Transplantation with unrelated bone marrow in leukaemic patients above 40 years of age

Bone Marrow Transplantation, (1998) 21, 43 49 1998 Stockton Press All rights reserved 0268 3369/98 $12.00 Transplantation with unrelated bone marrow in leukaemic patients above 40 years of age O Ringdén 1,2,3, M Remberger 2, J Mattsson 1,2,3, J Aschan 1,4, S Carlens 1,3,HHägglund 1,3, BLönnqvist 1,4, E Sparrelid 5 and P Ljungman 1,4 1 Bone Marrow Transplant Unit, Departments of 2 Clinical Immunology, 3 Transplantation Surgery, 4 Haematology and 5 Infectious Diseases, Huddinge Hospital, Huddinge, Sweden Summary: Twenty-seven patients above 40 years of age (range 40 55) with leukaemia underwent transplantation with haematopoietic stem cells from HLA-A, -B and -DR identical unrelated donors. They were compared to 69 younger patients, median age 23. In the older group, the diagnoses were acute myeloid leukaemia (AML) five, acute lymphoblastic leukaemia (ALL) three and chronic myeloid leukaemia (CML) 19. The corresponding figures in the younger patients were 21, 27 and 21, respectively. Conditioning consisted of cyclophosphamide (120 mg/kg) combined with 10 Gy total body irradiation. Immunosuppression was ATG or OKT3 for 5 days before transplantation and methotrexate combined with cyclosporin A. The probabilities of grades II IV acute graft-versus-host disease (GVHD) were 23 and 21%, and the cumulative incidences of chronic GVHD were 64 and 50% in the older and younger patient cohorts, respectively. Overall, 3-year transplantrelated mortality rates were 46% in patients 40 years of age and 32% in patients 40 years of age (P = 0.16). Three-year patient survival rates were 54 and 46% in the two groups, respectively. In patients with chronic phase CML, the corresponding figures were 67 and 68%, respectively. We conclude that patients above 40 years of age should be considered for transplantation with marrow from unrelated donors. Keywords: bone marrow transplantation; unrelated; leukaemia; age; ATG; graft-versus-host disease Bone marrow transplants from unrelated donors for leukaemias are increasing in number and greatly in proportion, compared to matched sibling donors. 1 7 The major obstacle to successful unrelated donor bone marrow transplantation (BMT) is the high incidence of acute graft-versus-host disease (GVHD) and other transplantation-related complications. The risks of acute GVHD and infectious complications have been more common in patients receiving bone marrow from unrelated donors than from HLA-identical siblings. 2,3,5,8 Correspondence: Prof O Ringdén, Dept of Clinical Immunology, Huddinge Hospital F79, S-141 86 Huddinge, Sweden Received 29 April 1997; accepted 2 September 1997 Age is an important factor following BMT, using HLAidentical sibling donors. 9 12 Age is also important for survival, using unrelated donors. 1,4,7 Because of the adverse association between age and outcome after BMT, many centres limit transplants with unrelated bone marrow to patients less than 40 years old. However, more than half of patients with leukaemia are above 40 years of age. Particularly chronic myeloid leukaemia (CML), otherwise incurable, is more common in older patients. CML is the major indication for BMT with unrelated donors. 1 5,8 16 With a fixed age below 40 years, the vast majority of patients with CML will not be considered for BMT with unrelated marrow. Therefore, it was of interest to analyse the outcome in our patients above 40 years of age with leukaemia, who had received bone marrow from unrelated donors. Patients and methods Patients and tissue typing From 1988 until July 1997, 96 leukaemic patients underwent BMT using unrelated donors at Huddinge Hospital. Twenty-seven were above 40 years of age, median 43 (range 40 55) and 69 were below 40 years of age. The characteristics of these two groups are shown in Table 1. There were more ALL patients in 2nd remission in those below 40 years of age (P = 0.05). All recipient donor pairs were HLA-A, -B and -DR identical. HLA-A and -B class I typing was performed serologically using commercially available reagents to define new subgroups. HLA-DR class II antigens were determined serologically until October 1989 and thereafter using genomic methods. Initially, the RFLP method was used with the help of cdna probes to determine DRB, DQA and DQB alleles. 14 Since July 1992, the PCR-SPP method (PCR method using sequence-specific primer pairs) has been used to define DRB1-5, DQA1, DQB and DPB1 alleles. 15 Mixed lymphocyte culture (MLC) was negative in the case where HLA-DR class II antigens were determined serologically. 16 In the other cases, MLC reactivity was not considered. In patients 40 years of age/ 40 years of age, class II typing was performed serologically in 1/0, by RFLP in 0/10 and by PCR- SPP in 26/59. In patients 40 years of age, the donor origin was Scandinavia in nine, and in the remaining, Europe in eight and USA in 10. Among patients 40 years of age,

