Neuroinflammation and Demyelination in Multiple Sclerosis After Allogeneic Hematopoietic Stem Cell Transplantation

ORIGINAL CONTRIBUTION Neuroinflammation and Demyelination in Multiple Sclerosis After Allogeneic Hematopoietic Stem Cell Transplantation

Jian-Qiang Lu, MD; Jeffrey T. Joseph, MD; Richard A. Nash, MD; Jan Storek, MD; Anne M. Stevens, MD; Luanne M. Metz, MD; Arthur W. Clark, MD; Edward S. Johnson, MD; V. Wee Yong, PhD

Objective: To evaluate the effects of allogeneic hema- Main Outcome Measures: Morphological features and topoietic stem cell transplantation (allo-HSCT) on the immunohistochemical features, including the quantita- brains of persons with and without multiple sclerosis (MS) tive measures of chronic inflammatory cells. by means of postmortem histopathological examination. Results: Demyelinating and inflammatory activities of MS persisted after allo-HSCT in all of the patients with MS. Ac- Design: Postmortem histopathology, case studies, and tive and chronic active MS lesions exhibited significantly case-control studies. higher numbers of CD3ϩ T cells and CD8ϩ cytotoxic T cells and significantly higher scores of CD68ϩ microglia/ Patients: Four patients with MS who died at a median macrophages than did chronic inactive lesions or normal- of 4.5 months (range, 3-9 months) after allo-HSCT for a appearing white matter. The normal-appearing brains of concomitant hematologic malignant neoplasm; 5 pa- allo-HSCT recipients who did not have MS were found to ϩ ϩ tients without MS who died at a median of 10.0 months have significantly higher numbers of CD3 T cells and CD8 ϩ (1-29 months) after allo-HSCT; and 5 control subjects cytotoxic T cells and higher scores of CD68 microglia/ without MS who did not undergo allo-HSCT. macrophages compared with the controls; however, no demyelination was identified in these non-MS samples.

Setting: Referral centers. Conclusion: Allo-HSCT fails to halt the demyelination and inflammation of MS. Intervention: Allogeneic hematopoietic stem cell transplantation. Arch Neurol. 2010;67(6):716-722

HE PATHOLOGIC PROCESSES egy in severe forms of MS. Based on the of multiple sclerosis (MS) published studies, autologous HSCT pro- are thought to be contrib- duces a broad range of patients with sur- uted to by T and B cells that vival free of MS progression (36%-95%) Author Affiliations: 1,6 Departments of Pathology and invade the central nervous and MS activity (33%-64%). The only Laboratory Medicine (Drs Lu, system (CNS) to cause oligodendrocyte published histopathological study of au- T 1-3 Joseph, and Clark), Internal damage, demyelination, and axonal loss. tologous HSCT in MS indicated a failure Medicine (Dr Storek), and Current therapies for MS consist of im- to arrest demyelination and neurodegen- Clinical Neurosciences and munomodulating agents such as inter- eration in 5 patients who died within 1.5 Hotchkiss Brain Institute feron beta and glatiramer acetate and anti- years after the transplantation.7 (Drs Metz and Yong), University inflammatory and immunosuppressive Allo-HSCT is inspired by case reports of Calgary, Calgary, Alberta, drugs such as glucocorticoids and mito- of MS patients who have undergone allo- Canada; Fred Hutchinson Cancer Research Center xantrone hydrochloride. Hematopoietic HSCT for a concomitant hematologic ma- (Dr Nash) and Department of stem cell transplantation (HSCT) is a form lignant neoplasm and subsequently has Pediatrics, University of of therapy in which immune suppression been supported by animal models of MS. Washington (Dr Stevens), is maximized to the point of transient im- Because allo-HSCT is designed to furnish Seattle; and Department of mune ablation.4 Selected patients with MS, a new immune system in primary lym- Laboratory Medicine and after preparation for the transplant by phoid tissues, it represents an approach dif- Pathology, University of Alberta, means of a potent immunosuppressive ferent from autologous HSCT for the eradi- Edmonton, Alberta, Canada regimen, undergo autologous HSCT or al- cation of autoimmune disease.6,8 This has (Dr Johnson). Dr Lu is now with the Department of Laboratory logeneic HSCT (allo-HSCT) to restore the not been explored in prospective studies, 5 Medicine and Pathology, host immune system. An increasing num- however, because treatment-related mor- University of Alberta Hospital, ber of studies have assessed autologous bidity in allo-HSCT is substantially higher Edmonton. HSCT as a potential new therapeutic strat- than that in autologous HSCT, mostly ow-

