VP1 protein, as well as inflammatory infiltrates of CD4-positive and CD8-positive T cells. In addition, restrictive JCV infection of rare cortical pyramidal neurons was demonstrated, consistent with previous observations (11). Immune reconstitution inflammatory syndrome greatly altered the characteristics of PML lesions while sparing MS lesions. Whether PML may alter the nature of active MS lesions remains to be determined. REFERENCES 1. Gheuens S, Wu¨thrich C, Koralnik IJ. Progressive multifocal leukoencephalopathy: Why gray and white matter. Annu Rev Pathol 2013;8: 189Y215 2. PML Incidence in Patients Receiving TYSABRI (natalizumab) [Internet]. BioGenIDEC. 2013. Available from: http://medinfo.biogenidec.com. Accessed on September 26, 2013 3. Clifford DB, De Luca A, Simpson DM, et al. Natalizumab-associated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: Lessons from 28 cases. Lancet Neurol 2010;9:438Y46
Autopsy Case Report of MS and PML
4. Dahlhaus S, Hoepner R, Chan A, et al. Disease course and outcome of 15 monocentrically treated natalizumab-associated progressive multifocal leukoencephalopathy patients. J Neurol Neurosurg Psychiatry 2013;84: 1068Y74 5. Kleinschmidt-DeMasters BK, Tyler KL. Progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis. N Engl J Med 2005;353:369Y74 6. Kuhle J, Gosert R, Buhler R, et al. Management and outcome of CSF-JC virus PCR-negative PML in a natalizumab-treated patient with MS. Neurology 2011;77:2010Y16 7. Lenhard T, Biller A, Mueller W, et al. Immune reconstitution inflammatory syndrome after withdrawal of natalizumab? Neurology 2010;75: 831Y33 8. Metz I, Radue EW, Oterino A, et al. Pathology of immune reconstitution inflammatory syndrome in multiple sclerosis with natalizumab-associated progressive multifocal leukoencephalopathy. Acta Neuropathol 2011; 123:235Y45 9. Schwab N, Hohn KG, Schneider-Hohendorf T, et al. Immunological and clinical consequences of treating a patient with natalizumab. Mult Scler 2012;18:335Y44 10. Kleinschmidt-DeMasters BK, Miravalle A, Schowinsky J, et al. Update on PML and PML-IRIS occurring in multiple sclerosis patients treated with natalizumab. J Neuropathol Exp Neurol 2012;71:604Y17 11. Wu¨thrich C, Koralnik IJ. Frequent infection of cortical neurons by JC virus in patients with progressive multifocal leukoencephalopathy. J Neuropathol Exp Neurol 2012;71:54Y65 12. Wu¨thrich C, Kesari S, Kim WK, et al. Characterization of lymphocytic infiltrates in progressive multifocal leukoencephalopathy: Colocalization of CD8(+) T cells with JCV-infected glial cells. J Neurovirol 2006;12: 116Y28 13. Wu¨thrich C, Dang X, Westmoreland S, et al. Fulminant JC virus encephalopathy with productive infection of cortical pyramidal neurons. Ann Neurol 2009;65:742Y48 14. Lucchinetti CF, Parisi J, Bru¨ck W. The pathology of multiple sclerosis. Neurol Clin 2005;23:77Y105 15. Gray F, Bazille C, Adle-Biassette H, et al. Central nervous system immune reconstitution disease in acquired immunodeficiency syndrome patients receiving highly active antiretroviral treatment. J Neurovirol 2005;11(Suppl 3):16Y22 16. Moll NM, Rietsch AM, Ransohoff AJ, et al. Cortical demyelination in PML and MS: Similarities and differences. Neurology 2008;70:336Y43 17. Lassmann H, Lucchinetti CF. Cortical demyelination in CNS inflammatory demyelinating diseases. Neurology 2008;70:332Y33 18. Bloomgren G, Richman S, Hotermans C, et al. Risk of natalizumab-associated progressive multifocal leukoencephalopathy. N Engl J Med 2012;366: 1870Y80 19. Berger JR, Kaszovitz B, Post MJ, et al. Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. A review of the literature with a report of sixteen cases. Ann Intern Med 1987;107:78Y87 20. Itoyama Y, Webster HD, Sternberger NH, et al. Distribution of papovavirus, myelin-associated glycoprotein, and myelin basic protein in progressive multifocal leukoencephalopathy lesions. Ann Neurol 1982;11: 396Y407 21. Munoz-Marmol AM, Mola G, Fernandez-Vasalo A, et al. JC virus early protein detection by immunohistochemistry in progressive multifocal leukoencephalopathy: A comparative study with in situ hybridization and polymerase chain reaction. J Neuropathol Exp Neurol 2004;63:1124Y30
