Non-myeloablative stem cell transplantation High stem cell dose will ...

Bone Marrow Transplantation (2002) 30, 267–271  2002 Nature Publishing Group All rights reserved 0268–3369/02 $25.00 www.nature.com/bmt

Non-myeloablative stem cell transplantation High stem cell dose will not compensate for T cell depletion in allogeneic non-myeloablative stem cell transplantation JR Passweg, S Meyer-Monard, M Gregor, G Favre, D Heim, M Ebnoether, A Tichelli and A Gratwohl Division of Hematology, Department of Internal Medicine, Basel University Hospital, Switzerland

Summary: The best strategies for non-myeloablative stem cell transplants (NST) are not known. We hypothesized that a high stem cell dose and post-transplant donor lymphocyte infusions (DLI) in a T cell-depleted NST setting may result in stable engraftment without severe GvHD. We used conditioning with 200 mg/kg cyclophosphamide, and ATG, a high peripheral stem cell dose of ⬎10 ⴛ 106 CD34+ cells/kg, T cell-depleted to ⬍1 ⴛ 105 CD3+ cells/kg followed by incremental DLI. Ten patients, 53 (42–61) years of age with hematological malignancy (CML in 3, MDS in 2, myeloma in 3 and CLL in 2) were included. All patients achieved initial engraftment, at a median 13.5 (10–20) days. Three patients achieved complete chimerism, four achieved a complete hematologic remission. In seven patients the graft ultimately failed. Acute GvHD grade II was seen in three patients after DLI. At a median follow-up of 28 months (range 15–35), eight patients are alive, none died of treatment-related complications. NST with T cell depletion to prevent GVHD results in a high graft failure rate. High stem cell dose (⭓10 ⴛ 106 CD34+ cells/kg) and post-transplant DLI will not compensate for the lack of T cells to ensure stable engraftment. Bone Marrow Transplantation (2002) 30, 267–271. doi:10.1038/sj.bmt.1703671 Keywords: stem cell dose; CD34+ cell dose; engraftment; T cell depletion; non-myeloablative stem cell transplant

Standard allogeneic hemopoetic stem cell transplants (HSCT), based on maximally tolerated doses of chemo- and radiotherapy, induce long-lasting bone marrow aplasia, are associated with considerable toxicity and are usually not considered in patients older than 50 years of age or patients with comorbidities.1,2 Alloreactivity of donor immune cells is essential for eradication of host tumor, ie the graft-versus-leukemia (GVL) effect. Donor lymphocyte infusions (DLI) were used Correspondence: Dr JR Passweg, Department of Internal Medicine, Kantonsspital Basel, Petersgraben 4, CH-4031, Basel, Switzerland Received 24 December 2001; accepted 11 June 2002

successfully for the treatment of relapsed leukemia after HSCT, providing direct evidence of a GVL effect of DLI.3 Advances in DLI have led to the development of non-myeloablative stem cell transplantation (NST), several groups4–14 have shown that engraftment after NST is feasible. In addition to regimen-related toxicities, graft-versus-host disease (GVHD) limits the widespread use of allogeneic HSCT. GVHD is in part induced by cytokine release related to the toxicity of the preparative regimen.15 Low intensity conditioning may result in a lower incidence and severity of GVHD. T cell depletion is effective for GVHD prophylaxis. T cell depletion is associated with a significant increase in leukemia relapse and graft rejection and has therefore not been adopted widely.16 Data from animal experiments and in vitro studies17,18 and from human transplantation19–25 have addressed the impact of stem cell dose. Previously, the achievable stem cell dose was limited by the amount of bone marrow that could be harvested. This has changed in the current era of allogeneic peripheral blood stem cell transplantation.26 This effect of high stem cell doses has been exploited in haplo-identical HSCT, where the term ‘mega-dose’ effect has been used. A high stem cell dose may compensate for the effects of T cell depletion. We used the cyclophosphamide + ATG low intensity conditioning regimen that has been studied extensively in the past for bone marrow failure states.27 We hypothesized, that a high stem cell dose supported by incremental doses of DLI, may promote engraftment, and maintain graft-versus-leukemia effects despite T cell depletion in a NST protocol using cyclophosphamide + ATG for conditioning. We report the results of this pilot study.

