T cell-depleted autologous hematopoietic stem cell transplantation for ...

Bone Marrow Transplantation, (1998) 21, 537–541  1998 Stockton Press All rights reserved 0268–3369/98 $12.00

T cell-depleted autologous hematopoietic stem cell transplantation for multiple sclerosis: report on the first three patients RK Burt1, AE Traynor1, B Cohen2, KH Karlin3, FA Davis3, D Stefoski3, C Terry4, L Lobeck4, EJ Russell5, C Goolsby1, S Rosen1, LI Gordon1, C Keever-Taylor6 M Brush2, M Fishman2 and WH Burns6 1

Department of Medicine, Division of Hematology/Oncology, 2Department of Neurology, 5Department of Radiology, Northwestern University Medical School and the Robert H Lurie Cancer Center, Chicago; 3Department of Neurology, Rush Presbyterian Medical Center, Chicago, IL; 4Department of Neurology, 6Department of Medicine, Division of Hematology/Oncology, Medical College of Wisconsin, Milwaukee, WI, USA

Summary: Multiple sclerosis (MS) is a disease of the central nervous system characterized by immune-mediated destruction of myelin. In patients with progressive deterioration, we have intensified immunosuppression to the point of myeloablation. Subsequently, a new hematopoietic and immune system is generated by infusion of CD34-positive hematopoietic stem cells (HSC). Three patients with clinical MS and a decline of their Kurtzke extended disability status scale (EDSS) by 1.5 points over the 12 months preceding enrollment and a Kurtzke EDSS of 8.0 at the time of enrollment were treated with hematopoietic stem cell (HSC) transplantation using a myeloablative conditioning regimen of cyclophosphamide (120 mg/kg), methylprednisolone (4 g) and total body irradiation (1200 cGy). Reconstitution of hematopoiesis was achieved with CD34-enriched stem cells. The average time of follow-up is 8 months (range 6–10 months). Despite withdrawal of all immunosuppressive medications, functional improvements have occurred in all three patients. We conclude that T cell-depleted hematopoietic stem cell transplantation can be performed safely in patients with severe and debilitating multiple sclerosis. Stem cell transplantation has resulted in modest neurologic improvements for the first time since onset of progressive disease although no significant changes in EDSS or NRS scales are evident at this time. Keywords: T cell depletion; autologous transplant; multiple sclerosis

Multiple sclerosis (MS) is a demyelinating disease of the central nervous system. The disease has relatively predictable clinical patterns which include relapsing remitting, primary progressive or secondary progressive. Immunemediated damage of central myelin is implicated in the pathogenesis, although the initiating events are unknown. Correspondence: Dr RK Burt, Allogeneic Bone Marrow Transplantation, Wesley Pavilion, Room 1416, 250 East Superior Street, Chicago, Illinois 60611-2950, USA Received 1 October 1997; accepted 5 November 1997

Acute MS plaques are characterized by infiltration with lymphocytes and macrophages.1,2 Because these cells arise from bone marrow progenitor cells, complete destruction of the immune system followed by hematopoietic progenitor (CD34+) cell rescue has been previously suggested or reported as therapy for multiple sclerosis.3–5 The CD34+ hematopoietic stem cells will proliferate and differentiate not only into new platelets, red blood cells and neutrophils but also into lymphocytes and macrophages. In an animal model of MS, experimental autoimmune encephalomyelitis, immune ablation and syngeneic bone marrow transplantation is capable of ameliorating disease.6–8

Methods Patient eligibility Patients with clinically definite MS are considered candidates if they fulfil all of the following: (1) age less than 55 at time of pretransplant evaluation; (2) failure to stabilize active clinical progression with intravenous methylprednisolone given for a minimum of 3 days at 1 g per day; (3) a Kurtzke EDSS of 5.0 to 8.0 with an increase in the EDSS by 1.5 points over the preceding 12 months in patients with an EDSS of 5.5 or less at the start of the evaluation period, or an increase of 1 point in patients with an EDSS of 6 or greater at the start of the evaluation period. Clinically definite MS is defined as a clinical diagnosis of MS by two independent neurologists supported by characteristic MRI changes and absence of serologic or clinical signs of other autoimmune diseases. If approved by the onsite neurologist and hematologist, the patient’s records and MRIs are forwarded to advisory panels at the National Institutes of Health and the University of Texas which must unanimously agree that the patient meets criteria before being enrolled. The protocol is approved and monitored by the institutional IRBs and the United States Food and Drug Administration. Hematopoietic stem cell collection Bone marrow is harvested from the posterior iliac crests under general anesthesia in the operating room. Peripheral