44 Table 1 Patient and donor characteristics Patients 40 Patients 40 P years of age years of age value Total numbers 27 69 Diagnosis CML 1st chronic phase 14 17 2nd chronic phase 2 1 accelerated phase 2 blast crisis 1 3 AML 1st CR 2 4 partial remission 3 5 relapse 7 2nd remission 5 ALL 1st CR a 2 11 2nd CR 1 16 0.05 Early/late a 18/9 32/37 NS Recipient age (years) 43 (40 55) b 23 (1 39) Donor age 34 (22 55) 34 (21 49) NS Recipient sex (female/male) 12/15 30/39 Donor sex (female/male) 13/14 23/46 Immune female donor to 4 6 NS male recipient ABO comp/minor/major 13/7/7 20/22/27 incomp. CMV serology, donor/ recipient +/+ 11 19 +/ 11 18 /+ 2 9 / 3 22 0.07 Cell dose c 10 8 /kg 2.0 (0.6 15.6) 3.0 (0.3 14.0) 0.05 Median follow-up (days) 688 (86 1793) 643 (68 2109) NS Median time diagnosis to 448 (177 1577) 384 (91 5224) BMT a CR = complete remission; early: 1st remission, 1st chronic phase; late: beyond these stages. b Median (range). c Bone marrow or peripheral blood progenitor cells in six cases. CSF. 17 As prophylaxis against GVHD, cyclosporin A (CsA) was combined with a short course of MTX. 16,18 At the first sign of grade I acute GVHD, prednisolone 2 mg/kg/day was given for 1 week. Thereafter, the dose was reduced, depending on response. Granulocyte transfusions were given to patients with septicaemia and high fever ( 40 C) who did not respond to antibiotics, or with disseminating local infections. Details regarding supportive care have been published. 16,19 IVIG 500 mg/kg was given once before BMT and once a week during the first 3 months after BMT to seven patients (26%) 40 years of age and to 21 (30%) of the younger patients 20 (Table 1). G-CSF 5 g/kg/day was given after BMT to all patients after May 1994, 16 (59%) and 47 (68%) in the two groups, respectively. Statistical analysis The data were analysed on 13 August 1997. Time to GVHD, relapse, survival rates etc, were analysed by the life table method with a log-rank (Mantel Haenszel) test, and sensor data were taken into account. The log-rank test was used for statistical comparisons of survival curves. For statistical comparisons of absolute numbers, the 2 test or Fisher s exact test was used. Cox s regression model was used for multivariate analysis. The following factors were included: recipient age, diagnosis, acute leukemia or CML, disease status, marrow cell dose, donor age, donor and recipient cytomegalovirus (CMV) serology. CMV disease was defined as CMV infection verified by biopsy, bronchoalveolar lavage, spinal fluid isolation or typical retinitis. 21 Chronic GVHD was described as limited or extensive. 22 Current leukaemia-free survival (LFS) was calculated such that patients who were given donor lymphocyte transfusions after a relapse and achieved complete cytogenetic and haematological remission were also counted as leukaemia-free. corresponding numbers were 19, 30 and 20, respectively. The incidence of grades II IV acute GVHD was 4/28 if the donor was from Scandinavia, 13/38 from Europe and 4/30 from the USA. All patients referred for BMT were considered if they fulfilled indication criteria and had no severe organ impairment that could hamper outcome. Conditioning and treatment All patients were treated with cyclophosphamide 60 mg/kg on days 5 and 4 or 4 and 3 before transplantation. Two 12 mg doses of intrathecal methotrexate (MTX) were given to patients with ALL and AML M4 and M5. This was followed by 10 Gy of total body irradiation with the lungs shielded to receive no more than 9 Gy. Most patients were given 5 days treatment with anti-thymocyte globulin (ATG) or OKT3 before transplantation, 26 in the older and 67 in the younger group, respectively. 