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Case No./ Time From Clinical Sex/Age at Conditioning/GVH MS Dx to Disease EDSS Before EDSS After Time of Death After Allo-HSCT, y Disease Prophylaxisa Allo-HSCT, y Course Allo-HSCT Allo-HSCT Allo-HSCT, mo Cause of Death 1/F/39 Busulfan, fludarabine phosphate, 19 RRMS 2.0 3.0 4 Multiple organ failure ATG/methotrexate sodium, cyclosporine 2/F/38 Cyclophosphamide, thiotepa, TBI, 9 RRMS Unknown Unknown 5 Pneumonia ATG/corticosteroids, cyclosporine 3/M/42 Busulfan, cyclophosphamide/ 5 PPMS Unknown Unknown 9 Myocardial methotrexate, cyclosporine infarction/pneumonia 4/F/59 Busulfan, fludarabine, Incidentalb NA NA NA 3 Pneumonia ATG/methotrexate, cyclosporine

Abbreviations: Allo-HSCT, allogeneic hematopoietic stem cell transplantation; ATG, antithymocyte globulin; Dx, diagnosis; EDSS, Expanded Disability Status Scale; GVH, graft-vs-host; MS, multiple sclerosis; NA, not applicable; PPMS, primary progressive multiple sclerosis; RRMS, relapse-remitting multiple sclerosis; TBI, total body irradiation. aMedications designated for conditioning were administered before HSCT; medications for GVH disease prophylaxis were administered after HSCT. bIndicates no documentation of MS in the clinical medical record.

Table 2. Demographic and Clinical Characteristics of non-MS Patients

Case No./ Conditioning/GVH Time of Death After Sex/Age, y Hematological Disease Disease Prophylaxis Allo-HSCT, mo Cause of Death 1/M/63 Idiopathic myelofibrosis Busulfan, fludarabine phosphate, 1 Multiple organ failure ATG/methotrexate sodium, cyclosporine, corticosteroids 2/F/58 Cutaneous T-cell CHOP/alemtuzumab (Campath), 12 Multiple organ failure lymphoma corticosteroids 3/F/56 Acute lymphoblastic Busulfan, fludarabine, ATG/methotrexate, 3 Multiple organ failure leukemia cyclosporine, corticosteroids 4/M/54 Acute myelogenous Idarubicin hydrochloride, 10 Diffuse alveolar damage leukemia cytarabine/methotrexate, cyclosporine, corticosteroids 5/M/33 Chronic myelogenous Hydroxyurea/corticosteroids, 29 Pneumonia leukemia cyclosporine 6-10/3 M, 2 F/26-65a NA NA NA NA

Abbreviations: Allo-HSCT, allogeneic hematopoietic stem cell transplantation; ATG, antithymocyte globulin; CHOP, cyclophosphamide, doxorubicin, vincristine sulfate, and prednisone; GVH, graft-vs-host; MS, multiple sclerosis; NA, not applicable. a Indicates a group of age-matched controls.

ing to graft-vs-host (GVH) disease and conditioning- METHODS related toxic effects.6,9,10 To date, allo-HSCT has not been performed with the in- SUBJECTS tention of treating MS.6 Reports of 5 MS patients who underwent allo-HSCT because of coincidental hematological This study was approved by local research ethics boards and cancer demonstrated variable effects of allo-HSCT on the performed on archival autopsy tissues from 4 MS (Table 1) and course of MS.11-15 Of these reports, 3 patients showed clini- 10 non-MS (Table 2) cases that were obtained from the de- 11-13 partments of pathology of the University of Calgary and the Uni- cal improvement or stabilization. Two patients exhib- versity of Alberta. A prior diagnosis of MS was made clini- ited clinical and radiological evidence of persistent MS cally16 in 3 of these MS patients, but a diagnosis of MS was not activities with follow-up times of 24 and 4 months after noted in the hospital records of the fourth patient. Postmor- allo-HSCT.14,15 To our knowledge, our recent case report15 tem neuropathological examination in all the MS patients re- was the only study with postmortem histopathological ex- vealed multiple lesions diagnostic of MS. The MS patients re- amination that demonstrated persistent inflammatory and ceived allo-HSCT for treatment of their concurrent hematological demyelinating activities of MS 4 months after allo-HSCT. diseases, including chronic myelogenous leukemia in case 1 and The present study examined the histopathological find- acute myelogenous leukemia in cases 2 through 4 (Table 1). ings of MS in 4 patients who received allo-HSCT. Our Their lengths of time to death after allo-HSCT ranged from 3 recently reported case15 was extended in the present study to 9 months (median, 4.5 months). Five non-MS patients from the Calgary archives (cases 1-5 (case 1 in Table 1) for the purpose of quantitative analy- in Table 2) who were described as having a normal brain and ses. To assess whether allo-HSCT in the absence of MS spinal cord on macroscopic and routine histopathological ex- could be associated with CNS inflammation, we further amination were included to investigate the effect of allo- examined a group of non-MS allo-HSCT recipients and HSCT on normal-appearing brains. Their lengths of time to death a group of non-MS, non–allo-HSCT control subjects. after allo-HSCT ranged from 1 to 29 months (median, 10.0