Patients and methods Study design This was a pilot study to test the aforementioned hypothesis. A predetermined number of 10 patients with hematologic malignancy were to be enrolled. Informed consent was obtained from patients and donors. Outcomes analyzed included engraftment, acute and chronic GVHD, tumor response and survival. Engraftment was defined as reaching 0.5 ⫻ 109/l neutrophils stable over 3 days, after a period of

High stem cell dose will not compensate for T Cell depletion JR Passweg et al.

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aplasia. The modified Glucksberg criteria were used to score acute graft-versus-host disease (GVHD). Short tandem repeat-RT-PCR analysis was used for monitoring posttransplant whole blood chimerism. Amplified alleles were separated by capillary electrophoresis and measured peak surface area was used to quantitate chimerism. Patients Characteristics of the 10 patients enrolled in this protocol are listed in Table 1. Six were male, median age was 53 years (range 42–61). The original disease was CML in three, MDS in two, multiple myeloma in three, and CLL in two. All had relative contraindications to standard transplants. Donors were HLA-identical siblings in nine and a one-antigen mismatched sibling in one. Methods Peripheral stem cells were mobilized by daily subcutaneous injection of 10 ␮g/kg of G-CSF. Donors underwent 15–25 l apheresis on days 5–7 of mobilization. The goal was to collect ⬎10 ⫻ 106 CD34+ cells/kg. Assuming a 50% loss with stem cell manipulation, ⬎20 ⫻ 106 CD34+ cells/kg were collected. If this goal could not be reached a second mobilization was performed.28 The apheresis product was T cell depleted using CD34+ positive selection followed by CD4/CD8 depletion steps (Isolex 300i, Baxter, Deerfield, IL, USA) to achieve ⬍1 ⫻ 105 CD3+ cells/kg. The final product was cryopreserved using 10% DMSO cryoprotectant and stored in the vapor phase of a liquid nitrogen tank at ⫺196°C. Pretransplant conditioning was 200 mg/kg cyclophosphamide divided in four daily doses and ATG 90 mg/kg (Lymphoglobuline; Sangstat, Lyon, France) over 3 days. The transplants were thawed at the bedside and infused rapidly on day 0. Cyclosporine was used to promote engraftment and tapered rapidly after day 14. Incremental doses of DLI were given starting on day 30 and continued at monthly intervals using the following scheme: 106/kg, 106/kg, 107/kg, 107/kg, 108/kg, 108/kg. DLI were discon-

Table 1

Patient and disease characteristics

UPN Disease Disease stage

Sex Age Donor match CD34+ CD3+ cell and sex cell dose dose (⫻106/kg) (⫻105/kg)

735 742 749 751 763 770 782 799 819 843

F M M F F M M F M M

MDS CML CLL MM CLL MDS CML MM CML MM

RAEB AP III III III RAEBt AP III AP III

56 53 49 58 50 59 42 61 50 53

id sib/m id sib/f id sib/f id sib/f id sib/f id sib/f 1mm sib/f id sib/f id sib/m id sib/m

11.7 12.2 13.7 10.9 14.3 10.7 13.2 20.2 9.8 11.0

0.55 0.47 0.17 0.18 0.20 0.09 0.35 0.09 0.89 0.61

MDS = myelodysplastic syndrome; CML = chronic myeloid leukemia; CLL = chronic lymphoid leukemia; MM = multiple myeloma; RAEB = refractory anemia with excess of blasts; ap = accelerated phase, id sib = HLA identical sibling, 1 mm sib = HLA one antigen mismatched sibling. Bone Marrow Transplantation