T cell-depleted autologous HSC transplant for MS RK Burt et al

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Definition of disease status

blood stem cells (PBSC) are mobilized with G-CSF (Amgen, Thousand Oaks, CA, USA) using 10 ␮g/kg subcutaneous administration daily for 5 or 6 days. On the 5th and 6th day, a 15 l leukapheresis using a continuous flow blood cell separator (Fenwal CS 3000 plus, Baxter, Deerfield, IL, USA) is performed to supplement the enriched CD34+ cell count to more than 2.0 × 106 CD34+ cells/kg. Depletion of lymphocytes is by CD34+ enrichment using the CEPRATE SC Stem Cell Concentrator (CellPro, Bothell, WA, USA).

Improvement is defined as a decrease in the Kurtzke EDSS9 by at least one point and increase in the Scripps NRS10 by at least 10 points. Deterioration is defined as an increase in the Kurtzke EDSS by at least one point or decrease in the Scripps NRS by at least 10 points. Stabilization of active disease is defined as absence of any new or progressive neurologic deficits and no significant change in the EDSS or NRS scores.

Conditioning regimen and supportive care

Results

Cyclophosphamide (120 mg/kg) in divided doses of 60 mg/kg/day is given intravenously over 2 h on days ⫺6 and ⫺5. Total body irradiation is given as 1200 cGy divided 150 cGy twice a day on days ⫺4, ⫺3, ⫺2 and ⫺1 in the AP/PA position with 50% lung and 30% kidney and right lobe of the liver transmission blocks. One gram of methylprednisolone is given intravenously on days ⫺4, ⫺3, ⫺2 and ⫺1. G-CSF (Amgen) is given at a dose of 5 ␮g/kg/day subcutaneously starting the day of stem cell reinfusion and continued until the absolute neutrophil is greater than 1000/␮l for 3 consecutive days.

Hematopoietic stem cell collection After CD34+ enrichment, marrow stem cell yield was a median of 1.0 × 106 CD34+ cells/kg. All patients had peripheral blood stem cells collected beginning 5 days after starting G-CSF. One to two 15 l aphereses were sufficient to collect another 1.1 × 106 CD34+ cells/kg after lymphocyte depletion. After enrichment, a median of 8 × 105 CD3+ cells/kg were present which is approximately a two log T cell reduction. Toxicity

MRI MRI studies are performed before enrollment and at approximately 1, 3 and 6 months after treatment, and then yearly. Magnetic resonance imaging with T1 weighted spin echo images are obtained with and without magnetization transfer (MT) suppression, and pre- and post-infusion of 0.1 mmol/kg gadolinium DPTA (magnevist, Berlex Imaging, Wayne, NJ, USA) contrast medium. Proton density and T2 weighted images are acquired using a dual echo turbo (fast) spin echo sequence. A T2 weighted CSF suppressed (fluid attenuated) inversion recovery (FLAIR) acquisition is obtained in a similar fashion.

Pre-transplant G-CSF was well tolerated without exacerbation of neurologic symptoms. Transient elevation of hepatic transaminases (5 × normal) occurred in two patients while receiving G-CSF for stem cell mobilization. These values normalized within 7 days without intervention. The transplant conditioning regimen was surprisingly well tolerated without notable mucositis, nausea, vomiting, liver, pulmonary or renal dysfunction. No neurologic deterioration or exacerbation occurred during high-dose chemotherapy, TBI or G-CSF. WBC engraftment defined as an ANC ⬎ 500/␮l (0.5 × 109/l) and platelet engraftment defined as a platelet count greater than 20 000/␮l (20 × 109/l) occurred on day 11. The median time to hospital discharge was day 14.