16 OKT-3 was used after June 1996 because it is cheaper. As an alternative to bone marrow, five of the patients 40 years of age and five of the patients 40 years of age were given peripheral blood progenitor cells (PBPC) from donors treated with G- Results Engraftment and transfusion Engraftment was achieved in 96% of the patients above or below 40 years of age (Table 2). Time to engraftment, 0.2 10 9 WBC/l and 0.5 10 9 /l, did not differ between the two groups. Requirements for transfusions did not differ significantly in the two groups. Infections and GVHD The incidence of bacteraemia during the first month (positive blood culture) was the same in the two groups (Table 2). Positive CMV PCR was more common in patients 40 years of age (P = 0.01). CMV disease occurred in four patients 40 years of age; two interstitial pneumonitis (IP), one enteritis and one retinitis. Among patients below 40 years of age were one CMV IP, one encephalitis and two retinitis. The cumulative incidence of acute GVHD grades II IV was 23% in patients 40 years of age, compared to 21% in patients 40 years of age. The

Table 2 Engraftment, tranfusions, infections and GVHD 45 Patients 40 years of age Patients 40 years of age P value Engraftment 26/27 (96%) 66/69 (96%) Days to WBC 0.2 10 9 /l 16 (10 24) a 15 (6 23) 0.27 Days to PMN 0.5 10 9 /l 20 (11 31) 16 (10 33) 0.21 Transfusions Erythrocyte units 5 (0 22) 4 (0 58) NS Platelet tranfusions 14 (1 40) 10 (2 57) NS Granulocytes Number of patients 8 13 0.38 Infections Bacteraemia 62% 51% -streptococci 4 16 Straphylococcus epidermidis 9 15 Other bacteraemia 1 3 Positive CMV PCR 21 (78%) 32 (46%) 0.01 CMV disease 4 4 0.18 CMV interstitial pneumonitis 2 1 Acute GVHD 0 2 11 GVHD I 18 44 GVHD II 4 11 GVHD III 2 2 GVHD IV 0 1 Cumulative incidence, grades II IV 23% 21% Chronic GVHD, limited/extensive 9/2 14/5 Cumulative incidence at 3 years 64% 50% 0.10 a Median (range). cumulative incidence of chronic GVHD was 64 and 50% in the two groups, respectively. In the other patients, one died from acute and two from chronic GVHD. Four of the younger patients died of acute and one from chronic GVHD. Survival and relapse Twelve of the older and 33 of the younger patients died. Causes of death in the patients 40 years/ 40 years were relapse 0/13, bronchopneumonia 4/2, interstitial pneumonitis 3/2, septicaemia 1/3, invasive fungal infection 1/3, obstructive broncheolitis 0/3, encephalitis 1/1, VOD 0/2, liver insufficiency 0/2, GVHD 1/2 and hemorrhage 1/0. Overall, transplant-related mortality (TRM) was not statistically different in patients 40 years of age, compared to patients 40 years of age (Table 3). The probability of relapse was lower in the older patients (P = 0.03) and the overall patient survival rates at 3 years were the same, around 50% in both groups (Figure 1). In patients with early leukaemia, first remission of acute leukaemia and first chronic phase of CML, 3 year survival and LFS were similar in the two groups (Table 3). Current LFS at 3 years was 53% in patients 40 years of age and 56% in those 40 years of age in patients with early leukaemia. In patients with more advanced disease, there were only nine above 40 years of age. However, the outcome parameters did not differ significantly or numerically from those in the younger patients. Multivariate analysis was performed regarding TRM, LFS and survival. Recipient age above or below 40 years was not significantly different. None of the other factors analysed were significant in multivariate analysis. Table 3 Outcome parameters, 3-year probability of TRM, relapse, survival and leukaemia-free survival in recipients of unrelated bone marrow 40 years of age and 40 years of age Patients 40 Patients 40 P years of age years of age value % % All patients, n 27 69 TRM 46 32 0.16 Relapse 7 43 0.03 Survival 54 46 LFS 50 37 NS Early leukaemia, n 18 32 TRM 47 24 0.06 Relapse 0 40 0.06 Survival 53 65 LFS 53 45 Advanced disease, n 9 37 TRM 44 39 Relapse 20 47 NS Survival 56 32 NS LFS 44 31 CML chronic phase, n 16 18 TRM 33 32 Relapse 0 45 0.06 Survival 67 68 LFS 67 37 NS Outcome in chronic phase CML The most homogeneous group comprises patients with CML in chronic phase. In those 40 of age, TRM was 33%, and none has so far had a relapse with a 3-year survival rate and LFS of 67% (Figure 2). In the younger patients with CML in chronic phase, TRM was 32% and

46 1 0.9 0.8 Probability of survival 0.7 0.6 0.5 0.4 0.3 P = NS 0.2 0.1 0 0 2 4 6 Years after BMT Figure 1 Probability of survival after BMT in recipients of marrow from HLA-A, -B and -DR identical, matched unrelated donors 40 years of age (n = 27, ), and patients 40 years of age (n = 69, ). 1 0.9 0.8 0.7 Probability of survival 0.6 0.5 0.4 0.3 0.2 0.1 P = NS 0 0 2 4 6 Years after BMT Figure 2 Probability of survival in patients with CML in chronic phase receiving marrow from unrelated donors 40 years of age (n = 16, ), and patients 40 years of age (n = 18, ).