tinued upon evidence of GVHD, or at the time of graft failure. Patients who had lost their grafts by day 30 did not receive DLI. Patients with graft failure were offered a second transplant using a T cell non-depleted NST protocol with TBI 2 Gy and fludarabine 3 ⫻ 30 mg/kg5 for conditioning and cyclosporine (for 35 days) and mycophenolate mofetil (for 28 days) for GVHD prophylaxis. Results Feasibility The median number of CD34+ cells was 11.95 (9.8– 20.2) ⫻ 106 cells/kg; the targeted stem cell dose of 10 ⫻ 106 CD34+ cells/kg was reached in all but one patient. The median number of CD3+ cells was 0.28 (0.09– 0.89) ⫻ 105 CD3+ cells/kg. The goal of T cell depletion to ⬍1 ⫻ 105 CD3 cells/kg was reached in all patients. Engraftment All patients went through a phase of aplasia and had initial neutrophil recovery, reaching 0.5 ⫻ 109/l neutrophils at a median 13.5 (10–20) days with documented mixed chimerism on day 14. Two patients had lost their grafts by day 30 and did not receive DLI. In spite of DLI given in incremental doses to 8/10 patients, graft failure occurred in five of these eight patients. Graft loss was a slow process in some patients with slowly decreasing amounts of donor alleles over a time period of up to 6 months. Three patients reversed to full donor chimeras after DLI. Details of quantitative chimerism are shown in Table 2. Outcome Outcome details are shown in Table 3. Acute GVHD grade I was seen in one patient occurring in the early post-transplant phase, three additional patients developed grade II GVHD after DLI. Those three patients achieved a state of complete chimerism, after DLI. None of the seven patients without acute GVHD achieved a complete chimerism (P = 0.008 by Fisher’s exact test). Four patients achieved a complete hematologic remission, three patients with stable engraftment and GVHD and one additional patient with CLL in spite of graft failure after conditioning with cyclophosphamide and ATG. Two of the responding patients relapsed, one died of rapidly progressive myeloma, one patient with CNS relapse was put back into remission with i.t. chemotherapy and radiation. Second T cell non-depleted NST Of the seven patients with graft failure, two are in complete remission in spite of graft failure, one with CLL without further treatment and one with CML on interferon. These two patients were not offered a second NST. The remaining five patients with graft failure underwent a second T cell non-depleted NST using 2 Gy of TBI and fludarabine for conditioning. Engraftment of the second NST was seen in


Table 2

269

Quantitative donor chimerism over time

No. DLI doses Day after HSCT +14 +30 +60 +90 +120 +150 +180 +210 +240 +270

UPN 735 6

UPN 742 6

UPN 749 6

UPN 751 4

UPN 763 3

UPN 770 5

UPN 782 0

— 75 66 50 33 25 10 33

100 66 40 25 10 0

75 50 0

50 50 10 5

75 50 50 66 100

0

100

0

100

0

100 90 90 80 90 75 66 95 100 100

50 0

0

100 85 75 75 90 100 100

0

0

0

UPN 799 UPN 819 UPN 843 3 0 6

100

10 0 0 0

91 64 18 16 14 12 7 13 0 ⬍5

No DLI doses = number of incremental doses of DLI infused; % donor alleles = % donor chimerism as described. Values are % donor alleles.

Table 3

Transplant characteristics and outcome

UPN

Days to aGVHD No. DLI aGVHD cGVHD Chimerism Best 2nd NST ANC 0.5 grade doses grade response 9 after DLI (10 /l) after NST

Last follow-up

735 742 749 751 763 770

15 13 20 11 14 11

0 0 0 0 0 0

6 6 6 4 3 5

0 0 0 II 0 II

0 0 0 m/h 0 s/m/h

mc mc-gf mc-gf cc mc-gf cc

NR NR NR CR CR CR

Y N Y

a/w, d+1051 a/w, d+1004 alive, d+960 died, d+460 a/w, d+862 a/w, d+829

782 799 819

16 10 11

I 0 0

0 3 0

— II —

0 m/h 0

gf cc gf

NR CR NR

Y Y

a/w, d+773 a/w, d+681 died, d+168

843

18

0

6

0

0

mc-gf

PR

Y

a, d+456

Outcome at last follow-up

cc, in CR after 2nd NST gf; CML in CR on interferon gf after 2nd NST, CLL PD cc, relapsed d+372, died of PD gf, CR of CLL in spite of gf cc, successfully treated for CNS relapse on day+500, CR cc, in CR after 2nd NST cc, CR mc, died after 2nd NST of PD and GVHD III (d+111 after 2nd HSCT) cc, in PD after 2nd NST

