- Email: [email protected]
In-vivo generation of leukaemia-derived dendritic cells
Best Practice & Research Clinical Haematology Vol. 17, No. 3, pp. 439–451, 2004 doi:10.1016/j.beha.2004.06.004 available online at http://www.sciencedirect.com
6 In-vivo generation of leukaemia-derived dendritic cells Hans-Jochem Kolb*
Dr Med
Professor, Head of Hematopoietic Transplantation
Andreas Rank Dr Med Xiao Chen Dr Med Anja Woiciechowsky
Dr Med
Marie Roskrow Dr Med Christoph Schmid Johanna Tischer
Dr Med
Dr Med
Georg Ledderose
Dr Med
Clinical Cooperative Group Haematopoietic Cell Transplantation, Department of Medicine III, University of Munich, GSF—National Research Centre for Environment and Health, Marchioninistr. 15, 81377 Munich, Germany
Adoptive immunotherapy with transfusion of donor lymphocytes in allogeneic stem cell chimeras has been successful in the treatment of recurrent chronic myelogenous leukaemia (CML) and some patients with acute myeloid leukaemia (AML). The hypothesis that the graft-vs-leukaemia effect (GVL) is promoted by leukaemia-derived dendritic cells has been supported by the concurrent treatment of patients with cytokines that are known to induce differentiation of leukaemia cells towards dendritic cells. In combination with donor lymphocyte transfusions, treatment with interferon-a and granulocyte-macrophage colony-stimulating factor has been studied in patients with recurrent CML and AML, and pre-emptively in patients with high-risk AML. Long-term remissions have been observed in cytokine-treated patients, indicating the beneficial effect of cytokine stimulation of GVL reactions. This is likely to be due to differentiation of leukaemia progenitor cells towards dendritic cells in vivo. Key words: cytokine; adoptive immunotherapy; donor lymphocyte transfusion; leukaemia.
* Corresponding author. Tel.: C49-897-095-4240; Fax: C49-89-7095-4242. E-mail address: [email protected] (H.-J. Kolb). 1521-6926/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
440 H. -J. Kolb et al
Allogeneic stem cell transplantation may induce long-term remission, even in advanced leukaemia.1 However, allogeneic stem cell transplantation is complicated by the risk of severe graft-vs-host disease (GVHD) produced by T lymphocytes contained in the graft. Depletion of T lymphocytes from the graft prior to transplantation, i.e. T-cell depletion, reduces the risk of GVHD.2 However, depletion of T cells from the graft increases the risk of graft rejection and recurrence of leukaemia. Transfusion of donor lymphocytes (DLT) early after transplantation improves engraftment3 without preventing recurrence of leukaemia.4 In contrast, DLT into stable canine chimeras does not abrogate tolerance and does not produce GVHD.5 GVHD could be avoided if DLT was delayed for at least 2 months following transplantation of T-cell-depleted marrow.6 Nevertheless, mixed chimerism could be converted into complete chimerism and immunity could be transferred from the donor to the chimera by delayed transfusion of lymphocytes.6 Unlike normal haematopoietic cells, leukaemia cells may escape immune mechanisms of elimination by donor lymphocytes. The response of recurrent leukaemia after allogeneic stem cell transplantation to DLT was best in chronic-phase chronic myelogenous leukaemia (CML), intermediate in transformedphase CML, myelodysplastic syndrome and acute myeloid leukaemia (AML), and poor in acute lymphoblastic leukaemia (ALL).7 We formulated the hypothesis that myeloid leukaemia responds better to DLT than lymphoid malignancies because of the generation of dendritic cells of leukaemia origin (Figure 1). However, blood cells of CML patients prior to treatment and AML blasts are poor stimulators in mixed lymphocyte reactions and cell-mediated toxicity. Cytokine treatment in vitro and in vivo may restore their stimulatory capacity. We and others have shown dendritic cells
PSC CFU-GEMM
BFU-E
LSC ?
CFU-MEG CFU-GM
Pre-B
CFU-G
Pre-T
CFU-M Dendritic cells
Figure 1. Schema of differentiation of chronic myeloid leukaemia to myeloid dendritic cells. PSC, pluripotent stem cell; LSC, lymphatic stem cell; CFU-GEMM, colony forming unit granulocyte, erythrocyte, monocyte, megakaryocyte; BFU-E, burst forming unit erythrocyte; CFU-GM, colony forming unit granulocyte, monocyte; CFU-G, colony forming unit granulocyte; CFU-M, colony forming unit monocyte; pre-B, B lymphocyte precursor cell; pre-T, T lymphocyte precursor cell. Dendritic cells of myeloid origin may be necessary to present peptides relevant for the graft-vs-leukaemia effect, these could be either minor histocompatibility antigens, differentiation antigens or leukaemia-specific antigens.
In-vivo generation of leukaemia-derived dendritic cells 441
carrying the karyotype of the leukaemia.8–10 This review will summarize current evidence on the cytokine-induced differentiation of leukaemia cells towards dendritic cells and cells with capacities of antigen presentation.
CHRONIC MYELOGENOUS LEUKAEMIA
% specific lysis
The best results of DLT have been observed in patients with recurrent CML after allogeneic transplantation.11–13 T lymphocytes appear to be responsible for control of the disease, as depletion of T cells had a deleterious effect on the relapse incidence after allogeneic transplantation.14 The response to interferon-a (IFN-a) and the response after allogeneic transplantation correlated well with the development of cytotoxic T lymphocytes recognizing a peptide (PR1) of myeloid lineage.15 Newly diagnosed CML contains a large proportion of DR-negative CD34-positive progenitor cells16, and stimulates allogeneic T cells poorly (Figure 2).10 After treatment with IFN-a, the proportion of CD34-positive and DR-negative cells is normalized. Cells of newly diagnosed CML do not stimulate allogeneic T cells efficiently, but they are sensitive to T-cell lysis (Figures 2 and 3). The antigen presentation of CML cells can be improved by treatment with a combination of IFN-a and granulocyte-monocyte colony stimulating factor (GM-CSF). This combination has been tested in vitro for the induction of molecules on the cell surface that are relevant for recognition, stimulation and killing. The combination of IFN-a and GM-CSF has been compared with that of GM-CSF and IL-4 described by Romani et al.17 This combination induced the expression of human leukocyte antigen (HLA) class I and class II antigens, CD80, CD86 and CD40 better than the combination of GM-CSF and IL-4 (Figure 4).10 Only CD1a was less well expressed on dendritic cells produced by the combination of IFN-a and GM-CSF. This molecule is expressed mainly on immature Langerhans cells and it may not be relevant for the stimulation of T cells against CML cells.
100 90 80 70 60 50 40 30 20 10 0 -10 D/A*-> A
E/T ratio 40 : 1 10 : 1 2.5: 1
D/B*-> B
D/B*-> A
D/B*-> Co
D/A*-> Co
A = CML patient (HLA-A1/2 B51/47 C2/6 DR11/13 DQ1/7) B = HLA-id. brother (HLA-A1/2 B51/47 C2/6 DR11/13 DQ1/7) D = HLA-haploid. brother (HLA-A2/28 B51/18 C2/7) Co = unrelated (HLA-A2/24 B12/44 C4/5 DR15/11DQ6/7)
Figure 2. Results of cytotoxic assays involving the cells of a patient with chronic myelogenous leukaemia (CML) before treatment as stimulators. Cells of the healthy human leukocyte antigen (HLA)-haploidentical brother did not lyse the patient’s cells after stimulation with the patient’s cells. They did lyse the patient’s cells after stimulation with cells of the brother that was HLA-identical to the patient. Lysis was measured by release by 51Cr as described by Chen et al (British Journal of Haematology2000; 111: 596–607).
442 H. -J. Kolb et al
Effec
tive k
Effec
tive p
illing
CTL
resen
tation
HLA-id. brother
ion
ntat
iv
fect
Inef
ese e pr
g
HLA-haploidentical brother
illin ve k
cti
Effe
CML patient, untreated Figure 3. Schema of ineffective antigen presentation by chronic myelogenous leukaemia (CML) cells. The cells of the patient are not able to raise an immune response against themselves, but they are sensitive to lysis by the sensitized cells of the brother.