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C D

Figure 1. Demyelinating and inflammatory activity in a patient with multiple sclerosis (case 1 in Table 1) 4 months after allogeneic hematopoietic stem cell transplantation. An active lesion in the frontal juxtacortical region shows inflammatory/demyelinating activity throughout the lesion with decreased intensity of Luxol fast blue staining for myelin, abundant macrophages phagocytosing myelin debris (A) (original magnification ϫ400), and colocalization of myelin with CD68ϩ macrophages/microglia on double immunofluorescence staining, indicative of ongoing demyelination (B) (myelin basic protein [green]/CD68 [red], original magnification ϫ400), as well as scattered acutely damaged axons immunoreactive for amyloid precursor protein (brown profiles) (C) (hematoxylin-eosin [blue], original magnification ϫ400). A chronic active lesion in the middle cerebral peduncle exhibits a hypocellular core and an inflammatory/demyelinating edge with focal remyelination (D) (hematoxylin-eosin and Luxol fast blue, original magnification ϫ100). Other features of inflammatory activity in this patient were described in our previously published study.15

months). Five neuropathologically normal subjects who did not The primary antibodies used were mouse antihuman CD68 undergo allo-HSCT (cases 6-10 in Table 2) served as the con- (macrophages/microglia; DAKO Denmark A/S, Glostrup, trols in the present study. All the non-MS patients who did or Denmark), anti-CD3 (T cells; Thermo Scientific, Rockford, did not undergo allo-HSCT were free of neurological symp- Illinois), anti-CD4 (helper T cells; Cell Marque Corporation, toms and abnormal radiologic findings of the CNS. Rocklin, California), anti-CD8 (cytotoxic T cells; DAKO Although complete chimerism of circulating leukocytes were Denmark A/S), anti-CD20 (B cells; DAKO Denmark A/S), anti- achieved by allo-HSCT in all the cases (data not shown), the CD138 (plasma cells; Thermo Scientific), anti-CD21 (profes- protocols for allo-HSCT differed by individual in the present sional antigen-presenting cells; DAKO Denmark A/S), anti- study (Tables 1 and 2). CD1a (professional antigen-presenting cells; Immunotech, Marseilles, France), anti–myelin basic protein (a myelin marker; ␤ HISTOPATHOLOGICAL AND Novocastra, New Castle Upon Tyne, England), and mouse anti- IMMUNOHISTOCHEMISTRY STUDIES amyloid precursor protein (a marker of axonal injury; Milli- pore, Billerica, Massachusetts). Double immunofluorescence staining was performed with primary antibodies against my- The postmortem delay was less than 24 hours in all cases. All elin basic protein and CD68 to detect myelin degradation prod- brain tissue was fixed in 10% formalin for at least 1 week. Neu- ucts in macrophages. ropathological examination was performed on sections of the The MS lesions were histopathologically classified using a cerebral hemispheres, brainstem, and cerebrum ranging from modification of the Bö-Trapp staging system17,18 into active, 0.5 to 1.0 cm in thickness. The tissue blocks containing MS chronic active, and chronic inactive lesions. lesions and control areas were sampled from various brain regions. In the non-MS cases, 3 sampling blocks were taken from the frontal lobe, brainstem, and temporal lobe (or basal gan- QUANTITATIVE AND SEMIQUANTITATIVE glia, depending on the availability) in the present study. The ANALYSES OF INFLAMMATORY ACTIVITY tissue samples were embedded in paraffin, cut into 6-µm- thick tissue sections, deparaffinized, and stained with hema- To assess inflammatory activity, the numbers of CD3ϩ and CD8ϩ toxylin-eosin and Luxol fast blue to locate the lesions. T cells were counted in a high-power field (original magnifi-

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 cation ϫ200, defined as 0.79 mm2), with separate counting in the perivascular and parenchymal areas. The frequency of CD68ϩ A B microglia/macrophages was scored as follows: 0 indicates none; 1, rare; 2, scattered; and 3, frequent per high-power field. For counting or scoring in the perivascular areas, the fields containing the vessels larger than 300 µm were excluded. The val- ues represent sums of 10 consecutive high-power microscopic fields.