Immunologic assays Peripheral blood lymphocytes from each patient are washed and resuspended in RPMI + 15% FBS and plated at 105 cells/well in 96-well, flat-bottom microtiter plates with either 0.04% phytohemagglutinin, or OKT3 (1 ␮g/ml) (Ortho Biotech, Raritan, NJ, USA) and interleukin-2 (Chiron, Emeryville, CA, USA) (100 U/ml). Cultures are incubated for 48–72 h at 37°C in 7.5% CO2, pulsed for the final 18 h with tritiated thymidine (3H-TdR), and incorporation quantitated by liquid scintillation counting. Pre- and post-transplant, two and three color immunophenotyping is performed on EDTA anticoagulated whole blood. The infused stem cell products are assessed by three color immunophenotyping. The panel of fluorescein isothiocyanate (FTIC), phycoerythrin (PE), PE-cyanin 5 (PECy5) or PerCp fluorochromes included antibodies to CD45 and CD34 (Becton Dickinson, San Jose, CA, USA), and CD3, CD4, CD8, CD29, CD45RA, CD19, CD16, and CD56 (Coulter Cytometry, Miami, FL, USA).

Clinical course All three patients were female with an EDSS of 8.0 at the time of enrollment and 8.0 to 8.5 at the time of treatment. The first patient was 43 years old with primary progressive MS, an EDSS of 8.5 and NRS of 25. In 24 months, she progressed from normal to confinement in a wheelchair with severe ataxia, head titubation, disabling hand tremors, urinary urgency, and cognitive impairment characterized by inability to calculate serial sevens. Symptoms had progressed despite previous treatment with oral prednisone, intravenous methylprednisolone, hyperbaric oxygen, interferon-beta and azathioprine. Following BMT, she has demonstrated the first improvement in neurologic disability since disease onset. Her hand tremor has decreased and cognitive abilities have improved. She is now able to perform serial sevens without difficulty. An MRI done 2 months before treatment showed extensive, diffuse, nonenhancing bilateral hemispheric lesions and severe brainstem


white matter loss with confluent lesions affecting the central medulla, pons and bilateral brachium pontis. A second MRI done immediately before starting therapy showed a new large lesion in the left periventricular white matter. No abnormal enhancement was noted. One month post transplant, an MRI showed abnormal enhancement in the left periventricular white matter lesion which was present before transplant. Three months after transplant the previously enhancing white matter lesion decreased 60% in size and no longer enhanced. Repeat MRI 8 months after transplantation is stable with no new lesions and no enhancement. The second patient, 34 years old, had secondary progressive MS. Diagnosis was made 27 months prior to transplantation. During this interval she became nonambulatory, confined to a wheelchair, unable to feed herself due to tremor, and had an indwelling catheter due to urinary incontinence. Neurologic deterioration continued despite therapy with intravenous and oral steroids and interferonbeta. At the time of transplantation, her EDSS was 8.5 and NRS was 30. Following transplantation, neurologic deficits have improved, although her EDSS and NRS are not significantly different. Three months after transplantation, she took 10 steps with bilateral assistance, although after suffering a fall, her response was not sustained. At 6 months, she began to feed herself with assistance. Her bladder sensation improved, and she no longer required an indwelling catheter. Improvement in urinary incontinence occurred without change in medications. Pre-transplant MRI revealed numerous supertentorial white matter lesions several of which enhanced. Post transplantation, there have been no new or enhancing lesions. The third patient is a 33-year-old female who had relapsing remitting MS since 1991 which became secondary progressive in 1993. Her EDSS was 8.0 and NRS was 37. At the time of transplantation, she was in a wheelchair but able to transfer without assistance. Neurologic deficits progressed despite methylprednisolone and interferon-beta. In the 6 months since transplantation, nightly urinary incontinence has resolved despite discontinuation of medications. Although still confined to a wheelchair, upper extremity strength has improved. She is now able to lift her right arm over her head and can now swim 10 laps in an olympicsized pool. Prior to transplantation, she was unable to swim. The pre-transplant MRI revealed numerous brainstem and periventricular lesions with two areas of abnormal enhancement (approximately 5 mm) in the left frontal and parietal hemispheric white matter. The cervical spinal cord appeared atrophic from the C1 to C4 level. After transplantation, MRI enhancement has resolved. No new lesions have appeared. Immune reconstitution assays Mitogen-induced proliferation of peripheral blood lymphocytes using either phytohemagglutinin or OKT3 and IL2 demonstrated diminished responses after transplantation (Figure 1). Two of the three patients had markedly reduced numbers of CD4 and CD8 cells during the first 3 months post transplant and all had inverted CD4/CD8 ratios (Figure 2). CD4+ cells were almost exclusively CD45RA negative