GM-CSF+IL4
GM-CSF+IFN
HLA-DR
HLA-DR
CD 86
CD 86
HLA-ABC
HLA-ABC
CD 83
CD 83
CD 1a
CD 1a
CD 11c
CD 11c
GM-CSF+IL4
GM-CSF+IFN
CD 40
CD 40
CD 80
CD 80
CD 54
CD 54
CD 123
CD 123
Chen Xiao et al., 2000 Figure 4. Expression of surface molecules on chronic myelogenous leukaemia cells after stimulation with GM-CSF and IFN-a compared with GM-CSF and IL-4. Reproduced from Chen et al (British Journal of Haematology 2000; 111: 596–607).
In-vivo generation of leukaemia-derived dendritic cells 443
Another beneficial effect is that of IFN-a on T cells. T cells of patients with CML tend to downregulate the expression of z- and 3-chains of the T-cell receptor.18 This not only depresses their immune reactivity, but also favours apoptosis of T cells. Immune reactivity and vitality of T cells can be recovered by treatment with either anti-CD3 antibody, interleukin (IL)-2 or IFN-a.
APPLICATION OF IN-VITRO FINDINGS TO THE TREATMENT OF PATIENTS WITH CML Historically, patients with recurrent CML after allogeneic transplantation were treated with IFN-a before DLT. A minority of patients responded and stayed in remission after treatment with IFN-a19, but the majority of patients did not respond to the treatment or relapsed. Some patients developed GVHD after discontinuation of immunosuppression and commencement of IFN-a, and they have remained in prolonged remission. A retrospective comparison20 found that the combination of IFN-a and DLTwas not better than DLTalone, but the comparison was biased by the fact that DLTwas only given after failure of IFN-a. A prospective randomized trial comparing DLT alone with DLT and IFN-a has not yet been performed. However, a study comparing very low doses of DLT alone and DLT combined with IFN-a revealed a faster response with IFN-a.21 The combination of IFN-a and GM-CSF with DLT has been used in patients with high-risk CML and patients not responding to DLT and IFN-a (Table 1). Four patients with haematological relapse responded to the combined treatment, but only one of three patients in the transformed phase of relapse responded. This patient had received Table 1. Adoptive immunotherapy for relapse in advanced chronic myelogenous leukaemia: GM-CSF and IFN-a.
Donor
Stage at diagnosis
Time of first relapse
Type of relapse
Response
BJ, 30 years, m
unr
CP
692
Haematol
CR
EH, 31 years, m
unr
AP
876
BC
PR
SH, 44 years, f
bro
CP
401
BC
CR
WR, 37 years, m
unr
CP2
1487
Haematol
CR
JM, 39 years, m
bro
CP
970
Haematol
CR
MA, 24 years, m
bro
CP
895
Haematol
CR
SF, 50 years, m
unr
BC
25
BC
NR
Patient
Outcome survival (days) mol CCR, d1171C IFN, GM-CSF dc STI d1244C Mol. CCR, death w. cGVH d1100 Mol. CCR, d1035C Cytogen, rel. d3648C Hep.B, a&w d94C Died leuk. D49
Patients were treated with a combination of GM-CSF, IFN-s and DLT for high-risk relapse of CML. CP, chronic phase; CP2, second chronic phase; AP, accelerated phase; BC, blast crisis; CR, complete cytogenetic remission; PR, partial cytogenetic remission; NR, no remission; mol, molecular; CCR, continued cytogenetic remission; d, day; rel, relapse; dc, discontinued; a&w, alive and well; m, male; f, female; unr, unrelated; bro, brother.
444 H. -J. Kolb et al
Table 2. Outcome of the treatment of chronic myelogenous leukaemia relapse after allogeneic stem cell transplantation with Imatinib. Relapse
Time post SCT
Best response PCR (day)
AF, 36 years, f MT, 37 years, m
Cytogenetic Haematol
683 2028
neg (858) neg. (2360)
ME, 59 years, f CL, 36 years, m
Cytogenetic Cytogenetic
233 706
pos. (329) neg. (960)
DM, 38 years, m IB, 54 years, f AW, 24 years, f
Cytogenetic Cytogenetic Cytogenetic
947 726 822
neg. (1542) pos. (1119) neg. (3231)
Patient
Outcome Res (942) PCR pos. (2505) after DC Res (566) PCR pos[ (1129) after DC Res (1720) Res. (1191) Res (3304)
Times are given in days after stem cell transplantation. SCT, stem cell transplantation; PCR, real-time RTPCR; Res, resistant to Imatinib; DC, discontinuation of Imatinib; m. male; f, female.
chemotherapy for reduction of the tumour load and she developed extended chronic GVHD after DLT. Imatinib is a selective inhibitor of the bcr/abl coded thymidine kinase that has been advocated for relapse of CML after allogeneic transplantation.22,23 Imatinib is highly effective in inducing haematological, cytogenetic and molecular remissions in these patients. However, in most patients, these remissions are not durable, and molecular relapses occur on drug treatment and after discontinuation of the drug (Table 2). Unfortunately, DLT following or concurrent with Imatinib has not induced durable remission in patients to date. These findings are in contrast to the good results of allogeneic stem cell transplantation in patients treated with Imatinib (M. Deininger, personal communication, 2003).24 The impact of Imatinib on subsequent adoptive immunotherapy with DLT is not yet clear. Single cases24 have shown a response to the combination of Imatinib and DLT, but durable responses were not observed in patients treated with the combination either concurrently or subsequently. The effect of Imatinib on antigen presentation is controversial.25,26 One group found excellent antigen presentation, whereas another group described missing cellular immunity to an endogenous peptide (myeloblastin). The differentiation to dendritic cells and the expression of co-stimulatory molecules (CD80, CD86) was inhibited by the presence of Imatinib in culture.27
ACUTE MYELOID LEUKAEMIA Recurrent AML is responsive to DLT in some cases7,28; the response rate to chemotherapy and G-CSF-mobilized blood cells of the donor (PBSCT) was 47%7,28 and that of patients not receiving chemotherapy or not responding to chemotherapy was 25%7 [European Group for Blood and Marrow Transplantation (EBMT) cohort]. The 2-year survival and the 4-year survival of the EBMT cohort were 25 and 15%, respectively7,29, and the survival of the chemotherapy and PBSCT group (US cohort) was 19% at 2 years.28 There are several possible reasons for the inferior response of AML compared with CML. AML blasts do not differentiate spontaneously to dendritic
In-vivo generation of leukaemia-derived dendritic cells 445
cells, whereas CML cells do. Fresh AML blasts rarely express the co-stimulatory molecules CD80 and CD86. As a rule, AML progresses more rapidly than CML, and the GVL effect of DLT is overgrown before it is building up. Both possibilities are taken into consideration in a regimen combining mild chemotherapy with low-dose cytosine arabinosid (AraC), transfusion of mobilized donor stem cells (PBSC) and GM-CSF. The capacity of myeloid leukaemia cells to differentiate in vitro has been known for some time30, and the induction of in-vivo differentiation by GM-CSF and IL-3 has been described in mice.31 Observations in patients with relapsed AML after allogeneic stem cell transplantation indicated that differentiation occurs in vivo at early stages of relapse.32 In recent years, differentiation has been induced towards dendritic cells by culturing AML blasts in vitro in the presence of GM-CSF and IL-4.33 The expression of HLA antigens and co-stimulatory molecules may be improved by the combination of IL-4 and GM-CSF with Flt-3L and TNF-a (Figure 5).34,35 Cytotoxic T cells with specificity towards autologous blasts could be produced by dendritic cells derived from AML blasts.33,35 Differentiation of AML blasts occurred in cases with favourable
cell count
Upregulation of costimulatory molecules in AML blasts 120
120
100
100
80
80
60
60
40
40
20
20
0 100
101
102
0
103
120
d0
100
101
102
103
120
d6 (GM-CSF and IL-4)
0 100
0 101
102 B7.1
103
104
100
101
102
103
104
B7.2
Figure 5. Expression of B7.1 and B7.2 on acute myeloid leukaemia blasts before and after 6-day culture in the presence of GM-CSF and IL-4.
446 H. -J. Kolb et al
Table 3. Differentiation of acute myeloid leukaemia blasts to dendritic cells (DCs) - influence of karyotype. Number of cases responding to GM-CSF, IL-4, TNF-aGFlt-3L. Generation of DCs in vitro
Favourable karyotype
Intermediate karyotype
Unfavourable karyotype
Total
3 1 4
10 1 11
5 3 8
18 5 23
Yes No Total
The differentiation of blasts to DCs was studied in 23 patients; it failed in five patients and was successful in 18. The response was independent of the karyotype. Favourable karyotypes were in 16, t(8;21), intermediate wereC8 and others, unfavourable were K7, K5 and complex karyotypes.
and unfavourable karyotypes35 (Table 3), in newly diagnosed and untreated cases, and in relapsed and chemotherapy-refractory cases. Similarly, AML blasts refractory to chemotherapy may be sensitive to immunotherapy with antibody conjugates.36 These findings indicate that immunotherapy may be effective in cases with resistance to chemotherapy.