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ORIGINAL ARTICLE
Natalizumab-Associated Progressive Multifocal Leukoencephalopathy in a Patient With Multiple Sclerosis: A Postmortem Study Christian Wu¨thrich, PhD, Bogdan F. Gh. Popescu, PhD, Sarah Gheuens, MD, Michael Marvi, MD, Ronald Ziman, MD, Stephen Pojen Denq, MD, Mylyne Tham, BS, Elizabeth Norton, BS, Joseph E. Parisi, MD, Xin Dang, PhD, Claudia F. Lucchinetti, MD, and Igor J. Koralnik, MD
Abstract Natalizumab, a monoclonal antibody directed against >4 integrins, has, to date, been associated with 399 cases of progressive multifocal leukoencephalopathy (PML) worldwide in patients receiving treatment for multiple sclerosis (MS). Because of the limited number of histologic studies, the possible interplay between MS and PML lesions has not been investigated. We report the clinical, radiologic, and histologic findings of an MS patient who developed PML after 32 months of natalizumab monotherapy. After withdrawal of natalizumab, she received plasma exchange, mefloquine, and mirtazapine but died soon thereafter. Postmortem examination was restricted to examination of the brain and spinal cord. Extensive PML lesions, characterized by the presence of JC virus DNA were found in the cerebral white matter and neocortex. Sharply demarcated areas of active PML lesions contained prominent inflammatory infiltrates composed of approximately equal numbers of CD4-positive and CD8-positive T cells, From the Division of Neurovirology, Department of Neurology (CW, SG, EN, XD, IJK), and Center for Virology and Vaccine Research, Department of Medicine (CW, SG, EN, XD, IJK), Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts; Department of Anatomy and Cell Biology (BFGhP, MT), and Cameco MS Neuroscience Research Center (BFGh.P, MT), University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Providence Saint Joseph Medical Center, The Hycy and Howard Hill Neuroscience Institute, Movement Disorder Center, Burbank (MM); David Geffen School of Medicine, University of California, Los Angeles, Los Angeles (RZ); Department of Medicine, Northridge Hospital Medical Center, Northridge (RZ); and Buddhist Tzu Chi Medical Center, Alhambra, California (SPD); and Department of Laboratory Medicine and Pathology (JEP), and Department of Neurology (CFL), Mayo Clinic, Rochester, Minnesota. Send correspondence and reprint requests to: Igor J. Koralnik, MD, Division of Neurovirology, Beth Israel Deaconess Medical Center, E/CLS-1005, 330 Brookline Ave, Boston, MA 02215; E-mail: [email protected] This study was supported in part by grants from the National Multiple Sclerosis Society and from Biogen Idec. This study was supported by NMSS grant RG 4523-A-1 and NIH grants R56 NS 041198, R01 NS 047029 and 074995, and K24 NS 060950 (to Igor Koralnik) and by grants from the Saskatchewan Health Research Foundation (2633; to Bogdan Popescu), the Canada Research Chairs program (to Bogdan Popescu), the National Multiple Sclerosis Society (NMSS RG3185-B-3; to Claudia Lucchinetti), and the National Institutes of Health (1R01NS049577; to Claudia Lucchinetti). The funding agencies had no input in the investigations or analysis of the results. Conflict of Interest: Sarah Gheuens and Elizabeth Norton are currently employees of Biogen Idec. The other authors declare that they have no competing interests.
consistent with an immune reconstitution inflammatory syndrome. Conversely, all MS lesions identified were hypocellular, long-standing inactive plaques characterized by myelin loss, relative axonal preservation, and gliosis and, importantly, were devoid of JC virus DNA and active inflammation. Chronic inactive MS lesions were separate and distinct from nearby PML lesions. This case demonstrates the coexistence and apparent lack of interplay between chronic inactive MS and PML lesions, and that immune reconstitution inflammatory syndrome seems to affect the shape and appearance of PML but not MS lesions. Key Words: Immune reconstitution inflammatory syndrome, JC virus, Multiple sclerosis, Natalizumab, Progressive multifocal leukoencephalopathy.