early post transplant and remained mostly CD45RA negative to 6 months in all patients (not shown). Nearly all CD4+ T cells persistently co-expressed CD29, a marker for the helper-inducer subset. Although B cells had reached the normal range for both patients tested at 6 months, it could be shown in one (patient No. 2) that more than 50% of the B cells co-expressed CD5, indicating an immature B cell phenotype. Discussion The conditioning agents of cyclophosphamide and TBI were selected in order to maximize immunosuppression. In addition, TBI could penetrate to lymphocytes sequestered within the central nervous system without regard for permeability of the blood–brain barrier. However, in conceptualizing this therapy, there were several concerns. It was unclear whether patients with MS could tolerate myeloablative doses of TBI and immune ablative doses of cyclophosphamide without exacerbation of neurologic deficits. Independent of the conditioning regimen, if MS is due to an ongoing occult viral infection, total immune ablation could have deleterious effects by enhancing viral replication and aggravating the clinical disease. Finally, damage to the blood–brain barrier from the conditioning regimen may increase lymphocyte trafficking to the CNS which could also exacerbate immune-mediated damage. However, all three patients tolerated treatment without adverse neurologic events. Both bone marrow and peripheral blood stem cell sources were used for hematologic reconstitution because the marrow stem cell yield after CD34+ enrichment was low in all three patients. The stem cells were enriched for CD34+ cells in order to decrease reinfusion of potential disease-causing lymphocytes. With the CEPRATE column, a 2.0 log reduction of lymphocytes was obtained. In these initial cases, more aggressive lymphocyte depletion was not attempted. Although of potential benefit in purging any lymphocytes already primed and reactive to myelin, more aggressive lymphocyte depletion could increase subsequent viral and fungal infections even after adequate neutrophil recovery. An autologous stem cell source was selected over allogeneic stem cells from an unaffected sibling due to the lower mortality and morbidity of an autologous compared to allogeneic transplant. In addition, there is no method available to guarantee that a clinically asymptomatic sibling does not have subclinical MS or will not eventually develop multiple sclerosis. Indeed, asymptomatic siblings of patients with MS may have subclinical disease on MRI.11 MRI was used to follow disease activity since gadolinium enhancement signifies disruption of the blood–brain barrier in areas of active disease.12–14 However, breakdown in vascular endothelial cell integrity in the first few weeks after TBI and high-dose cyclophosphamide could result in enhancement without immune-mediated inflammation. Thus, gadolinium enhancement shortly after BMT may not necessarily indicate active disease. Future monitoring with serial MRI scans in these patients should clarify whether active MS persists. Determination of IgG synthesis rates by

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Figure 1 Mitogen-induced proliferative responses of peripheral blood performed before hematopoietic stem cell transplantation and at 1, 3 and 6 months after transplantation. 3H-TdR = tritiated thymidine, c.p.m. = counts per minute, IL-2 = interleukin-2, PHA = phytohemagglutinin.

evaluation of cerebral spinal fluid albumin and immunoglobulin in relation to levels in serum may also help in distinguishing blood–brain barrier breakdown due to the conditioning regimen from active disease. To evaluate immunologic function after T cell-depleted stem cell transplantation, peripheral lymphocytes were evaluated for mitogen-induced proliferation and flow cytometric surface membrane characteristics. As is the case after allogeneic and autologous BMT for hematologic or oncologic diseases, an immunosuppressive phenotype with diminished mitogen responses predominated.15,16 This can be expected to be present for at least 12–18 months after transplantation.15,16 The absence of naive T cells (CD45RA+) until at least 3 months after treatment suggests that immediately after transplantation, immune recovery may be regenerated by peripheral expansion of memory lymphocytes infused with the graft. Whether skewing of the peripheral T cell receptor repertoire in the early posttransplant course is associated with early neurologic outcome is under investigation. Multiple sclerosis has a variable relapse and progression rate and fluctuating clinical course with spontaneous remissions which could limit the determination of treatment

efficacy. To prevent inherent bias in outcome due to spontaneous fluctuation of disease, we chose patients with severe impairments who had significant and progressive deterioration in their baseline neurologic function for the 12 months prior to enrollment. We also terminated all immunosuppressive medications after transplantation. In the short interval following transplantation, no new neurologic deficits or symptoms have occurred and all three patients have had some neurologic improvements. The EDSS in its upper scoring range is heavily weighted by ambulation and is insensitive to other changes. Therefore, despite these improvements, the patients’ extended disability status scales have remained unchanged. In summary, hematopoietic stem cell transplantation using high-dose cyclophosphamide and TBI may be performed with minimal nonhematopoietic toxicity in patients with MS. In patients who have rapid progressive deterioration that is unresponsive to standard therapy and who have severe neurologic deficits, stem cell transplantation has resulted not only in stabilization of disease but also mild to modest neurologic improvements. This has occurred despite stopping all immunosuppressive and immune modulating medications. In patients with chronic