STATISTICAL ANALYSIS C D E

We used the Mann-Whitney test to assess the difference in various measures of inflammation between groups. PϽ.05 was re- garded as significant.

RESULTS

The clinical features of the MS patients who underwent F allo-HSCT are summarized in Table 1. After allo-HSCT, MS in 1 patient progressed clinically (represented by a change in the Expanded Disability Status Scale score from 2.0 to 3.0)15; 2 MS patients did not have information of assessments specifically for MS in their medical records; and the last patient had no clinical documentation of MS as a concurrent condition (MS was an incidental neuropathological finding). Besides the MS lesions, histopathological examinations revealed no other abnormality in case 1, disclosed multiple acute and chronic infarcts in cases 2 and 3, and found a focus of leukoencephalopathy in Figure 2. Demyelinating and inflammatory activity in a patient with multiple case 4. The non-MS patients who underwent allo-HSCT sclerosis (MS) (case 3 in Table 1) 9 months after allogeneic hematopoietic and the controls died of various causes (Table 2), but the stem cell transplantation. A chronic active lesion in the periventricular white routine neuropathological examination exhibited nor- matter shows a hypocellular core and inflammatory/demyelinating edge (A) (Luxol fast blue, original magnification ϫ400) containing abundant CD68ϩ mal brain and spinal cord. macrophages/microglia (brown profiles) (B) (hematoxylin-eosin [blue], original magnification ϫ400), scattered CD3ϩ T cells (brown) (C) DEMYELINATING ACTIVITY IN MS PATIENTS (hematoxylin-eosin [blue], original magnification ϫ400), CD8ϩ T cells (brown) (D) (hematoxylin-eosin [blue], original magnification ϫ400), and WHO UNDERWENT ALLO-HSCT occasional acutely damaged axons immunoreactive for amyloid precursor protein (brown) (E) (hematoxylin-eosin [blue], original magnification ϫ400). All 4 MS patients showed varying degrees of demyelinat- Double immunofluorescence staining reveals colocalization of myelin with ϩ ing activity, that is, with active lesions (case 1 in Table 1) CD68 macrophages/microglia, indicative of ongoing demyelination at the edge of this MS lesion (F) (myelin basic protein [green]/CD68 [red], original and/or chronic active lesions (cases 1-4 in Table 1). Ac- magnification ϫ400). tive lesions exhibited demyelinating activity throughout the lesions, containing abundant macrophages/microglia (Figure 1A and B) and frequent T cells.15 Chronic active sions predominantly at their edges (Figures 1D and 2). lesions were hypocellular in their cores but inflammatory Inflammatory cells were mainly macrophages/microglia and demyelinating at their edges, with frequent macro- immunoreactive for CD68 (Figures 1A and B and 2B and Figure 2 F); T cells (lymphocytes) were variably immunoreac- phages/microglia and T cells (Figure 1D and ). 15 7,19 tive for CD3, CD4, and CD8 (Figure 2C and D). These The demyelinating activity was demonstrated by di- ϩ ϩ minishing intensity of Luxol fast blue staining (Figures 1A lesions had rare CD138 plasma cells but no CD20 B and 2A) and macrophages phagocytosing myelin debris cells. The markers for professional antigen-presenting (Figure 1A) and was confirmed by colocalization of my- cells, CD1a and CD21, yielded negative results. elin basic protein with the CD68-immunoreactive mac- The analysis of 13 lesions (active or chronic active) with rophages on double immunofluorescence staining inflammatory/demyelinating activities disclosed that the (Figures 1B and 2F). Focal remyelination was suggested lesions with the activities had significantly higher numbers of CD3ϩ T cells and CD8ϩ cytotoxic T cells, as well in some of the chronic active lesions (Figure 1D). Rela- ϩ tively mild axonal injury was associated with the demy- as significantly higher scores of CD68 microglia/macro- elinating activity and shown by immunoreactivity for amy- phages than chronic inactive lesions (n=13) or normal- loid precursor protein (Figures 1C and 2E). appearing white matter (n=14) from the same patients (PϽ.001; Table 3). INFLAMMATORY ACTIVITY One MS patient (case 4 in Table 1) had focal leukoen- IN MS PATIENTS WITH ALLO-HSCT cephalopathy mainly in the posterior cerebral hemispheres, which was presumably owing to immunosup- Inflammatory activity was identified throughout the ac- pressant treatment with methotrexate sodium and/or tive lesions (Figure 1A and B)15 and in chronic active le- cyclosporine.20,21 This additional leukoencephalopathy was