INTRODUCTION Progressive multifocal leukoencephalopathy (PML) is a fulminant inflammatory demyelinating disease caused by the reactivation of the polyomavirus JC (JCV) in the setting of immunosuppression (1). As of September 3, 2013, 399 cases of PML have been diagnosed in multiple sclerosis (MS) patients who received immunomodulatory treatment with natalizumab (Tysabri) (2). Natalizumab is a humanized monoclonal antibody against >4 integrins that is administered monthly and prevents egress of all leukocytes from the bloodstream into brain parenchyma, thereby decreasing immune surveillance. Therefore, T lymphocytes are sequestered in the blood vessels and cannot prevent reactivation and spread of JCV in the CNS. Because the biologic activity of natalizumab can extend to up to 3 months, MS patients who develop PML usually are treated with plasma exchange. This decreases blood levels of natalizumab and hastens the return of lymphocytes into the CNS (3). This phenomenon usually is associated with an immune reconstitution inflammatory syndrome (IRIS), which by itself can be damaging to the brain. Immune reconstitution inflammatory syndrome is characterized clinically by paradoxic worsening of neurologic signs and symptoms at the time of immune recovery, and magnetic resonance imaging (MRI) may show enlargement of PML lesions, contrast enhancement, and swelling (3, 4). Of the 399 MS-PML patients, 23% have died (2), but histologic postmortem examinations of the brain have been limited. In the initial report of natalizumab-associated PML
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occurring in an MS patient, no residual MS lesions were identified in the CNS (5). More recently, a few reports have described limited results of stereotactic biopsies (6Y9). An additional detailed postmortem examination of a natalizumabassociated PML patient failed to demonstrate JCV infection in brain lesions (10). The coexistence of lesions of JCV and MS and their possible interactions have not been described. We present detailed clinical, radiologic, and pathologic findings in an MS patient who developed PML during natalizumab therapy and who, at autopsy, demonstrated lesions of both diseases.
slightly modified technique in which streptavidin-AP was replaced by streptavidin-HRP (DAB detection) and an additional tyramide amplification step (12). Polymerase chain reaction amplification of JCV DNA extracted from brain samples was performed using T AgYspecific primers, as previously described (13).
Characterization of the MS Lesions Demyelinating activities in MS lesions based on myelin degradation products in macrophages were classified according to previously published criteria (14).
Characterization of Inflammatory Infiltrates in the MS and PML Lesions
MATERIALS AND METHODS Histopathologic Methods Formalin-fixed, paraffin-embedded sections were studied using routine neuropathologic methods (i.e. hematoxylin and eosin, Luxol fast blueYperiodic acid-Schiff, and Bielschowsky impregnation), as well as single and double labeled immunohistochemistry (IHC) and immunofluorescence using antibodies listed in the Table, as previously described (11).
Characterization of PML Lesions Viral protein immunoreactivity for JCV T Antigen (T Ag) (v-300) or VP1 (PAB597) was correlated with the extent and pattern of demyelination using double labeling for myelin proteins, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase, myelinassociated glycoprotein, and myelin oligodendrocyte glycoprotein. Polyomavirus JCYinfected cells were identified by double labeling of specific cells (i.e. neurons and glia). In situ hybridization (ISH) for JCV DNA was performed using a
CD3-positive and CD8-positive T lymphocytes in both PML and MS lesions were examined relative to JCV VP1 (PAB597) expression. CD4-positive T cells were inferred based on double labeling for CD3 and CD8 (double positive consistent with CD8-positive T cells; CD3-positive/CD8-negative cells were considered CD4-positive T cells).
Case History A 52-year-old woman diagnosed as having relapsingremitting MS of 14 years’ duration had been treated with several immunomodulatory and immunosuppressive drugs, including interferon-A, glatiramer acetate, methotrexate, and cyclophosphamide. In November 2006, she began intravenous natalizumab monotherapy consisting of 300 mg every 4 weeks for refractory exacerbations. One year later, MRI showed stable MS lesions in the left periventricular area and the splenium of the corpus callosum (Fig. 1A, E). By June 2008, she was tetraplegic because of accumulating disability
TABLE. Antibodies Used for Immunohistochemistry and Immunofluorescence Staining Antigen; Product Name PAB597 SV40 T Ag (v-300); sc-20800 MAP-2; HM-2; M4403 MAP-2; ab5622 MAP-2; LS-B290 GFAP; M0761; 6F2 GFAP; Z0334 GFAP; ab4674 CNPase; C5922; 11-5B CNP; HPA023266 MOG; EP4281; ab109746 MAG; ab89780 CD8; 1A5; VP-C325 CD3; CD3-12; OASA11552 PLP NF KiM1P
Host
Isotype
Target
Source
m r m r ch m r ch m r r m m rat m m m
NA IgG IgG1 IgG IgY IgG1 IgG IgY IgG1 IgG IgG IgG1 IgG1 IgG
JCV VP1 JCV T Ag Neurons Neurons Neurons Astrocytes Astrocytes Astrocytes Myelin/Oligo Myelin/Oligo Myelin/Oligo Myelin/Oligo CD8 CD3 PLP NF KiM1P
Santa Cruz Biotechnology, Santa Cruz, CA Sigma, St. Louis, MO Chemicon, Temecula, CA Lifespan Bioscience, Seattle, WA Dako, Carpinteria, CA Dako Abcam, Cambridge, MA Sigma Sigma Abcam Abcam Vector Laboratories, Burlingame, CA Aviva Systems Biology, San Diego, CA Serotec, USA Dako, Glostrup, Denmark Dr. Radzun, Go¨ttingen, Germany
For double staining, primary antibodies (Abs) from different species (mouse, rat, rabbit, or chicken) were used with appropriately matched (species and isotypes) secondary Abs. These Abs were conjugated to either alkaline phosphatase (AP) or horseradish peroxidase (HRP) for immunohistochemistry (IHC) or Alexa Fluor 350, 488, and 568 according to the manufacturer’s instructions for immunofluorescence (IF). Single and double IHC were performed with the Vectastain Elite ABC Kits (Vector, Burlingame, CA) according to the manufacturer’s instructions. Negative controls, for IHC and IFA, included omission of the primary Abs, use of isotype matched controls, and use of sections from individuals known to be free of JCV-infected cells. ch, chicken; CNPase, CNP, 2¶,3¶-cyclic-nucleotide 3¶-phosphodiesterase; GFAP, glial fibrillary acidic protein; KiM1P, macrophage marker; m, mouse; MAG, myelin-associated glycoprotein; MAP-2, microtubule-associated protein 2; MOG, myelin-oligodendrocyte glycoprotein; NF, neurofilament; oligo, oligodendrocytes; PLP, myelin proteolipid protein; r, rabbit; SV40 T Ag, Simian virus 40 T antigen; NA, not available.