four patients had a relapse. Three had a relapse of their CML, 1 year, 1 year and 9 months and 2 years after BMT, respectively. All three were treated with donor lymphocytes and are now in haematological and cytogenetic remission 2 years, 2 years and 3 years and 1 month after donor lymphocyte treatment, respectively. The fourth patient became Philadelphia chromosome-positive 2 years after BMT, started interferon treatment in November 1996 and received donor lymphocyte transfusions in December 1996, 3 years after BMT. This patient did not enter remission and died of relapse. The 3-year patient survival rate in patients 40 years of age with CML in chronic phase is 67% and similar to that of 40 years of age (Figure 2). The current LFS at 3 years was 67% in those 40 years and 53% in patients 40 years of age. Discussion In this comparison between patients 40 years of age and younger patients receiving unrelated haematopoietic stem cells, more ALL patients with advanced disease were found in the younger group. Furthermore, there was a trend for more CMV seronegative donor/recipient pairs in the younger patients, who also received a significantly higher bone marrow cell dose. This may be expected, since ALL and CMV seronegativity are more common in younger patients. Furthermore, all donors were adults and the younger group also included children of low weight, who therefore received a higher cell dose. High bone marrow cell dose was associated with a lowered risk of dying from GVHD in a previous study from our unit. 10 A recent study from Seattle also showed improved survival in recipients who received a high cell dose of unrelated bone marrow. 23 In the present analysis, all were consecutive patients from a single institution. Therefore, treatment, conditioning and GVHD prophylaxis were the same in the two groups. Because of the small number of patients, the data should be interpreted with caution. The incidence of septicaemia (above 50%) was not significantly different between the two groups. The incidence of septicaemia is significantly higher in recipients of unrelated bone marrow, than in HLA-identical siblings. 8,16 The reasons for this may be more pronounced immunosuppressive therapy, delayed engraftment, minor HLA-incompatibility and high donor age. High donor age had previously been found to prolong immunological recovery after BMT. 24 Positive CMV PCR was more common in the older patients, 78 vs 46% (P = 0.01). This may be expected since there tended to be fewer CMV seronegative donor/recipient pairs in the older patients. 25 With early diagnosis using PCR and pre-emptive therapy with ganciclovir or foscarnet, CMV disease only occurred in four and three patients each in the two groups, respectively. 21 PBPC instead of bone marrow was given to five of the older patients and five of the younger ones. 17 It has rarely been used in recipients of unrelated stem cells, since the increased number of T cells in the graft may increase the risk of severe GVHD. However, using unmanipulated bone marrow in HLA-identical siblings, we found no correlation with the number of T cells in the graft and the consequent risk of acute GVHD. 10 Only three patients in this study, who received PBPC developed grade II III acute GVHD. Therefore, although experimental, PBPC from unrelated donors may be used as an alternative to bone marrow. Generally, age has not been associated with an increased risk of acute GVHD. 26,27 However, advanced age is associated with an increased risk of dying of acute GHVD. 9,10 This is one reason why older adults have not been considered for BMT from unrelated donors in all centres. In the present study, patients above and below 40 years of age had a probability of around 20% of developing moderate-to-severe acute GVHD. Mortality from GVHD was low in both groups. The reason for this may be the use of ATG, which may reduce the risk not only of rejection, but also of acute GVHD. 16 Bär et al 28 reported that T cell depletion, using elutriation, had a favourable outcome in HLA-identical siblings above 40 years of age. Therefore, T cell depletion in vivo and in vitro may be important in older adults undergoing BMT from unrelated donors. Chronic GVHD is strongly correlated with acute GVHD. 29,30 Chronic GVHD has also been shown to increase with age. 13,29,30 However, in this study, age above 40 years did not increase the risk of chronic GVHD. Overall, the risk of chronic GVHD in these patients was higher than that seen in HLA-identical siblings. 