APPLICATION OF IN-VITRO FINDINGS TO THE TREATMENT OF PATIENTS WITH RECURRENT AML AFTER ALLOGENEIC STEM CELL TRANSPLANTATION In the EBMT cohort, complete remissions were induced in 42% of all patients and in 25% of those not given chemotherapy or not responding to chemotherapy (Table 4). In a more recent EBMT study involving 120 chemotherapy patients prior to DLT, the development of acute GVHD and longer duration of remission (O194 days) post transplantation were favourable factors influencing the response.29 However, the duration of remission and survival after DLT were independent of prior chemotherapy. Complete remissions were obtained in cases with favourable and unfavourable karyotypes (Table 5). In the majority of cases, the remissions were of limited duration.
Table 4. Response to donor lymphocyte transfusion (DLT) for recurrent acute myeloid leukaemia (AML)/myelodysplastic syndrome after bone marrow transplant (EBMT Database). Chemotherapy
No. of patients in CR/studied
No chemo. Chemo—CR
9/36 14/15
Chemo—no CR
6/22
Survival time (days) 112C, 155C, 291, 439, 503, 598C, 1007, 1014C, 2374 60, 96, 159, 312C, 372C, 438C, 527, 646, 1173, 1245, 1416C, 1453C, 1463C, 1563C 152, 617C, 800, 977, 1234C, 1263C
Seventy-three patients were treated with DLT for recurrent AML after allogeneic stem cell transplantation. Prior to DLT, they were either not treated with chemotherapy or treated with chemotherapy. DLTwas given to patients treated with chemotherapy while they were in remission (CR) or after chemotherapy had failed (no CR). Survival time is given in days. C, alive at last day of contact.
In-vivo generation of leukaemia-derived dendritic cells 447
Table 5. Response of recurrent acute myeloid leukaemia to donor lymphocyte transfusion (DLT) according to cytogenetic groups. Cytogenetic groups Response
Favourable
Intermediate
Unfavourable
Total
2 1 1
8 1 8
5 1 6
15 3 15
CR PR NR
The response of patients given DLT is shown with regard to the karyotype. Favourable karyotypes were inv 16 and t(8;21), intermediate were C8 and others, unfavourable were K7, complex karyotypes. CR, complete remission; PR, partial remission; NR, no remission.
Low-dose AraC is a mild form of chemotherapy and it can be given on an outpatient basis. G-CSF mobilized stem cells not only provide stem cells for rescue of haematopoiesis, but are also a source of donor-derived dendritic cells and accessory cells as monocytes and macrophages. GM-CSF given to the patient after transfusion of donor cells induces differentiation of residual AML blasts as well as donor-derived dendritic cells. T cells were given in patients without GVHD and with residual AML blasts on Day 35. This regimen induced complete remissions in 67% of patients and a full analysis has been performed in 24 patients with recurrent AML after allogeneic stem cell transplantation (Figure 6).37 The disease could be controlled by low-dose AraC in 11 of 24 patients, and 13 patients required more intensive chemotherapy consisting of higher doses of AraC, fludarabin and anthracyclin or amsacryl. Thirty days after Treatment withlow-doseAraC n=24
Disease controlled n=11
Progression intensive chemotherapy n=13
MDBC and GM-CSF, n=24
Evaluation at day +30 Early death
---
n=3
Treatment failure
n=1
n=6
Response
n=10
n=4
Figure 6. Flow diagram for the treatment of recurrent acute myeloid leukaemia after allogeneic stem cell transplantation with low-dose cytosine arabinosid (AraC), G-CSF mobilized donor blood cells (MDBC) and GM-CSF. Low-dose AraC (20 mg) was administered twice per day subcutaneously for at least 2 weeks followed by a brief recovery period. At least two cycles were administered.
448 H. -J. Kolb et al
10.0
Probabiliy of survival
0.8
0.6
Low-dose AraCresponder: n=11 0.4
0.2 Low-dose AraCnon-responder: n=13
0.0 0
12
24
36
48
60
72
84
96
Months from MDBC transfusion Figure 7. Survival of patients with recurrent acute myeloid leukaemia after allogeneic stem cell transplantation following treatment with low-dose cytosine arabinosid (AraC), transfusion of mobilized donor blood cells (MDBC) and GM-CSF. Response to low-dose AraC is favourable for subsequent response to donor lymphocyte transfusion.
transfusion of donor cells, 10 of 11 responders to low-dose AraC and four of 10 nonresponders were alive and in remission. Three patients who did not respond to lowdose AraC died early. Only one of 13 patients who did not respond to low-dose AraC survived for more than 1 year, whereas 36% of the responding patients survived for more than 2 years (Figure 7). Unfortunately, the leading patient died of leukaemia meningiosis after more than 7 years without systemic relapse. In an analysis of risk factors (Table 6), response to low-dose AraC and the development of GVHD after transfusion favourably influenced the response. Monocytic subtype of the leukaemia Table 6. Response to mobilized donor blood cells (MDBC) and GM-CSF of acute myeloid leukaemia risk factors: univariate analysis. Factor Monocytic subtype Cytogenetic group HLA-identical family donor vs other Response to low-dose AraC GVHD after MDBC
Complete remission
Disease-free survival
PZ0.044 n.s. n.s.
n.s. n.s. PZ0.035
PZ0.024 PZ0.002
PZ0.22 PZ0.016
Factors influencing the response of recurrent acute myeloid leukaemia after allogeneic stem cell transplantation. Response rate was evaluated by Chi-square test and disease-free survival by log rank test. AraC, cytosine arabinosid; GVHD, graft-vs-host disease.
In-vivo generation of leukaemia-derived dendritic cells 449
favourably influenced the response, but not the duration of the response. Conversely, a histocompatible sibling donor favourably influenced survival after relapse and treatment. Two patients have received repeated treatments with low-dose AraC, donor cells and GM-CSF, and have been in remission for more than 2 years.
CONCLUSIONS AND OUTLOOK The spectrum of possible forms of treatment of CML has been enlarged enormously. IFN-a has induced cytogenetic remissions in a minority of patients with CML38, and Imatinib has induced cytogenetic remission in the majority of patients with CML.39 Allogeneic stem cell transplantation may be curative in the majority of patients with remission at the molecular level. T-cell depletion and DLT have demonstrated that the major effect of allogeneic transplantation is immune mediated. The effects of IFN-a are also primarily immune mediated, as shown by cytotoxic T cells directed against the PR1 peptide.15 The effect of Imatinib is most likely to be direct inhibition of the growth of CML precursor cells, and the immune effects of Imatinib are presently unclear. Treatment with Imatinib has induced remission in patients with advancedstage CML, allowing allogeneic stem cell transplantation at lower risk.24 The effects of Imatinib on the GVL reaction of DLT are not yet well defined. Moreover, it is not clear whether Imatinib can be discontinued without risking relapse of CML. IFN-a and GM-CSF may enforce the GVL effect of DLT by improving antigen presentation on leukaemia cells. However, late relapses have been observed in some patients with CML, indicating that the immune-mediated effects may not guarantee life-long protection. In AML, results of immunotherapy have been improved by low-dose AraC, donor cells and GM-CSF. The beneficial effect of GM-CSF may be more evident in an allogeneic GVL reaction than in chemotherapy studies because T cells of the healthy donor may be more likely to respond to improved antigen presentation. The response rate of AML relapse to the combined treatment with low-dose AraC, mobilized donor stem cells and GM-CSF is similar to that of CML. However, the duration of the response is shorter. Repeat treatment may prolong the duration of remission. Acute GVHD remains a problem in most AML patients, particularly in those patients requiring more intensive chemotherapy. Recognition of the relevant peptides in the responding patients may allow more specific interventions in AML. Adoptive immunotherapy has become an indispensable part of the armamentarium in the treatment of myeloid leukaemia, and cytokines and haematopoietic growth factors are part of immunotherapy. Adoptive immunotherapy promises success even in unfavourable cases for chemotherapy. The preconditions and the dynamics of the response will soon become clear.