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Autopsy Case Report of MS and PML
FIGURE 1. Magnetic resonance imaging evolution of multiple sclerosis (MS) and progressive multifocal leukoencephalopathy (PML) lesions. Axial (AYD) and corresponding sagittal (EYH) FLAIR images. After 1 year of natalizumab monotherapy, MS lesions are present in the left periventricular area and the splenium of the corpus callosum (A, E, arrows). There is a new left subcortical lesion (B, arrowhead) in the left frontal lobe, remote from the old MS lesions 20 months later (B, F, arrows); 2.5 months later, the left frontal lobe lesion has extended posteriorly and contralaterally through the genu of the corpus callosum to the right frontal lobe white matter (C, G, arrowheads), whereas the MS lesions (arrows) seem stable. One month later, the PML lesions (D, H, arrowheads) have spread further in the right frontal lobe and the left frontal and parietal lobes, close, but separate from the MS lesions (D, H, arrows).
and required admission to a skilled nursing facility. She missed 2 injections of natalizumab in January and February 2009. In June 2009, she presented with cognitive decline. An MRI performed in July 2009, 32 months after onset of natalizumab therapy, showed a new left frontal subcortical nonenhancing white matter lesion in addition to stable chronic MS lesions (Fig. 1B, F). A few weeks later, she developed a speech deficit and worsening cognitive difficulties. She was treated with a short course of dexamethasone and received her last injection of natalizumab in early September 2009. Repeat MRI performed in September 2009 showed marked progression of the left frontal lesion, extending across the genu of the corpus callosum into the right frontal lobe, associated with a mild mass effect on the left lateral ventricle (Fig. 1C, G) and enhancement. A lumbar puncture performed in October 2009 showed 36 red blood cells/KL, 7 white blood cells/KL, glucose level of 67 mg/dL, and protein level of 77 mg/dL. Polyomavirus JC cerebrospinal fluid polymerase chain reaction was positive, showing 9900 JCV DNA copies/mL, thereby confirming the diagnosis of PML. She had a detectable cellular immune response against JCV, mediated by both CD4-positive and CD8-positive T cells, and was treated with 5 alternate day courses of plasma exchange, mirtazapine 30 mg/day, and mefloquine 250 mg/week. Later that month, cerebrospinal fluid showed undetectable JCV DNA, and MRI showed progression of the PML lesions in the right frontal, left frontal, and
parietal lobes (Fig. 1D, H) with minimal associated enhancement. Despite these interventions, there was no clinical improvement. She developed aspiration pneumonia and died in November 2009, 4 months after PML onset, 2 months after the last natalizumab injection, 3.5 weeks after onset of plasma exchange, and 12 days after the last MRI. Postmortem examination was restricted to the brain and spinal cord.
RESULTS Macroscopic Neuropathology The formalin-fixed brain showed mild generalized swelling and was sectioned in the axial plane to correlate with the recent MRI. Corresponding to the axial FLAIR images, the brain slices showed that the MS lesions were well demarcated and somewhat retracted firm areas of brown discoloration, whereas the PML lesions were softened, granular, and ‘‘motheaten’’ in appearance. Several MS lesions were identified in the white matter, including 5 periventricular lesions (3 around lateral ventricles, 1 in the midbrain tectum around the cerebral aqueduct, and 1 in the medulla around the fourth ventricle), 2 lesions in the cerebellar peduncles, 1 in subpial in the basis pontis, 1 in the medullary pyramid, 2 in the white matter of the spinal cord, and 2 in the optic tract. Also identified were 5 lesions in deep
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gray matter structures: 1 each in the amygdala, the tail of the caudate, putamen, claustrum, and inferior olivary nucleus.