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Median (Range) No. of Cells

CD3؉ T Cells CD8؉ T Cells CD68؉ Microglia/Macrophagesa

Lesion Type Parenchyma Perivascular Parenchyma Perivascular Parenchyma Perivascular Active (n=13)b 50 (10-134)c 62 (23-129)d 42 (12-133)c 49 (15-130)c 26 (22-30)c 19 (15-27)c Inactive (n=13) 15 (6-41) 28 (12-65) 16 (3-41) 21 (9-36) 18 (12-25) 12 (3-17) NAWM (n=14) 6 (0-26) 16 (5-32) 6 (3-25) 12 (5-37) 19 (14-22) 14 (8-16)

Abbreviations: Allo-HSCT, allogeneic hematopoietic stem cell transplantation; MS, multiple sclerosis; NAWM, normal-appearing white matter from the same patients. a Frequency of CD68ϩ microglia/macrophages was scored from 0 to 3 by summing 10 consecutive high-power (ϫ200) microscopic fields. b PϽ.001 by Mann-Whitney test vs NAWM. c PϽ.001 by Mann-Whitney test vs inactive. d PϽ.01 by Mann-Whitney test vs inactive.

and 3 chronic inactive lesions) apart from the foci of leu- A B koencephalopathy were included in the present analysis; the other lesions abutting the focus of leukoencephalopathy were excluded.

INFLAMMATION IN NON-MS PATIENTS WHO UNDERWENT ALLO-HSCT

Because persistent inflammatory/demyelinating activities C D of MS were present after allo-HSCT, further investigation was focused on whether inflammation/demyelination can result from allo-HSCT in the absence of MS. Five neuropathologically normal non-MS allo-HSCT recipients were examined (Figure 3A-C, E, and G) and compared with 5 neuropathologically normal subjects who did not re- ceive allo-HSCT (Figure 3D, F, and H). The analysis of 3 brain regions in each subject demonstrated that the pa- E F tients who underwent allo-HSCT had significantly higher numbers of CD3ϩ (Figure 3C) and CD8ϩ (Figure 3E) cytotoxic T cells and higher scores of CD68ϩ microglia/ macrophages (Figure 3G) compared with the controls (Figure 3D, F, and H, respectively; PϽ.05) in both the parenchyma and perivascular areas (except for CD8ϩ cytotoxic T cells in perivascular areas; Table 4). However, no obvious demyelination was identified by the Luxol fast blue stain (Figure 3B) or by double immunostains of my- G H elin basic protein/CD68 in these non-MS brains that did not undergo allo-HSCT (not shown).

COMMENT

The present study is, to our knowledge, the first to ex- amine CNS histopathological findings in MS patients af- Figure 3. Mild inflammation in normal-appearing brains of the patients ter allo-HSCT. Despite the procedure, active demyelin- without multiple sclerosis after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The white matter in the frontal lobe appears ation and inflammation persisted, indicating the failure normal on routine microscopic examination (A and B) (hematoxylin-eosin of allo-HSCT to halt the MS disease activity, at least dur- and Luxol fast blue, respectively, original magnification ϫ200) but exhibits ing the follow-up period in these patients. This failure increased numbers of CD68ϩ macrophages/microglia (brown profiles) (C), ϩ ϩ may be attributed to several possibilities. CD3 T cells (brown) (E), and CD8 T cells (brown) (G), compared with the control subjects without allo-HSCT (D, F, and H, respectively) (for C-H, First, the persistence of recipient immune cells in the hematoxylin-eosin [blue], original magnification ϫ400). The inset in C brain may fuel the MS activities. This possibility is sup- demonstrates the morphological features of immunoreactive cells in higher ϫ ported by our previous finding in which fluorescent in situ detail (original magnification 1000). hybridization analysis of sex chromosomes of cells in a female MS patient who received allo-HSCT from a male do- characteristically necrotic and distinct from the MS le- nor (case 1 in Table 1) revealed that, although only do- sions. In this case, 4 MS lesions (1 chronic active lesion nor blood cells were present in the peripheral circulation,