ACKNOWLEDGEMENTS Supported by EU Contracts, MedNet Leuka¨mie, DFG Sonderforschungsbereich 455, and Deutsche Jose´ Carreras Leuka¨mie Stiftung e.V.
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REFERENCES 1. Thomas ED, Storb R, Clift RA et al. Bone marrow transplantation. New England Journal of Medicine 1975; 292: 832–843. 2. Rodt H, Kolb HJ, Netzel B et al. GVHD suppression by incubation of bone marrow grafts with anti-T- cell globulin: effect in the canine model and application to clinical bone marrow transplantation. Transplantation Proceedings 1979; 11: 962–966. 3. Storb R, Rudolph RH, Kolb H-J et al. Marrow grafts between DLA matched canine littermates. Transplantation 1973; 15: 92–100. 4. Sullivan KM, Storb R, Buckner CD et al. Graft-versus-host disease as adoptive immunotherapy in patients with advanced hematologic neoplasms. New England Journal of Medicine 1989; 320: 828–834. 5. Weiden PL, Storb R, Tsoi M-S, Graham TC, Lerner KG & Thomas ED. Infusion of donor lymphocytes into stable canine radiation chimeras: implications for mechanism of transplantation tolerance. Journal of Immunology 1976; 116: 1212–1219. 6. Kolb HJ, Gu¨nther W, Schumm M, Holler E, Wilmanns W & Thierfelder S. Adoptive immunotherapy in canine chimeras. Transplantation 1997; 63: 430–436. 7. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995; 86: 2041–2050. 8. Smit WM & Rijnbeck M. Dendritic cells generated from FACS sorted chronic myeloid leukemia (CML) precursor cells express BCR/ABL, and are potent stimulators for allogeneic T cells. British Journal of Haematology 1996; 93(supplement 2): 313 abstr. 1186. 9. Eibl B, Ebner S, Duba Ch et al. Philadelphia-chromosome positive dendritic cells (DC) of chronic myelocytic leukemia (CML) patients induce primary cytotoxic T-cell responses to CML cells. Bone Marrow Transplantion 1997; 19(supplement 1): S33. 10. Chen X, Regn S, Raffegerst S, Kolb HJ & Roskrow M. Interferon alpha in combination with GM-CSF induces the differentiation of leukaemic antigen-presenting cells that have the capacity to stimulate a specific anti-leukaemic cytotoxic T-cell response from patients with chronic myeloid leukaemia. British Journal of Haematology 2000; 111: 596–607. 11. Kolb HJ, Mittermueller J, Clemm C et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: 2462–2465. 12. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia. Blood 1995; 86: 2041–2050. 13. Collins RH, Shpilberg O, Drobyski WR et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. Journal of Clinical Oncology 1997; 15: 433–444. 14. Horowitz MM, Gale RP, Sondel PM et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75: 555–562. 15. Molldrem JJ, Lee PP, Wang C et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Natural Medicine 2000; 6: 1018–1023. 16. Schneider EM, Chen ZZ, Ellwart J, Wilmanns W & Kolb HJ. Immune phenotype of chronic myelogenous leukemia progenitor cells. Bone Marrow Transplantion 1996; 17: S69–S319. 17. Romani N, Gruner S, Brang D et al. Proliferating dendritic cell progenitors in human blood. Journal of Experimental Medicine 1994; 180: 83–93. 18. Chen X, Woiciechowsky A, Raffegerst S, Schendel D, Kolb HJ & Roskrow M. Impaired expression of the CD3-zeta chain in peripheral blood T cells of patients with chronic myeloid leukaemia results in an increased susceptibility to apoptosis. British Journal of Haematology 2000; 111: 817–825. 19. Arcese W, Goldman JM, DArcangelo E et al. Outcome for patients who relapse after allogeneic bone marrow transplantation for chronic myeloid leukemia. Blood 1993; 82: 3211–3219. 20. Kolb HJ, Mittermueller J, Hertenstein B, The Chronic Leukemia Working Party et al. Adoptive immunotherapy in human and canine chimeras: the role of interferon-alpha. Seminars in Hematology 1993; 30(supplement 3): 37–39. 21. Posthuma EF, Marijt EW, Barge RM et al. Alpha-interferon with very-low-dose donor lymphocyte infusion for hematologic or cytogenetic relapse of chronic myeloid leukemia induces rapid and durable
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22. 23. 24.
25.
26. 27. 28.
29.
30. 31. 32.
33. 34. 35.
36.
37.
38.
39.
complete remissions and is associated with acceptable graft-versus-host disease. Biol Blood Marrow Transplantion 2004; 10: 204–212. Olavarria E, Craddock C, Dazzi F et al. Imatinib mesylate (STI571) in the treatment of relapse of chronic myeloid leukemia after allogeneic stem cell transplantation. Blood 2002; 99: 3861–3862. Kantarjian HM, O’Brien S, Cortes JE et al. Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood 2002; 100: 1590–1595. Shimoni A, Kroger N, Zander AR et al. Imatinib mesylate (STI571) in preparation for allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusions in patients with Philadelphiapositive acute leukemias. Leukemia 2003; 17: 290–297. Burchert A, Wolfl S, Schmidt M et al. Interferon-alpha, but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia. Blood 2003; 101: 259–264. Sato N, Narita M, Takahashi M et al. The effects of STI571 on antigen presentation of dendritic cells generated from patients with chronic myelogenous leukemia. Hematological Oncology 2003; 21: 67–75. Appel S, Boehmler AM, Grunebach F et al. Imatinib mesylate affects the development and function of dendritic cells generated from CD34Cperipheral blood progenitor cells. Blood 2004; 103: 538–544. Levine JE, Braun T, Penza SL et al. Prospective trial of chemotherapy and donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic stem-cell transplantation. Journal of Clinical Oncology 2002; 20: 405–412. Schmid C, Alessandrino EP, Bunjes D et al. Treatment of relapsed AML and MDS after allogeneic stem cell transplantation with donor lymphocyte transfusion: a retrospective analysis of EBMT results. Blood 2000; 96: 477a. Sachs L. Control of normal cell differentiation and the phenotypic reversion of malignancy in myeloid leukemia. Nature 1978; 274: 535. Lotem J & Sachs L. In vivo control of differentiation of myeloid leukemic cells by recombinant granulocytemacrophage colony-stimulating factor and interleukin 3. Blood 1988; 71: 375–382. Mittermueller J, Kolb HJ, Gerhartz HH & Wilmanns W. In vivo differentiation of leukemic blasts and effect of low dose ara-c in a marrow grafted patient with leukemic relapse. British Journal of Haematology 1986; 62: 757–762. Choudhury BA, Liang J, Thomas EK et al. Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous anti-leukemic T-cell responses. Blood 1999; 93: 780–786. Boyer MW, Waller EK, Bray RA et al. Cytokine upregulation of the antigen presenting function of acute myeloid leukemia cells. Leukemia 2000; 14: 412–418. Woiciechowsky A, Regn S, Kolb HJ & Roskrow M. Leukemic dendritic cells generated in the presence of FLT3 ligand have the capacity to stimulate an autologous leukaemia-specific cytotoxic T cell response from patients with acute myeloid leukaemia. Leukemia 2001; 15: 246–255. Jedema I, Barge RM, Frankel AE, Willemze R & Falkenburg JH. Acute myeloid leukemia cells in G0 phase of the cell cycle that are unresponsive to conventional chemotherapy are sensitive to treatment with granulocyte-macrophage colony-stimulating factor/diphtheria toxin fusion proteins. Experimental Hematology 2004; 32: 188–194. Schmid C, Schleuning M, Aschan J et al. Low dose ara-c, donor cells and GM-CSF for treatment of recurrent acute myeloid leukemia after allogeneic stem cell transplantation: a pilot study. Leukemia 2004; in press. Talpaz M, Kantarjian H, Kurzrock R, Trujillo JM & Gutterman JU. Interferon-alpha produces sustained cytogenetic responses in chronic myelogenous leukemia. Philadelphia chromosome-positive patients. Annual Internal Medicine 1991; 114: 532–538. Druker B, Tamura S, Buchdunger E et al. Effects of selective inhibitor of the ABL tyrosine kinase on the growth of BCR-ABL positive cells. Natural Medicine 1996; 2: 561–566.