Histology, IHC, and ISH Microscopically, all white and deep gray matter MS lesions were chronic, inactive, demyelinated, and hypocellular plaques, characterized by mild axonal loss, gliosis, and largely devoid of inflammatory T-cell infiltrates. All lesions showed mild microglial activation without macrophages, except for 1 spinal cord lesion that contained periodic acid-SchiffYpositive macrophages, consistent with more recent demyelination. Cortical MS lesions included 16 intracortical and subpial chronic lesions. Extensive PML lesions were present in the subcortical and deep white matter of both cerebral hemispheres, the corpus callosum, and the brainstem; they were characterized at the active edge of the lesion by relative myelin preservation and a high density of infected oligodendrocytes. Myelin loss was more apparent toward the centers of these lesions, which contained few infected cells. In situ hybridization showed abundance of JCV DNA localized mostly to sharply demarcated areas at the periphery of large lesions and involving the gray-white matter junction and the gray matter (Fig. 2A, C, E). Immunohistochemistry staining of sequential sections with an antibody against SV40 (which cross-reacts against JCV major capsid protein VP1) showed a similar staining pattern at the gray-white junction, indicating the presence of a productive lytic infection of glial cells (Fig. 2B, D). The extent of PML-associated demyelination in the white matter and the cortex is illustrated in Figure 2B inset. Only a few infected cells were present in gray matter (Fig. 2F). A particular feature of these lesions was the well-demarcated aspect of JCV-infected areas. CD3-positive T cells were present at the edge of actively infected areas (Fig. 2B, D, F) and were often in close contact with JCV-infected cells (Fig. 2D, F). Topographic relationships between PML and MS lesions were studied by comparing ISH and IHC for JCV DNA. Progressive multifocal leukoencephalopathy lesions were more obvious and extensive, as demonstrated by ISH than by IHC and Luxol fast blue stain. Infected cells within PML lesions contained JCV T Ag (which is typically expressed early in the viral replication cycle), as well as JCV VP1 capsid protein (indicating the presence of mature viral particles). Chronic inactive MS lesions showed no evidence of JCV DNA or JCV proteins. The juxtaposition of PML and MS lesions is illustrated in an area of the left occipital lobe containing PML lesions adjacent to a cortical MS plaque (Fig. 3). A chronic subpial MS plaque is adjacent to PML lesions that are in the subcortical white matter and the lower layers of the cortex (Fig. 3A). The boxed areas (d and e in Fig. 3B and C, respectively) magnified in (Fig. 3D and E, respectively) show JCV-infected cells located immediately adjacent to the MS lesion (Fig. 3D) and a readily identified inflammatory infiltrate of CD3-positive T cells (Fig. 3E) in the PML lesions but not in the MS lesion. Higher-magnification images show that the MS lesion is entirely devoid of JCV infection or CD3-positive T-cell infiltrates, whereas CD3-positive T cells (Fig. 3G, I) are located in close proximity to JCV-infected cells (Fig. 3FYI).
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To determine the nature of the inflammatory T-cell infiltrate, double immunofluorescence staining for CD3 (green) and CD8 (red) in the PML lesions was performed (Fig. 4). As shown in Figure 4A, an approximately equal number of cells are CD3 positive/CD8 negative (green) or CD3 positive/CD8 positive (yellow). No CD3-negative/CD8-positive (red) cells were present. Higher-magnification shows a CD3-positive/ CD8-positive T-cell (B) and a CD3-positive/CD8-negative T cell (C), the latter corresponding to a CD4-positive T cell. The magnitude of the CD4-positive and CD8-positive T-cell infiltrates in the PML lesions is consistent with IRIS. In addition, occasional JCV-infected neurons expressing JCV T Ag, but not JCV VP1, were identified in lesions at the gray-white junction and in gray matter (Fig. 5A), findings consistent with nonproductive infection of cortical neurons by JCV, as in other PML cases (11). Extensive PML lesions were found in the corpus callosum, some extending into the septum pellucidum (Fig. 5B). A small PML focus also involved hippocampal white matter (not shown).
DISCUSSION Coexistence of Active PML and Chronic Inactive MS Lesions To our knowledge, this is the first histopathologically confirmed report showing coexistent MS and PML lesions in a natalizumab-treated patient. It provides a unique opportunity for comparing radiologic and histologic findings in natalizumabassociated PML as well as examining the early manifestations of superimposed IRIS. Progressive multifocal leukoencephalopathy lesions were extensive, corresponding to the MRI findings, and harbored ongoing productive JCV infection. Conversely, MS lesions were chronic inactive plaques devoid of active inflammatory infiltrates. Although PML lesions displayed a large number of cells containing JCV DNA or proteins, MS lesions were entirely devoid of JCV infection, even when they abutted PML lesions. This may be explained by the relative absence of remaining glial cells that could potentially become infected by JCV in chronic MS lesions. As such, the MS plaques and PML lesions were distinct and pathologically independent.