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Median (Range) No. of Cells

CD3؉ T Cells CD8؉ T Cells CD68؉ Microglia/Macrophagesa

Group Parenchyma Perivascular Parenchyma Perivascular Parenchyma Perivascular Allo-HSCT 35 (3-72)b 35 (2-96)c 11 (1-93)b 18 (2-90)d 19 (11-21)c 11 (5-18)e Controlf 4 (0-14) 18 (4-27) 3 (0-16) 16 (2-38) 11 (10-19) 5 (3-14)

a Frequency of CD68ϩ microglia/macrophages was scored from 0 to 3 by summing 10 consecutive high-power (ϫ200) microscopic fields. b PϽ.001 by Mann-Whitney test vs controls. c PϽ.01 by Mann-Whitney test vs controls. d PϾ.05 by Mann-Whitney test vs controls. e PϽ.05 by Mann-Whitney test vs controls. f Indicates neuropathologically normal subjects who did not undergo allo-HSCT.

most CD45ϩ and CD68ϩ cells within the brain were still Fourth, although the CNS showed no evidence of in- of the female recipient’s origin.15 Although complete chi- fection, a systemic viral infection after allo-HSCT in some merism of circulating leukocytes can be optimally achieved of the patients could trigger the relapse of MS and con- by allo-HSCT, the conditioning immunosuppressive treat- tribute to the progression of MS.31-33 This possibility can- ment may have much less effect on the cells trapped or not be excluded in the present study, although at least resident within the CNS.22-24 In support, Mondria et al25 1 of the patients (case 1 in Table 1) exhibited increased examined 2 markers indicative of lymphocyte activation lesion burden in magnetic resonance images and clinical in the cerebrospinal fluid, sCD27, and oligoclonal IgG progression of MS several weeks before the occurrence of bands, and found that these were still evident 6 to 9 months viral infection.15 after whole-body immune ablation in MS subjects. The au- Finally, the beneficial effect of allo-HSCT may take a thors concluded that complete eradication of activated lym- longer time to manifest in the CNS. Studies on the inter- phocytes from the CNS had not been achieved despite in- mitotic life span of lymphocytes have yielded widely dis- tense immunoablation. parate results. The reason for the discrepancy in these es- Second, GVH reaction could theoretically induce allo- timates is unclear. Most of the studies have demonstrated immune damage to the brain9,10,25 and may complicate the an average life span of months or years for T cells and of pathologic processes of MS after allo-HSCT.26,27 In the pres- several weeks or months for B cells.34-36 In the present study, ent study, all the patients (including the MS patients and the longest survival after allo-HSCT was 9 months (with non-MS patients with normal-appearing brains) devel- a median survival of 4.5 months). It is possible that allo- oped histopathologically confirmed GVH disease after allo- HSCT may not yet exhibit optimal effects on the brains HSCT, involving the skin, gut, or liver. Graft-vs-host dis- by the time of histopathological examination. Neverthe- ease is associated with a cytokine storm and T-cell activation, less, the fatal outcome after allo-HSCT in these MS pa- which induces the complex immune reactions that inter- tients implies that the complications of allo-HSCT (along act with the pathological processes of MS.27,28 The present with its conditioning regimen) and potential progression study demonstrated increased diffuse infiltration of T cells of MS dominate the prognosis of allo-HSCT. and macrophages/microglia within the normal-appearing In comparison with autologous HSCT, which has been brains of non-MS patients after allo-HSCT, which would reported to suppress the inflammatory activity of MS,7 allo- be consistent with the GVH reaction after allo-HSCT. Nev- HSCT in our patients failed to halt the demyelinating and ertheless, the contribution of GVH disease to the demy- inflammatory activities of MS. Because the conditioning elinating activity of MS may be minor in the present study treatment of allo-HSCT is similar to that of autologous because (1) at least 1 patient (case 1 in Table 1) showed HSCT, the difference in the effect on MS inflammatory ac- clinical evidence of MS progression before the develop- tivity between the 2 kinds of transplantation presumably ment of GVH disease15; (2) there was no obvious demy- lies in the nature (autologous vs allogeneic) of the cells trans- elination in the non-MS allo-HSCT recipients, although their planted into the patients. This presumption is compatible brains showed mild and diffuse inflammation; and (3) de- with the possibility (discussed in the preceding para- myelination and neurodegeneration were active even with graphs) that the persistence of inflammation after allo- marked suppression of the inflammatory activity in MS pa- HSCT is likely to result from complications of allo-HSCT, tients who received autologous HSCT.7 especially of GVH reaction. We cannot completely ex- Third, the failure to arrest MS progression has been clude other possibilities, however, such as the difference often attributed to studying patients in whom MS has pro- in the selection of MS patients between the present study gressed into advanced stages.7,29,30 This possibility is un- and the only histopathological study on autologous HSCT.7 likely in the present study because one of our patients Although the non-MS brains that underwent allo- (case 1 in Table 1) had a pretransplantation Expanded HSCT contained diffuse but mild inflammation, no ob- Disability Status Scale score of 2.0 and another patient vious demyelination was identified in the present study. (case 4 in Table 1) was asymptomatic before allo-HSCT. The hypotheses for the preservation of myelin sheaths Therefore, the failure of allo-HSCT may occur even in in these brains are the following: (1) possible dissocia- less advanced MS. tion of inflammation with demyelination after HSCT,7