31 This may be due to mismatched minor HLA antigens and the high proportion of patients with CML. We have found an increased risk of chronic GVHD in patients with CML, compared to other diagnoses receiving HLA-identical sibling marrow and also unrelated marrow, respectively. 32 Most studies showing an effect of age on transplant outcome have compared children to adults. 9 12 However, in this analysis, we investigated whether BMT using unrelated donors is feasible in patients above 40 years of age. In our study of consecutive leukaemic patients receiving marrow from unrelated donors, survival was above 50% in recipients above and below 40 years of age (Table 3). In the most homogeneous cohort, patients with CML in chronic phase, 3-year survival rates were almost identical in those above and below 40 years of age, 67 and 68% respectively (Figure 2). This figure should be viewed with caution, because the patients were few (n = 16 and 18) and the observation time short. However, LFS was only 37% in the younger patients. With the use of donor lymphocyte transfusions, most CML patients with recurrent disease after BMT have a remission. 33 Therefore, it may be more relevant to compare survival or current LFS rather than LFS in CML patients after BMT. One explanation why patients 40 years of age and older benefited as much as the younger patients, may be that these patients have been more carefully selected. Nevertheless, this study suggests that age above 40 years should not be a determining factor for BMT from unrelated donors. Several investigations of HLA-identical siblings have indicated that patients above 40, 45 or 50 years of age should be considered for BMT. 28,34 36 A report from the International Bone Marrow Transplant Register showed that patients, 50 years of age and older, with early and intermediate leukaemia, benefited as much as those between 30 47

48 and 49 years of age. 35 Therefore, it is also reasonable that patients above 50 years of age with early leukaemia should be considered for BMT from unrelated donors. However, we cannot determine the upper age limit for receiving an unrelated BMT. Acknowledgements We are indebted to the nursing staff at the BMT Unit and the Depts of Transplantation Surgery, and Haematology at Huddinge Hospital for excellent patient care. We thank Inger Hammarberg for expert preparation of this manuscript. This study was supported by grants from the Swedish Cancer Foundation (0070-B95-09XCC), the Children s Cancer Foundation (1994 060), the Belvén Foundation, the Swedish Medical Research Council (B96-16X-05971-16C), FRF Foundation, the Tobias Foundation and the Ellen Bachrach Foundation. References 1 McGlave PB, Beatty P, Ash R, Hows JM. Therapy for chronic myelogenous leukemia with unrelated donor bone marrow transplantation: results in 102 cases: published erratum appears in Blood 1990; 76: 654. 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29 Storb R, Prentice RL, Sullivan KM et al. Predictive factors in chronic graft-versus-host disease in patients with aplastic anemia treated by marrow transplantation from HLA-identical siblings. Ann Intern Med 1983; 98: 461 466. 30 Ringdén O, Paulin T, Lönnqvist B, Nilsson B. An analysis of factors predisposing for chronic graft-versus-host disease. Exp Hematol 1985; 13: 1062 1067. 31 Ringdén O, Horowitz MM, Sondel P et al. Methotrexate, cyclosporine or both to prevent graft-versus-host disease after HLA-identical sibling bone marrow transplants for early leukemia? Blood 1993; 81: 1094 1101. 32 Carlens S, Ringdén O, Aschan J et al. Risk-factors for chronic graft-versus-host disease after bone marrow transplantation: a retrospective single center analysis. (Submitted). 33 Kolb HJ, Mittermüller J, Clemm CH et al. Donor leukocyte transfusions for treatment of recurrent chronic myelogenous leukemia in marrow transplant patients. Blood 1990; 76: 2462 2465. 34 Klingemann H-G, Storb R, Fefer A et al. Bone marrow transplantation in patients aged 45 years and older. Blood 1986; 67: 770 776. 35 Ringdén O, Horowitz M, Gale RP et al. Outcome after allogeneic bone marrow transplant for leukemia in older adults. JAMA 1993; 270: 57 60. 36 Rapoport AP, DiPersio JF, Martin BA et al. Patients age 40 years undergoing autologous or allogeneic BMT have regimen-related mortality rates and event-free survivals comparable to patients age 40 years. Bone Marrow Transplant 1995; 15: 523 530. 49