IRIS Affected PML but Spared MS Lesions Immune reconstitution inflammatory syndrome was originally described in HIV-infected patients treated with combination antiretroviral therapy, in which the recovering immune system was able to mount a cellular response. Progressive multifocal leukoencephalopathyYIRIS is a common consequence of plasma exchange immunomodulatory treatment and is thought to be caused by the return of lymphocytes to the brain parenchyma. The influence of IRIS may be heightened when natalizumab is withdrawn and the action of the drug is diminished, leading to catastrophic inflammation, with swelling, mass effect, and herniation, often requiring high doses of corticosteroids. In this case, the clinical manifestations of IRIS were limited to mild enhancement of PML lesions on MRI. Histologic examination of PML lesions revealed prominent T-cell infiltrates, characterized by equal numbers of CD4-positive Ó 2013 American Association of Neuropathologists, Inc.
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Autopsy Case Report of MS and PML
FIGURE 2. Progressive multifocal leukoencephalopathy (PML) lesions are sharply demarcated and harbor CD3-positive T cells. (A, B) In situ hybridization for JC virus (JCV) DNA (cresyl violet counterstaining) (A) and immunohistochemistry for JCV VP1 and CD3 (B) show that JCV-infected cells (arrowheads) are mostly restricted to the junction between white matter (WM) and gray matter (GM) and the lower layers of the cortex. Conversely, the deep WM is almost free of JCV-infected cells and totally demyelinated as shown by myelin proteolipid protein (PLP) immunostaining (brown [B inset]), but not cavitary. (CYF) Magnifications of rectangles c and e from (A) show a sharp demarcation of the areas containing JCV-infected cells (arrowheads) at the gray-white junction (C). The edge of JCV infection spreads in the lower layer of the cortex (E, arrowheads). Magnification of rectangles d and f from (B) show that cells expressing JCV VP1 protein (arrowheads) are surrounded by CD3-positive T cells (arrows in D and F). Most of these T cells (D and F insets, arrow) are located in close proximity to the JCV-infected cells (D and F insets, arrowheads). Overall, the T cells are mostly in areas where the cell nuclei contain JCV DNA (C) and express JCV VP1 (D). Ó 2013 American Association of Neuropathologists, Inc.
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and CD8-positive T cells within and at the active edge of lesions. This differs from PML-IRIS lesions in AIDS patients, who typically have low CD4-positive T-cell counts in peripheral
blood and a predominance of CD8-positive T cells in brain parenchyma (15). Inasmuch as JCV-specific CD4-positive and CD8-positive T cells were detected in the blood of our patient
FIGURE 3. Proximity of multiple sclerosis (MS) and progressive multifocal leukoencephalopathy (PML) lesions in the left occipital lobe. (AYC) Immunohistochemistry (IHC) for myelin proteolipid protein (A) shows a subpial MS lesion (delineated in red) and demyelination lesions of PML involving the subcortical white matter (WM) and lower layers of the cortical gray matter (GM) that contain numerous cells harboring JC virus (JCV) DNA ([B] JCV in situ hybridization with cresyl violet counterstaining) and expressing JCV VP1 protein in (C) (Luxol fast blue counterstaining). (D, E) Higher magnifications of boxes d and e in (B) and (C) show that cells harboring JCV DNA (D, arrowheads), those expressing JCV VP1 and CD3-positive T cells, are not present in the MS lesion (E). The distribution of the CD3-positive T cells is shaded orange (arrows). (FYI) Higher magnifications of the MS-PML interface (boxes f, g) and the lower GM side of the PML lesion (boxes h, i) show that cells harboring JCV DNA (F, arrowhead), those expressing JCV VP1 (G, arrowheads) and CD3-positive T cells (G, arrows), are present at the border of the MS lesion. Polyomavirus JC infection of the GM is shown in (H, I, arrowheads). Insets in G and I at higher magnification show CD3-positive T cells in close proximity to cells expressing JCV VP1 protein.
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Autopsy Case Report of MS and PML
FIGURE 4. CD8-positive and CD4-positive T cells are present in progressive multifocal leukoencephalopathy (PML) lesions. Double immunofluorescence staining for CD3 (Alexa Fluor 488, green) and CD8 (Alexa Fluor 568, red) and autofluorescence (blue). (A) Similar numbers of CD8-positive T cells (CD3-positive/CD8-positive yellow-red, arrows) and CD4-positive T cells (CD3-positive/CD8-negative, green, arrowheads) are present at the border of this PML lesion. (B) Higher magnification with insets show CD8-positive T cells (arrows) in both red (CD8) and green (CD3) channel. (C) Conversely, a CD4-positive T cell (arrowhead) is characterized by absence of staining in the red channel (CD8) but staining in the green channel (CD3). Ó 2013 American Association of Neuropathologists, Inc.