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©2010 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 (2) insufficient severity of inflammatory activity to pro- 6. Van Wijmeersch B, Sprangers B, Dubois B, Waer M, Billiau AD. Autologous and allogeneic hematopoietic stem cell transplantation for multiple sclerosis: per- duce demyelination, (3) individual vulnerabilities in- spective on mechanisms of action. J Neuroimmunol. 2008;197(2):89-98. cluding genetic predisposition,37,38 or (4) different anti- 7. Metz I, Lucchinetti CF, Openshaw H, et al. Autologous haematopoietic stem cell trans- gen specificities of the T cells in the MS vs non-MS cohorts. plantation fails to stop demyelination and neurodegeneration in multiple sclerosis. Brain. 2007;130(pt 5):1254-1262. As well, the CNS milieu of non-MS subjects is likely dif- 8. Griffith LM, Pavletic SZ, Tyndall A, et al; Workshop participants. Feasibility of ferent from that of MS subjects, as evident by increase allogeneic hematopoietic stem cell transplantation for autoimmune disease. Biol in the levels of chemokines, cytokines, free radicals, pro- Blood Marrow Transplant. 2005;11(11):862-870. 9. Padovan CS, Gerbitz A, Sostak P, et al. Cerebral involvement in graft-versus-host teases, and other molecules in the MS brains, and may disease after murine bone marrow transplantation. Neurology. 2001;56(8):1106- not be able to promote the activation and pathogenic po- 1108. tential of infiltrating T cells. Finally, although we did not 10. Siegal D, Keller A, Xu W, et al. Central nervous system complications after allogeneic hematopoietic stem cell transplantation: incidence, manifestations, and look for this, the increased cell content in the normal brain clinical significance. Biol Blood Marrow Transplant. 2007;13(11):1369-1379. after transplant without evidence of damage to histologi- 11. McAllister LD, Beatty PG, Rose J. Allogeneic bone marrow transplant for chronic cal structures may be related to increased regulatory myelogenous leukemia in a patient with multiple sclerosis. Bone Marrow Transplant. 1997;19(4):395-397. T-cell content, which could prevent alloreactivity to brain 12. Mandalfino P, Rice G, Smith A, Klein JL, Rystedt L, Ebers GC. Bone marrow trans- tissues. In transplant models, protection of organs from plantation in multiple sclerosis. J Neurol. 2000;247(9):691-695. alloreactive immune cells is associated with an in- 13. La Nasa G, Littera R, Cocco E, Battistini L, Marrosu MG, Contu L. Allogeneic he- 39,40 matopoietic stem cell transplantation in a patient affected by large granular lym- creased regulatory T-cell content in that organ. phocyte leukemia and multiple sclerosis. Ann Hematol. 2004;83(6):403-405. In conclusion, the demyelinating and inflammatory 14. Jeffery DR. Failure of allogeneic bone marrow transplantation to arrest disease activities of MS persist after allo-HSCT. The demyelin- activity in multiple sclerosis. Mult Scler. 2007;13(8):1071-1075. 15. Lu JQ, Storek J, Metz L, et al. Continued disease activity in a patient with multiple ating activity is presumably due to the persistence of re- sclerosis after allogeneic hematopoietic cell transplantation. Arch Neurol. 2009; cipient immune cells in the MS brain, whereas the in- 66(1):116-120. flammatory activity is more likely the result of GVH 16. McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis. Ann Neurol. 2001;50(1):121-127. reaction after allo-HSCT. The findings of the present small 17. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, Bo L. Axonal transec- series of MS patients indicate that allo-HSCT fails to stop tion in the lesions of multiple sclerosis. N Engl J Med. 1998;338(5):278-285. 18. van der Valk P, De Groot CJ. Staging of multiple sclerosis (MS) lesions: pathol- the demyelination and inflammation of MS. ogy of the time frame of MS. Neuropathol Appl Neurobiol. 