6 In-vivo generation of leukaemia-derived dendritic cells Hans-Jochem Kolb*
Dr Med
Professor, Head of Hematopoietic Transplantation
Andreas Rank Dr Med Xiao Chen Dr Med Anja Woiciechowsky
Dr Med
Marie Roskrow Dr Med Christoph Schmid Johanna Tischer
Dr Med
Dr Med
Georg Ledderose
Dr Med
Clinical Cooperative Group Haematopoietic Cell Transplantation, Department of Medicine III, University of Munich, GSF—National Research Centre for Environment and Health, Marchioninistr. 15, 81377 Munich, Germany
Adoptive immunotherapy with transfusion of donor lymphocytes in allogeneic stem cell chimeras has been successful in the treatment of recurrent chronic myelogenous leukaemia (CML) and some patients with acute myeloid leukaemia (AML). The hypothesis that the graft-vs-leukaemia effect (GVL) is promoted by leukaemia-derived dendritic cells has been supported by the concurrent treatment of patients with cytokines that are known to induce differentiation of leukaemia cells towards dendritic cells. In combination with donor lymphocyte transfusions, treatment with interferon-a and granulocyte-macrophage colony-stimulating factor has been studied in patients with recurrent CML and AML, and pre-emptively in patients with high-risk AML. Long-term remissions have been observed in cytokine-treated patients, indicating the beneficial effect of cytokine stimulation of GVL reactions. This is likely to be due to differentiation of leukaemia progenitor cells towards dendritic cells in vivo. Key words: cytokine; adoptive immunotherapy; donor lymphocyte transfusion; leukaemia.
* Corresponding author. Tel.: C49-897-095-4240; Fax: C49-89-7095-4242. E-mail address: [email protected] (H.-J. Kolb). 1521-6926/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
440 H. -J. Kolb et al
Allogeneic stem cell transplantation may induce long-term remission, even in advanced leukaemia.1 However, allogeneic stem cell transplantation is complicated by the risk of severe graft-vs-host disease (GVHD) produced by T lymphocytes contained in the graft. Depletion of T lymphocytes from the graft prior to transplantation, i.e. T-cell depletion, reduces the risk of GVHD.2 However, depletion of T cells from the graft increases the risk of graft rejection and recurrence of leukaemia. Transfusion of donor lymphocytes (DLT) early after transplantation improves engraftment3 without preventing recurrence of leukaemia.4 In contrast, DLT into stable canine chimeras does not abrogate tolerance and does not produce GVHD.5 GVHD could be avoided if DLT was delayed for at least 2 months following transplantation of T-cell-depleted marrow.6 Nevertheless, mixed chimerism could be converted into complete chimerism and immunity could be transferred from the donor to the chimera by delayed transfusion of lymphocytes.6 Unlike normal haematopoietic cells, leukaemia cells may escape immune mechanisms of elimination by donor lymphocytes. The response of recurrent leukaemia after allogeneic stem cell transplantation to DLT was best in chronic-phase chronic myelogenous leukaemia (CML), intermediate in transformedphase CML, myelodysplastic syndrome and acute myeloid leukaemia (AML), and poor in acute lymphoblastic leukaemia (ALL).7 We formulated the hypothesis that myeloid leukaemia responds better to DLT than lymphoid malignancies because of the generation of dendritic cells of leukaemia origin (Figure 1). However, blood cells of CML patients prior to treatment and AML blasts are poor stimulators in mixed lymphocyte reactions and cell-mediated toxicity. Cytokine treatment in vitro and in vivo may restore their stimulatory capacity. We and others have shown dendritic cells
PSC CFU-GEMM
BFU-E
LSC ?
CFU-MEG CFU-GM
Pre-B
CFU-G
Pre-T
CFU-M Dendritic cells
Figure 1. Schema of differentiation of chronic myeloid leukaemia to myeloid dendritic cells. PSC, pluripotent stem cell; LSC, lymphatic stem cell; CFU-GEMM, colony forming unit granulocyte, erythrocyte, monocyte, megakaryocyte; BFU-E, burst forming unit erythrocyte; CFU-GM, colony forming unit granulocyte, monocyte; CFU-G, colony forming unit granulocyte; CFU-M, colony forming unit monocyte; pre-B, B lymphocyte precursor cell; pre-T, T lymphocyte precursor cell. Dendritic cells of myeloid origin may be necessary to present peptides relevant for the graft-vs-leukaemia effect, these could be either minor histocompatibility antigens, differentiation antigens or leukaemia-specific antigens.
In-vivo generation of leukaemia-derived dendritic cells 441
carrying the karyotype of the leukaemia.8–10 This review will summarize current evidence on the cytokine-induced differentiation of leukaemia cells towards dendritic cells and cells with capacities of antigen presentation.
CHRONIC MYELOGENOUS LEUKAEMIA
% specific lysis
The best results of DLT have been observed in patients with recurrent CML after allogeneic transplantation.11–13 T lymphocytes appear to be responsible for control of the disease, as depletion of T cells had a deleterious effect on the relapse incidence after allogeneic transplantation.14 The response to interferon-a (IFN-a) and the response after allogeneic transplantation correlated well with the development of cytotoxic T lymphocytes recognizing a peptide (PR1) of myeloid lineage.15 Newly diagnosed CML contains a large proportion of DR-negative CD34-positive progenitor cells16, and stimulates allogeneic T cells poorly (Figure 2).10 After treatment with IFN-a, the proportion of CD34-positive and DR-negative cells is normalized. Cells of newly diagnosed CML do not stimulate allogeneic T cells efficiently, but they are sensitive to T-cell lysis (Figures 2 and 3). The antigen presentation of CML cells can be improved by treatment with a combination of IFN-a and granulocyte-monocyte colony stimulating factor (GM-CSF). This combination has been tested in vitro for the induction of molecules on the cell surface that are relevant for recognition, stimulation and killing. The combination of IFN-a and GM-CSF has been compared with that of GM-CSF and IL-4 described by Romani et al.17 This combination induced the expression of human leukocyte antigen (HLA) class I and class II antigens, CD80, CD86 and CD40 better than the combination of GM-CSF and IL-4 (Figure 4).10 Only CD1a was less well expressed on dendritic cells produced by the combination of IFN-a and GM-CSF. This molecule is expressed mainly on immature Langerhans cells and it may not be relevant for the stimulation of T cells against CML cells.
100 90 80 70 60 50 40 30 20 10 0 -10 D/A*-> A
E/T ratio 40 : 1 10 : 1 2.5: 1
D/B*-> B
D/B*-> A
D/B*-> Co
D/A*-> Co
A = CML patient (HLA-A1/2 B51/47 C2/6 DR11/13 DQ1/7) B = HLA-id. brother (HLA-A1/2 B51/47 C2/6 DR11/13 DQ1/7) D = HLA-haploid. brother (HLA-A2/28 B51/18 C2/7) Co = unrelated (HLA-A2/24 B12/44 C4/5 DR15/11DQ6/7)
Figure 2. Results of cytotoxic assays involving the cells of a patient with chronic myelogenous leukaemia (CML) before treatment as stimulators. Cells of the healthy human leukocyte antigen (HLA)-haploidentical brother did not lyse the patient’s cells after stimulation with the patient’s cells. They did lyse the patient’s cells after stimulation with cells of the brother that was HLA-identical to the patient. Lysis was measured by release by 51Cr as described by Chen et al (British Journal of Haematology2000; 111: 596–607).
442 H. -J. Kolb et al
Effec
tive k
Effec
tive p
illing
CTL
resen
tation
HLA-id. brother
ion
ntat
iv
fect
Inef
ese e pr
g
HLA-haploidentical brother
illin ve k
cti
Effe
CML patient, untreated Figure 3. Schema of ineffective antigen presentation by chronic myelogenous leukaemia (CML) cells. The cells of the patient are not able to raise an immune response against themselves, but they are sensitive to lysis by the sensitized cells of the brother.
GM-CSF+IL4
GM-CSF+IFN
HLA-DR
HLA-DR
CD 86
CD 86
HLA-ABC
HLA-ABC
CD 83
CD 83
CD 1a
CD 1a
CD 11c
CD 11c
GM-CSF+IL4
GM-CSF+IFN
CD 40
CD 40
CD 80
CD 80
CD 54
CD 54
CD 123
CD 123
Chen Xiao et al., 2000 Figure 4. Expression of surface molecules on chronic myelogenous leukaemia cells after stimulation with GM-CSF and IFN-a compared with GM-CSF and IL-4. Reproduced from Chen et al (British Journal of Haematology 2000; 111: 596–607).