ANC = absolute neutrophil count; aGVHD = acute graft-versus-host disease; cGVHD = chronic graft-versus-host disease; m = oral mucosa; h = hepatic; s = skin; mc = mixed chimerism; gf = graft failure; cc = complete chimerism; NR = no response; CR = complete remission; PR = partial remission; PD = progressive disease; HSCT = stem cell transplant; a/w = alive and well; i.t. = intrathecal

four of five patients, three are currently in remission with stable graft function. One patient had T cell engraftment only but no myeloid engraftment with grade III GVHD associated with persistent and progressive CML. Two of these patients with uncomplicated stable engraftment of the second NST had previously been exposed to large doses of donor cells including the T cell depleted NST using a high stem cell dose in addition to six doses of DLI. Final outcome None of the patients on the high stem cell dose–T cell depletion NST protocol died of treatment-related complications. At a median follow-up of 28 months (range 15– 35) eight patients are alive, one patient had died of the original disease and one patient had died with relapse and simultaneous GVHD after the second T cell non-depleted NST.

Discussion These data show that T cell-depleted NST in patients with hematologic malignancy to reduce treatment-related mortality is feasible but is associated with high graft failure rates. We designed our protocol to answer the question whether a high peripheral stem cell dose, will overcome host-versus-graft reactions, and will promote graft-versusleukemia. There is evidence from animal and human studies that high (‘mega’) stem cell doses may promote engraftment and will maintain graft-versus-leukemia effects in spite of T cell depletion, when transplanted in HLAdisparate donor–recipient pairs after myeloablative conditioning.20 We failed to demonstrate an effect of stem cell dose on host-versus-graft reactivity in the current setting of HLA-identical sibling NST. Typically, NST is associated with low regimen-related toxicities, higher graft failure rates of up to 20% compared Bone Marrow Transplantation


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to conventional HSCT and probabilities of ⭓grade II acute GVHD of up to 50%.5 These rates may be modified, depending on conditioning intensity and amount of immunosuppressive drugs. We observed a high rate of graft failure in this study. The mechanisms of graft failure are not known and include graft rejection but also competitive repopulation of host hematopoetic cells. We used cyclophosphamide and ATG as the conditioning regimen. This regimen has been used in the past mainly in patients with nonmalignant disease without T cell depletion and it is not known whether our results apply to other NST regimens. It is possible that the rate of engraftment could have been improved by adding more immunosuppressive drugs, such as fludarabine, to the conditioning regimen. Although many groups are working on the optimization of NST few have used T cell depletion. In a recent study on T cell-depleted NST in chronic granulomatous disease the authors found an association of engraftment and response with graft-versus-host disease (GVHD). Four of six patients with ⬎75% donor chimerism developed GVHD vs 0 of four patients with lower degrees of donor chimerism. Two of these four patients had graft failure. The lower graft failure rate in these patients as compared to the present series is possibly related to the non-malignant nature of the disease treated.11 Other groups have reported high graft failure rates in small series of patients with malignancy undergoing T celldepleted NST.29,30 The close association of stable engraftment and tumor response with acute GVHD has been described by us and others.31 Apparently, patients with malignancy undergoing NST do require some, hopefully manageable degree, of GVHD. Of note, engraftment of a second T cell non-depleted NST in four of five recipients with graft failure after a first transplant is of interest. This contradicts the concept that graft failure may be overcome only with high intensity conditioning and that sensitization against donor antigens will inevitably result in rejection. This is exemplified by two patients exposed to high doses of donor cells including a ‘mega-dose’ T cell-depleted transplant and six doses of DLI. Both had uneventful engraftment of the second T cell non-depleted NST using only 2 Gy of TBI and fludarabine for conditioning. This suggests that NST may be administered repetitively achieving more stable engraftment and tumor response. The issue of a ‘mega-dose’ effect remains open. The stem cell dose achieved with currently available technologies is limited. Stem cell doses higher than 10 ⫻ 106 CD34+ cells/kg may be achieved in small children, but are difficult to reach in adults. Increasing the stem cell dose by an additional log will require new avenues of stem cell mobilization, harvesting and engineering, such as novel growth factor combinations or ex vivo expansion of peripheral stem cells. In conclusion, we show that a high peripheral stem cell dose in the context of a T cell-depleted NST is associated with a high rate of graft failure. Completely avoiding GVHD may not be the avenue to pursue. Some degree of GVHD may be essential for stable engraftment and tumor response.

Bone Marrow Transplantation

Acknowledgements In part supported by the Swiss national research foundation grant NF No. 32-52756.97

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