2000;26(1):2-10. 19. Brück W, Porada P, Poser S, et al. Monocyte/macrophage differentiation in early Accepted for Publication: January 11, 2010. multiple sclerosis lesions. Ann Neurol. 1995;38(5):788-796. 20. Rzeski W, Pruskil S, Macke A, et al. Anticancer agents are potent neurotoxins in Correspondence: V. Wee Yong, PhD, Hotchkiss Brain In- vitro and in vivo. Ann Neurol. 2004;56(3):351-360. stitute, University of Calgary, 3330 Hospital Dr, Cal- 21. Erbetta A, Salmaggi A, Sghirlanzoni A, et al. Clinical and radiological features of gary, Alberta, Canada T2N 4N1 ([email protected]). brain neurotoxicity caused by antitumor and immunosuppressant treatments. Neurol Sci. 2008;29(3):131-137. Author Contributions: Study concept and design: Lu, Nash, 22. Motl S, Zhuang Y, Waters CM, Stewart CF. Pharmacokinetic considerations in Storek, and Yong. Acquisition of data: Lu, Stevens, Clark, the treatment of CNS tumours. Clin Pharmacokinet. 2006;45(9):871-903. Johnson, and Yong. Analysis and interpretation of data: 23. Coisne C, Lyck R, Engelhardt B. Therapeutic targeting of leukocyte trafficking across the blood-brain barrier. Inflamm Allergy Drug Targets. 2007;6(4):210-222. Lu, Joseph, Nash, Stevens, Metz, Clark, Johnson, and 24. Ma M, Barnes G, Pulliam J, Jezek D, Baumann RJ, Berger JR. CNS angiitis in Yong. Drafting of the manuscript: Lu, Joseph, Nash, Stevens, graft vs host disease. Neurology. 2002;59(12):1994-1997. and Yong. Critical revision of the manuscript for impor- 25. Mondria T, Lamers CH, te Boekhorst PA, Gratama JW, Hintzen RQ. Bone-marrow transplantation fails to halt intrathecal lymphocyte activation in multiple sclerosis. tant intellectual content: Joseph, Nash, Storek, Stevens, J Neurol Neurosurg Psychiatry. 2008;79(9):1013-1015. Metz, Clark, Johnson, and Yong. Statistical analysis: Lu. 26. Welniak LA, Blazar BR, Murphy WJ. Immunobiology of allogeneic hematopoi- Obtained funding: Yong. Administrative, technical, and ma- etic stem cell transplantation. Annu Rev Immunol. 2007;25:139-170. 27. Duran-Struuck R, Reddy P. Biological advances in acute graft-versus-host dis- terial support: Joseph, Johnson, and Yong. Study super- ease after allogeneic hematopoietic stem cell transplantation. Transplantation. vision: Joseph, Nash, Storek, Clark, and Yong. 2008;85(3):303-308. Financial Disclosure: None reported. 28. Openshaw H, Stuve O, Antel JP, et al. Multiple sclerosis flares associated with re- combinant granulocyte colony-stimulating factor. Neurology. 2000;54(11):2147- Funding/Support: This study was supported by an op- 2150. erating grant from the Canadian Institutes of Health Re- 29. Nash RA, Stuve O, Bowen JD, et al. Autologous HSCT for advanced MS. Brain. 2008; search (Dr Yong) and in part by grant PP1251 from the 131(pt 2):e89. doi:10.1093/brain/awm180. 30. Metz I, Lucchinetti CF, Openshaw H, et al. Autologous hematopoietic stem cell National Multiple Sclerosis Society (Dr Nash). Dr Yong transplantation. Brain. 2008;131(pt 2):e90. doi:10.1093/brain/awm181. is a Canada Research Chair (tier 1) in neuroimmunol- 31. Andersen O, Lygner PE, Bergstrom T, Andersson M, Vahlne A. Viral infections trig- ogy and an Alberta Heritage Foundation for Medical ger multiple sclerosis relapses. J Neurol. 1993;240(7):417-422. 32. Monteyne P, Bureau JF, Brahic M. Viruses and multiple sclerosis. Curr Opin Neurol. Research (AHFMR) scientist. Dr Storek is a Canada Re- 1998;11(4):287-291. search Chair (tier 2) in transplant immunology and an 33. Steiner I, Nisipianu P, Wirguin I. Infection and the etiology and pathogenesis of multiple sclerosis. Curr Neurol Neurosci Rep. 2001;1(3):271-276. AHFMR clinical investigator. 34. Mackay CR, Marston WL, Dudler L. Naive and memory T cells show distinct pathways of lymphocyte recirculation. J Exp Med. 1990;171(3):801-817. REFERENCES 35. von Boehmer H, Hafen K. 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