In-vivo generation of leukaemia-derived dendritic cells 443
Another beneficial effect is that of IFN-a on T cells. T cells of patients with CML tend to downregulate the expression of z- and 3-chains of the T-cell receptor.18 This not only depresses their immune reactivity, but also favours apoptosis of T cells. Immune reactivity and vitality of T cells can be recovered by treatment with either anti-CD3 antibody, interleukin (IL)-2 or IFN-a.
APPLICATION OF IN-VITRO FINDINGS TO THE TREATMENT OF PATIENTS WITH CML Historically, patients with recurrent CML after allogeneic transplantation were treated with IFN-a before DLT. A minority of patients responded and stayed in remission after treatment with IFN-a19, but the majority of patients did not respond to the treatment or relapsed. Some patients developed GVHD after discontinuation of immunosuppression and commencement of IFN-a, and they have remained in prolonged remission. A retrospective comparison20 found that the combination of IFN-a and DLTwas not better than DLTalone, but the comparison was biased by the fact that DLTwas only given after failure of IFN-a. A prospective randomized trial comparing DLT alone with DLT and IFN-a has not yet been performed. However, a study comparing very low doses of DLT alone and DLT combined with IFN-a revealed a faster response with IFN-a.21 The combination of IFN-a and GM-CSF with DLT has been used in patients with high-risk CML and patients not responding to DLT and IFN-a (Table 1). Four patients with haematological relapse responded to the combined treatment, but only one of three patients in the transformed phase of relapse responded. This patient had received Table 1. Adoptive immunotherapy for relapse in advanced chronic myelogenous leukaemia: GM-CSF and IFN-a.
Donor
Stage at diagnosis
Time of first relapse
Type of relapse
Response
BJ, 30 years, m
unr
CP
692
Haematol
CR
EH, 31 years, m
unr
AP
876
BC
PR
SH, 44 years, f
bro
CP
401
BC
CR
WR, 37 years, m
unr
CP2
1487
Haematol
CR
JM, 39 years, m
bro
CP
970
Haematol
CR
MA, 24 years, m
bro
CP
895
Haematol
CR
SF, 50 years, m
unr
BC
25
BC
NR
Patient
Outcome survival (days) mol CCR, d1171C IFN, GM-CSF dc STI d1244C Mol. CCR, death w. cGVH d1100 Mol. CCR, d1035C Cytogen, rel. d3648C Hep.B, a&w d94C Died leuk. D49
Patients were treated with a combination of GM-CSF, IFN-s and DLT for high-risk relapse of CML. CP, chronic phase; CP2, second chronic phase; AP, accelerated phase; BC, blast crisis; CR, complete cytogenetic remission; PR, partial cytogenetic remission; NR, no remission; mol, molecular; CCR, continued cytogenetic remission; d, day; rel, relapse; dc, discontinued; a&w, alive and well; m, male; f, female; unr, unrelated; bro, brother.
444 H. -J. Kolb et al
Table 2. Outcome of the treatment of chronic myelogenous leukaemia relapse after allogeneic stem cell transplantation with Imatinib. Relapse
Time post SCT
Best response PCR (day)
AF, 36 years, f MT, 37 years, m
Cytogenetic Haematol
683 2028
neg (858) neg. (2360)
ME, 59 years, f CL, 36 years, m
Cytogenetic Cytogenetic
233 706
pos. (329) neg. (960)
DM, 38 years, m IB, 54 years, f AW, 24 years, f
Cytogenetic Cytogenetic Cytogenetic
947 726 822
neg. (1542) pos. (1119) neg. (3231)
Patient
Outcome Res (942) PCR pos. (2505) after DC Res (566) PCR pos[ (1129) after DC Res (1720) Res. (1191) Res (3304)
Times are given in days after stem cell transplantation. SCT, stem cell transplantation; PCR, real-time RTPCR; Res, resistant to Imatinib; DC, discontinuation of Imatinib; m. male; f, female.
chemotherapy for reduction of the tumour load and she developed extended chronic GVHD after DLT. Imatinib is a selective inhibitor of the bcr/abl coded thymidine kinase that has been advocated for relapse of CML after allogeneic transplantation.22,23 Imatinib is highly effective in inducing haematological, cytogenetic and molecular remissions in these patients. However, in most patients, these remissions are not durable, and molecular relapses occur on drug treatment and after discontinuation of the drug (Table 2). Unfortunately, DLT following or concurrent with Imatinib has not induced durable remission in patients to date. These findings are in contrast to the good results of allogeneic stem cell transplantation in patients treated with Imatinib (M. Deininger, personal communication, 2003).24 The impact of Imatinib on subsequent adoptive immunotherapy with DLT is not yet clear. Single cases24 have shown a response to the combination of Imatinib and DLT, but durable responses were not observed in patients treated with the combination either concurrently or subsequently. The effect of Imatinib on antigen presentation is controversial.25,26 One group found excellent antigen presentation, whereas another group described missing cellular immunity to an endogenous peptide (myeloblastin). The differentiation to dendritic cells and the expression of co-stimulatory molecules (CD80, CD86) was inhibited by the presence of Imatinib in culture.27
ACUTE MYELOID LEUKAEMIA Recurrent AML is responsive to DLT in some cases7,28; the response rate to chemotherapy and G-CSF-mobilized blood cells of the donor (PBSCT) was 47%7,28 and that of patients not receiving chemotherapy or not responding to chemotherapy was 25%7 [European Group for Blood and Marrow Transplantation (EBMT) cohort]. The 2-year survival and the 4-year survival of the EBMT cohort were 25 and 15%, respectively7,29, and the survival of the chemotherapy and PBSCT group (US cohort) was 19% at 2 years.28 There are several possible reasons for the inferior response of AML compared with CML. AML blasts do not differentiate spontaneously to dendritic
In-vivo generation of leukaemia-derived dendritic cells 445
cells, whereas CML cells do. Fresh AML blasts rarely express the co-stimulatory molecules CD80 and CD86. As a rule, AML progresses more rapidly than CML, and the GVL effect of DLT is overgrown before it is building up. Both possibilities are taken into consideration in a regimen combining mild chemotherapy with low-dose cytosine arabinosid (AraC), transfusion of mobilized donor stem cells (PBSC) and GM-CSF. The capacity of myeloid leukaemia cells to differentiate in vitro has been known for some time30, and the induction of in-vivo differentiation by GM-CSF and IL-3 has been described in mice.31 Observations in patients with relapsed AML after allogeneic stem cell transplantation indicated that differentiation occurs in vivo at early stages of relapse.32 In recent years, differentiation has been induced towards dendritic cells by culturing AML blasts in vitro in the presence of GM-CSF and IL-4.33 The expression of HLA antigens and co-stimulatory molecules may be improved by the combination of IL-4 and GM-CSF with Flt-3L and TNF-a (Figure 5).34,35 Cytotoxic T cells with specificity towards autologous blasts could be produced by dendritic cells derived from AML blasts.33,35 Differentiation of AML blasts occurred in cases with favourable
cell count
Upregulation of costimulatory molecules in AML blasts 120
120
100
100
80
80
60
60
40
40
20
20
0 100
101
102
0
103
120
d0
100
101
102
103
120
d6 (GM-CSF and IL-4)
0 100
0 101
102 B7.1
103
104
100
101
102
103
104
B7.2
Figure 5. Expression of B7.1 and B7.2 on acute myeloid leukaemia blasts before and after 6-day culture in the presence of GM-CSF and IL-4.
446 H. -J. Kolb et al
Table 3. Differentiation of acute myeloid leukaemia blasts to dendritic cells (DCs) - influence of karyotype. Number of cases responding to GM-CSF, IL-4, TNF-aGFlt-3L. Generation of DCs in vitro
Favourable karyotype
Intermediate karyotype
Unfavourable karyotype
Total
3 1 4
10 1 11
5 3 8
18 5 23
Yes No Total
The differentiation of blasts to DCs was studied in 23 patients; it failed in five patients and was successful in 18. The response was independent of the karyotype. Favourable karyotypes were in 16, t(8;21), intermediate wereC8 and others, unfavourable were K7, K5 and complex karyotypes.
and unfavourable karyotypes35 (Table 3), in newly diagnosed and untreated cases, and in relapsed and chemotherapy-refractory cases. Similarly, AML blasts refractory to chemotherapy may be sensitive to immunotherapy with antibody conjugates.36 These findings indicate that immunotherapy may be effective in cases with resistance to chemotherapy.