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FIGURE 5. Nonproductive infection of hemispheric cortical neurons and productive infection of glial cells in the septum pellucidum. (A) Triple immunohistochemistry (IHC) for JC virus (JCV) T Antigen (T Ag) (blue), MAP-2 (orange), and 2¶,3¶-cyclicnucleotide 3¶-phosphodiesterase (CNPase) (brown). Rare JCV-infected cortical neurons expressing T Ag are present at the edge of a progressive multifocal leukoencephalopathy (PML) lesion in the gray matter (GM) (inset). (B) IHC for JCV VP1 (blue). Frequent glial cells expressing JCV VP1 can be seen in the corpus callosum (upper inset) and in the white matter (WM) of the septum pellucidum (lower inset). LFB, Luxol fast blue.
4 weeks before death, it is possible that T cells in the brain were actively involved in lysing JCV-infected cells. In contrast, inflammatory infiltrates spared chronic MS lesions. Because the patient did not develop an MS exacerbation during her brief course off natalizumab, it seems that the inflammatory reaction associated with IRIS did not lead to an MS-like autoimmunemediated response directed against myelin. These data suggest that, in MS patients with PML-IRIS, T cells are driven by JCV antigen specificity and do not cause an exuberant response as in HIV-infected patients (15).
PML Lesions Involve Both White and Gray Matter Progressive multifocal leukoencephalopathy lesions were extensive in white matter, including in the septum pellucidum, an area that is not classically regarded as a target of JCV infection. Such involvement might result in dissemination of infection to the fornix and hippocampus. There were numerous leukocortical and intracortical PML lesions that were occa-
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sionally in close proximity to subpial MS lesions, but no subpial lesions of PML were identified, consistent with previous reports (16, 17). This suggests that this location may be more characteristic of MS. In addition, some cortical neurons sustained nonproductive infection by JCV, as in other PML cases (11). This case exemplifies the risks of natalizumab therapy and provides further insights in PML pathogenesis in the context of MS. This patient’s aggressive relapsing form of MS required multiple immunomodulatory and immunosuppressive therapies before switching to natalizumab, which she received as monotherapy for 32 months. If, like 98% of natalizumab-associated PML patients (2), she had been JCV seropositive, the risk of her developing PML in the setting of prior immunosuppression use and natalizumab treatment for more than 24 months is estimated to be 1/90, according to a recent algorithm (18). This is significant and second only to the risk in patients with advanced AIDS not receiving antiretroviral treatment, which is 1/20 (19). The patient’s clinical presentation with progressive cognitive decline and speech difficulties superimposed on her Ó 2013 American Association of Neuropathologists, Inc.
Copyright © 2013 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
J Neuropathol Exp Neurol Volume 72, Number 11, November 2013
neurologic deficits attributed to MS was commensurate with the location of the PML lesions, as reported in other series (3, 4).
Lack of Concomitant Demonstration of JCV Infection and MS Lesions in Other Cases Our study expands previous biopsy and autopsy findings of natalizumab-associated PML. Earlier biopsy studies found no evidence of JCV infection (6, 7, 9) or only rare single-infected cells in 4 of 5 cases (8). In the original autopsy case report by Kleinschmidt-DeMasters and Tyler (5), no residual MS lesions were identified histologically among widespread PML lesions containing numerous cells harboring JCV DNA, suggesting the possibility that PML may have obscured entirely the MS lesions. This also may have occurred in some of the MS lesions in our case, given the extent of PML that was observed. In contrast to our patient, another reported case demonstrated no detectable JCV-infected cells by IHC for JCV T Ag within extensive demyelinated lesions attributed to PMLIRIS (10). Failure to demonstrate JCV T Ag by IHC may have been caused by technical factors related to the suboptimal sensitivity of PAb416 (DP02) for detection of JCV in formalinfixed, paraffin-embedded tissue (20, 21). Alternatively, it is possible that PML lesions were ‘‘burned out’’ in that case because the postmortem examination was performed 9 months after the clinical onset of PML (10).