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Months post transplant Figure 2 Shown are the absolute cell counts of lymphocyte subsets for the three transplanted patients. The first testing was performed on peripheral blood from all three patients between 1.5 and 3 months post transplant. Patient 1 (closed circle) and patient 2 (closed square) were tested a second time at 5.5 and 6.0 months, respectively. Normal control ranges for each subset are shown by the large open diamonds.

and progressive disease, further clinical improvement may be hindered by glial scarring and axonal degeneration. Only long-term observation will determine if stem cell transplantation can induce durable stabilization of the disease course and prevent further neurologic deterioration, consistent with the arrest of progressive MS. We are continuing to follow these patients to document immune regeneration and its association with disease course.

References 1 Katz D, Taubenberger JK, Cannella B et al. Correlation between magnetic resonance imaging findings and lesion development in chronic active multiple sclerosis. Ann Neurol 1993; 34: 661–669. 2 Prineas JW, Barnard RO, Revesz T et al. Multiple sclerosis. Pathology of recurrent lesions. Brain 1993; 116: 681–693. 3 Burt RK, Burns W, Hess A. Bone marrow transplantation for multiple sclerosis. Bone Marrow Transplant 1995; 16: 1–6. 4 Marmont A, Tyndall A, Gratwohl A et al. Haematopoietic precursor-cell transplants for autoimmune diseases. Lancet 1995; 345: 978. 5 Fassas A, Annagnostopoulos A, Kazis A et al. Peripheral blood stem cell transplantation in the treatment of progressive multiple sclerosis: first results of a pilot study. Bone Marrow Transplant 1997; 20: 631–638. 6 Karussis DM, Vaurka-Karussis U, Lehmann D et al. Prevention and reversal of adoptively transferred, chronic relapsing experimental autoimmune encephalomyelitis with a single high dose cytoreductive treatment followed by syngeneic bone marrow transplantation. J Clin Invest 1993; 92: 765–772.

7 Burt RK, Hess A, Burns W et al. Syngeneic bone marrow transplantation eliminates v␤8.2T lymphocytes from the spinal cord of Lewis rats with experimental allergic encephalomyelitis. J Neurosci Res 1995; 41: 526–531. 8 Burt RK, Padilla J, Dal Canto MC, Miller SD. Remission of relapsing experimental autoimmune encephalomyelitis by immune ablation and bone marrow transplantation. Blood 1996; 88 (Suppl. 1): 598a. 9 Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology (Cleveland) 1983; 33: 1444–1452. 10 Sipe JC, Knobler RL, Braheny SL et al. A neurologic rating scale (NRS) for use in multiple sclerosis. Neurology (Cleveland) 1994; 34: 1368–1372. 11 Tienari PJ, Salonen O, Wikstrom J et al. Familial multiple sclerosis: MRI findings in clinically affected and unaffected siblings. J Neurol Neurosurg Psychiat 1992; 55: 883–886. 12 Miller DH. Magnetic resonance in monitoring the treatment of multiple sclerosis. Ann Neurol 1994; 36 (Suppl.): S91–S94. 13 Frank JA, Stone LA, Smith ME et al. Serial contrast-enhanced magnetic resonance imaging in patients with early relapsingremitting multiple sclerosis: implications for treatment trials. Ann Neurol 1994; 36 (Suppl.): S86–S90. 14 Francis GS, Evans AC, Aronold DL. Neuroimaging in multiple sclerosis. Neurol Clin 1995; 13: 147–171. 15 Forman SJ, Nocker P, Gallagher M. Pattern of T cell reconstitution following allogeneic bone marrow transplantation for acute hematologic malignancy. Transplantation 1982; 34: 96–98. 16 Olsen GA, Gockerman JP, Bast RC et al. Altered immunologic reconstitution after standard dose chemotherapy or high dose chemotherapy with autologous bone marrow support. Transplantation 1988; 46: 57–60.