APPLICATION OF IN-VITRO FINDINGS TO THE TREATMENT OF PATIENTS WITH RECURRENT AML AFTER ALLOGENEIC STEM CELL TRANSPLANTATION In the EBMT cohort, complete remissions were induced in 42% of all patients and in 25% of those not given chemotherapy or not responding to chemotherapy (Table 4). In a more recent EBMT study involving 120 chemotherapy patients prior to DLT, the development of acute GVHD and longer duration of remission (O194 days) post transplantation were favourable factors influencing the response.29 However, the duration of remission and survival after DLT were independent of prior chemotherapy. Complete remissions were obtained in cases with favourable and unfavourable karyotypes (Table 5). In the majority of cases, the remissions were of limited duration.
Table 4. Response to donor lymphocyte transfusion (DLT) for recurrent acute myeloid leukaemia (AML)/myelodysplastic syndrome after bone marrow transplant (EBMT Database). Chemotherapy
No. of patients in CR/studied
No chemo. Chemo—CR
9/36 14/15
Chemo—no CR
6/22
Survival time (days) 112C, 155C, 291, 439, 503, 598C, 1007, 1014C, 2374 60, 96, 159, 312C, 372C, 438C, 527, 646, 1173, 1245, 1416C, 1453C, 1463C, 1563C 152, 617C, 800, 977, 1234C, 1263C
Seventy-three patients were treated with DLT for recurrent AML after allogeneic stem cell transplantation. Prior to DLT, they were either not treated with chemotherapy or treated with chemotherapy. DLTwas given to patients treated with chemotherapy while they were in remission (CR) or after chemotherapy had failed (no CR). Survival time is given in days. C, alive at last day of contact.
In-vivo generation of leukaemia-derived dendritic cells 447
Table 5. Response of recurrent acute myeloid leukaemia to donor lymphocyte transfusion (DLT) according to cytogenetic groups. Cytogenetic groups Response
Favourable
Intermediate
Unfavourable
Total
2 1 1
8 1 8
5 1 6
15 3 15
CR PR NR
The response of patients given DLT is shown with regard to the karyotype. Favourable karyotypes were inv 16 and t(8;21), intermediate were C8 and others, unfavourable were K7, complex karyotypes. CR, complete remission; PR, partial remission; NR, no remission.
Low-dose AraC is a mild form of chemotherapy and it can be given on an outpatient basis. G-CSF mobilized stem cells not only provide stem cells for rescue of haematopoiesis, but are also a source of donor-derived dendritic cells and accessory cells as monocytes and macrophages. GM-CSF given to the patient after transfusion of donor cells induces differentiation of residual AML blasts as well as donor-derived dendritic cells. T cells were given in patients without GVHD and with residual AML blasts on Day 35. This regimen induced complete remissions in 67% of patients and a full analysis has been performed in 24 patients with recurrent AML after allogeneic stem cell transplantation (Figure 6).37 The disease could be controlled by low-dose AraC in 11 of 24 patients, and 13 patients required more intensive chemotherapy consisting of higher doses of AraC, fludarabin and anthracyclin or amsacryl. Thirty days after Treatment withlow-doseAraC n=24
Disease controlled n=11
Progression intensive chemotherapy n=13
MDBC and GM-CSF, n=24
Evaluation at day +30 Early death
---
n=3
Treatment failure
n=1
n=6
Response
n=10
n=4
Figure 6. Flow diagram for the treatment of recurrent acute myeloid leukaemia after allogeneic stem cell transplantation with low-dose cytosine arabinosid (AraC), G-CSF mobilized donor blood cells (MDBC) and GM-CSF. Low-dose AraC (20 mg) was administered twice per day subcutaneously for at least 2 weeks followed by a brief recovery period. At least two cycles were administered.
448 H. -J. Kolb et al
10.0
Probabiliy of survival
0.8
0.6
Low-dose AraCresponder: n=11 0.4
0.2 Low-dose AraCnon-responder: n=13
0.0 0
12
24
36
48
60
72
84
96
Months from MDBC transfusion Figure 7. Survival of patients with recurrent acute myeloid leukaemia after allogeneic stem cell transplantation following treatment with low-dose cytosine arabinosid (AraC), transfusion of mobilized donor blood cells (MDBC) and GM-CSF. Response to low-dose AraC is favourable for subsequent response to donor lymphocyte transfusion.
transfusion of donor cells, 10 of 11 responders to low-dose AraC and four of 10 nonresponders were alive and in remission. Three patients who did not respond to lowdose AraC died early. Only one of 13 patients who did not respond to low-dose AraC survived for more than 1 year, whereas 36% of the responding patients survived for more than 2 years (Figure 7). Unfortunately, the leading patient died of leukaemia meningiosis after more than 7 years without systemic relapse. In an analysis of risk factors (Table 6), response to low-dose AraC and the development of GVHD after transfusion favourably influenced the response. Monocytic subtype of the leukaemia Table 6. Response to mobilized donor blood cells (MDBC) and GM-CSF of acute myeloid leukaemia risk factors: univariate analysis. Factor Monocytic subtype Cytogenetic group HLA-identical family donor vs other Response to low-dose AraC GVHD after MDBC
Complete remission
Disease-free survival
PZ0.044 n.s. n.s.
n.s. n.s. PZ0.035
PZ0.024 PZ0.002
PZ0.22 PZ0.016
Factors influencing the response of recurrent acute myeloid leukaemia after allogeneic stem cell transplantation. Response rate was evaluated by Chi-square test and disease-free survival by log rank test. AraC, cytosine arabinosid; GVHD, graft-vs-host disease.
In-vivo generation of leukaemia-derived dendritic cells 449
favourably influenced the response, but not the duration of the response. Conversely, a histocompatible sibling donor favourably influenced survival after relapse and treatment. Two patients have received repeated treatments with low-dose AraC, donor cells and GM-CSF, and have been in remission for more than 2 years.
CONCLUSIONS AND OUTLOOK The spectrum of possible forms of treatment of CML has been enlarged enormously. IFN-a has induced cytogenetic remissions in a minority of patients with CML38, and Imatinib has induced cytogenetic remission in the majority of patients with CML.39 Allogeneic stem cell transplantation may be curative in the majority of patients with remission at the molecular level. T-cell depletion and DLT have demonstrated that the major effect of allogeneic transplantation is immune mediated. The effects of IFN-a are also primarily immune mediated, as shown by cytotoxic T cells directed against the PR1 peptide.15 The effect of Imatinib is most likely to be direct inhibition of the growth of CML precursor cells, and the immune effects of Imatinib are presently unclear. Treatment with Imatinib has induced remission in patients with advancedstage CML, allowing allogeneic stem cell transplantation at lower risk.24 The effects of Imatinib on the GVL reaction of DLT are not yet well defined. Moreover, it is not clear whether Imatinib can be discontinued without risking relapse of CML. IFN-a and GM-CSF may enforce the GVL effect of DLT by improving antigen presentation on leukaemia cells. However, late relapses have been observed in some patients with CML, indicating that the immune-mediated effects may not guarantee life-long protection. In AML, results of immunotherapy have been improved by low-dose AraC, donor cells and GM-CSF. The beneficial effect of GM-CSF may be more evident in an allogeneic GVL reaction than in chemotherapy studies because T cells of the healthy donor may be more likely to respond to improved antigen presentation. The response rate of AML relapse to the combined treatment with low-dose AraC, mobilized donor stem cells and GM-CSF is similar to that of CML. However, the duration of the response is shorter. Repeat treatment may prolong the duration of remission. Acute GVHD remains a problem in most AML patients, particularly in those patients requiring more intensive chemotherapy. Recognition of the relevant peptides in the responding patients may allow more specific interventions in AML. Adoptive immunotherapy has become an indispensable part of the armamentarium in the treatment of myeloid leukaemia, and cytokines and haematopoietic growth factors are part of immunotherapy. Adoptive immunotherapy promises success even in unfavourable cases for chemotherapy. The preconditions and the dynamics of the response will soon become clear.
ACKNOWLEDGEMENTS Supported by EU Contracts, MedNet Leuka¨mie, DFG Sonderforschungsbereich 455, and Deutsche Jose´ Carreras Leuka¨mie Stiftung e.V.