CONCLUSIONS This case demonstrates the coexistence of chronic inactive MS and PML lesions harboring active JCV infection characterized by the presence of JCV DNA, T Ag, and VP1 protein, as well as inflammatory infiltrates of CD4-positive and CD8-positive T cells. In addition, restrictive JCV infection of rare cortical pyramidal neurons was demonstrated, consistent with previous observations (11). Immune reconstitution inflammatory syndrome greatly altered the characteristics of PML lesions while sparing MS lesions. Whether PML may alter the nature of active MS lesions remains to be determined. REFERENCES 1. Gheuens S, Wu¨thrich C, Koralnik IJ. Progressive multifocal leukoencephalopathy: Why gray and white matter. Annu Rev Pathol 2013;8: 189Y215 2. PML Incidence in Patients Receiving TYSABRI (natalizumab) [Internet]. BioGenIDEC. 2013. Available from: http://medinfo.biogenidec.com. Accessed on September 26, 2013 3. Clifford DB, De Luca A, Simpson DM, et al. Natalizumab-associated progressive multifocal leukoencephalopathy in patients with multiple sclerosis: Lessons from 28 cases. Lancet Neurol 2010;9:438Y46
Autopsy Case Report of MS and PML
4. Dahlhaus S, Hoepner R, Chan A, et al. Disease course and outcome of 15 monocentrically treated natalizumab-associated progressive multifocal leukoencephalopathy patients. J Neurol Neurosurg Psychiatry 2013;84: 1068Y74 5. Kleinschmidt-DeMasters BK, Tyler KL. Progressive multifocal leukoencephalopathy complicating treatment with natalizumab and interferon beta-1a for multiple sclerosis. N Engl J Med 2005;353:369Y74 6. Kuhle J, Gosert R, Buhler R, et al. Management and outcome of CSF-JC virus PCR-negative PML in a natalizumab-treated patient with MS. Neurology 2011;77:2010Y16 7. Lenhard T, Biller A, Mueller W, et al. Immune reconstitution inflammatory syndrome after withdrawal of natalizumab? Neurology 2010;75: 831Y33 8. Metz I, Radue EW, Oterino A, et al. Pathology of immune reconstitution inflammatory syndrome in multiple sclerosis with natalizumab-associated progressive multifocal leukoencephalopathy. Acta Neuropathol 2011; 123:235Y45 9. Schwab N, Hohn KG, Schneider-Hohendorf T, et al. Immunological and clinical consequences of treating a patient with natalizumab. Mult Scler 2012;18:335Y44 10. Kleinschmidt-DeMasters BK, Miravalle A, Schowinsky J, et al. Update on PML and PML-IRIS occurring in multiple sclerosis patients treated with natalizumab. J Neuropathol Exp Neurol 2012;71:604Y17 11. Wu¨thrich C, Koralnik IJ. Frequent infection of cortical neurons by JC virus in patients with progressive multifocal leukoencephalopathy. J Neuropathol Exp Neurol 2012;71:54Y65 12. Wu¨thrich C, Kesari S, Kim WK, et al. Characterization of lymphocytic infiltrates in progressive multifocal leukoencephalopathy: Colocalization of CD8(+) T cells with JCV-infected glial cells. J Neurovirol 2006;12: 116Y28 13. Wu¨thrich C, Dang X, Westmoreland S, et al. Fulminant JC virus encephalopathy with productive infection of cortical pyramidal neurons. Ann Neurol 2009;65:742Y48 14. Lucchinetti CF, Parisi J, Bru¨ck W. The pathology of multiple sclerosis. Neurol Clin 2005;23:77Y105 15. Gray F, Bazille C, Adle-Biassette H, et al. Central nervous system immune reconstitution disease in acquired immunodeficiency syndrome patients receiving highly active antiretroviral treatment. J Neurovirol 2005;11(Suppl 3):16Y22 16. Moll NM, Rietsch AM, Ransohoff AJ, et al. Cortical demyelination in PML and MS: Similarities and differences. Neurology 2008;70:336Y43 17. Lassmann H, Lucchinetti CF. Cortical demyelination in CNS inflammatory demyelinating diseases. Neurology 2008;70:332Y33 18. Bloomgren G, Richman S, Hotermans C, et al. Risk of natalizumab-associated progressive multifocal leukoencephalopathy. N Engl J Med 2012;366: 1870Y80 19. Berger JR, Kaszovitz B, Post MJ, et al. Progressive multifocal leukoencephalopathy associated with human immunodeficiency virus infection. A review of the literature with a report of sixteen cases. Ann Intern Med 1987;107:78Y87 20. Itoyama Y, Webster HD, Sternberger NH, et al. Distribution of papovavirus, myelin-associated glycoprotein, and myelin basic protein in progressive multifocal leukoencephalopathy lesions. Ann Neurol 1982;11: 396Y407 21. Munoz-Marmol AM, Mola G, Fernandez-Vasalo A, et al. JC virus early protein detection by immunohistochemistry in progressive multifocal leukoencephalopathy: A comparative study with in situ hybridization and polymerase chain reaction. J Neuropathol Exp Neurol 2004;63:1124Y30
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Copyright © 2013 by the American Association of Neuropathologists, Inc. Unauthorized reproduction of this article is prohibited.
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