450 H. -J. Kolb et al
REFERENCES 1. Thomas ED, Storb R, Clift RA et al. Bone marrow transplantation. New England Journal of Medicine 1975; 292: 832–843. 2. Rodt H, Kolb HJ, Netzel B et al. GVHD suppression by incubation of bone marrow grafts with anti-T- cell globulin: effect in the canine model and application to clinical bone marrow transplantation. Transplantation Proceedings 1979; 11: 962–966. 3. Storb R, Rudolph RH, Kolb H-J et al. Marrow grafts between DLA matched canine littermates. Transplantation 1973; 15: 92–100. 4. Sullivan KM, Storb R, Buckner CD et al. Graft-versus-host disease as adoptive immunotherapy in patients with advanced hematologic neoplasms. New England Journal of Medicine 1989; 320: 828–834. 5. Weiden PL, Storb R, Tsoi M-S, Graham TC, Lerner KG & Thomas ED. Infusion of donor lymphocytes into stable canine radiation chimeras: implications for mechanism of transplantation tolerance. Journal of Immunology 1976; 116: 1212–1219. 6. Kolb HJ, Gu¨nther W, Schumm M, Holler E, Wilmanns W & Thierfelder S. Adoptive immunotherapy in canine chimeras. Transplantation 1997; 63: 430–436. 7. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. Blood 1995; 86: 2041–2050. 8. Smit WM & Rijnbeck M. Dendritic cells generated from FACS sorted chronic myeloid leukemia (CML) precursor cells express BCR/ABL, and are potent stimulators for allogeneic T cells. British Journal of Haematology 1996; 93(supplement 2): 313 abstr. 1186. 9. Eibl B, Ebner S, Duba Ch et al. Philadelphia-chromosome positive dendritic cells (DC) of chronic myelocytic leukemia (CML) patients induce primary cytotoxic T-cell responses to CML cells. Bone Marrow Transplantion 1997; 19(supplement 1): S33. 10. Chen X, Regn S, Raffegerst S, Kolb HJ & Roskrow M. Interferon alpha in combination with GM-CSF induces the differentiation of leukaemic antigen-presenting cells that have the capacity to stimulate a specific anti-leukaemic cytotoxic T-cell response from patients with chronic myeloid leukaemia. British Journal of Haematology 2000; 111: 596–607. 11. Kolb HJ, Mittermueller J, Clemm C et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: 2462–2465. 12. Kolb HJ, Schattenberg A, Goldman JM et al. Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients. European Group for Blood and Marrow Transplantation Working Party Chronic Leukemia. Blood 1995; 86: 2041–2050. 13. Collins RH, Shpilberg O, Drobyski WR et al. Donor leukocyte infusions in 140 patients with relapsed malignancy after allogeneic bone marrow transplantation. Journal of Clinical Oncology 1997; 15: 433–444. 14. Horowitz MM, Gale RP, Sondel PM et al. Graft-versus-leukemia reactions after bone marrow transplantation. Blood 1990; 75: 555–562. 15. Molldrem JJ, Lee PP, Wang C et al. Evidence that specific T lymphocytes may participate in the elimination of chronic myelogenous leukemia. Natural Medicine 2000; 6: 1018–1023. 16. Schneider EM, Chen ZZ, Ellwart J, Wilmanns W & Kolb HJ. Immune phenotype of chronic myelogenous leukemia progenitor cells. Bone Marrow Transplantion 1996; 17: S69–S319. 17. Romani N, Gruner S, Brang D et al. Proliferating dendritic cell progenitors in human blood. Journal of Experimental Medicine 1994; 180: 83–93. 18. Chen X, Woiciechowsky A, Raffegerst S, Schendel D, Kolb HJ & Roskrow M. Impaired expression of the CD3-zeta chain in peripheral blood T cells of patients with chronic myeloid leukaemia results in an increased susceptibility to apoptosis. British Journal of Haematology 2000; 111: 817–825. 19. Arcese W, Goldman JM, DArcangelo E et al. Outcome for patients who relapse after allogeneic bone marrow transplantation for chronic myeloid leukemia. Blood 1993; 82: 3211–3219. 20. Kolb HJ, Mittermueller J, Hertenstein B, The Chronic Leukemia Working Party et al. Adoptive immunotherapy in human and canine chimeras: the role of interferon-alpha. Seminars in Hematology 1993; 30(supplement 3): 37–39. 21. Posthuma EF, Marijt EW, Barge RM et al. Alpha-interferon with very-low-dose donor lymphocyte infusion for hematologic or cytogenetic relapse of chronic myeloid leukemia induces rapid and durable
In-vivo generation of leukaemia-derived dendritic cells 451
22. 23. 24.
25.
26. 27. 28.
29.
30. 31. 32.
33. 34. 35.
36.
37.
38.
39.
complete remissions and is associated with acceptable graft-versus-host disease. Biol Blood Marrow Transplantion 2004; 10: 204–212. Olavarria E, Craddock C, Dazzi F et al. Imatinib mesylate (STI571) in the treatment of relapse of chronic myeloid leukemia after allogeneic stem cell transplantation. Blood 2002; 99: 3861–3862. Kantarjian HM, O’Brien S, Cortes JE et al. Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood 2002; 100: 1590–1595. Shimoni A, Kroger N, Zander AR et al. Imatinib mesylate (STI571) in preparation for allogeneic hematopoietic stem cell transplantation and donor lymphocyte infusions in patients with Philadelphiapositive acute leukemias. Leukemia 2003; 17: 290–297. Burchert A, Wolfl S, Schmidt M et al. Interferon-alpha, but not the ABL-kinase inhibitor imatinib (STI571), induces expression of myeloblastin and a specific T-cell response in chronic myeloid leukemia. Blood 2003; 101: 259–264. Sato N, Narita M, Takahashi M et al. The effects of STI571 on antigen presentation of dendritic cells generated from patients with chronic myelogenous leukemia. Hematological Oncology 2003; 21: 67–75. Appel S, Boehmler AM, Grunebach F et al. Imatinib mesylate affects the development and function of dendritic cells generated from CD34Cperipheral blood progenitor cells. Blood 2004; 103: 538–544. Levine JE, Braun T, Penza SL et al. Prospective trial of chemotherapy and donor leukocyte infusions for relapse of advanced myeloid malignancies after allogeneic stem-cell transplantation. Journal of Clinical Oncology 2002; 20: 405–412. Schmid C, Alessandrino EP, Bunjes D et al. Treatment of relapsed AML and MDS after allogeneic stem cell transplantation with donor lymphocyte transfusion: a retrospective analysis of EBMT results. Blood 2000; 96: 477a. Sachs L. Control of normal cell differentiation and the phenotypic reversion of malignancy in myeloid leukemia. Nature 1978; 274: 535. Lotem J & Sachs L. In vivo control of differentiation of myeloid leukemic cells by recombinant granulocytemacrophage colony-stimulating factor and interleukin 3. Blood 1988; 71: 375–382. Mittermueller J, Kolb HJ, Gerhartz HH & Wilmanns W. In vivo differentiation of leukemic blasts and effect of low dose ara-c in a marrow grafted patient with leukemic relapse. British Journal of Haematology 1986; 62: 757–762. Choudhury BA, Liang J, Thomas EK et al. Dendritic cells derived in vitro from acute myelogenous leukemia cells stimulate autologous anti-leukemic T-cell responses. Blood 1999; 93: 780–786. Boyer MW, Waller EK, Bray RA et al. Cytokine upregulation of the antigen presenting function of acute myeloid leukemia cells. Leukemia 2000; 14: 412–418. Woiciechowsky A, Regn S, Kolb HJ & Roskrow M. Leukemic dendritic cells generated in the presence of FLT3 ligand have the capacity to stimulate an autologous leukaemia-specific cytotoxic T cell response from patients with acute myeloid leukaemia. Leukemia 2001; 15: 246–255. Jedema I, Barge RM, Frankel AE, Willemze R & Falkenburg JH. Acute myeloid leukemia cells in G0 phase of the cell cycle that are unresponsive to conventional chemotherapy are sensitive to treatment with granulocyte-macrophage colony-stimulating factor/diphtheria toxin fusion proteins. Experimental Hematology 2004; 32: 188–194. Schmid C, Schleuning M, Aschan J et al. Low dose ara-c, donor cells and GM-CSF for treatment of recurrent acute myeloid leukemia after allogeneic stem cell transplantation: a pilot study. Leukemia 2004; in press. Talpaz M, Kantarjian H, Kurzrock R, Trujillo JM & Gutterman JU. Interferon-alpha produces sustained cytogenetic responses in chronic myelogenous leukemia. Philadelphia chromosome-positive patients. Annual Internal Medicine 1991; 114: 532–538. Druker B, Tamura S, Buchdunger E et al. Effects of selective inhibitor of the ABL tyrosine kinase on the growth of BCR-ABL positive cells. Natural Medicine 1996; 2: 561–566.