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Perioperative donor bone marrow infusion in cadaver kidney transplant recipients
Perioperative Donor Bone Marrow Infusion in Cadaver Kidney Transplant Recipients R.O. Garcia-Morales, G. Ciancio, J. Mathew, Y. Jin, A. Rosen, C. Ricordi, G.W. Burke III, B. Blomberg, L. Fuller, A.G. Tzakis, V. Esquenazi, and J. Miller
P
ERIOPERATIVE infusion of donor-specific bone marrow cells (DBMC) at the time of organ transplantation has had a somewhat renewed interest using clinical trials with novel immunosuppressive strategies.1–5 These have evolved from animal work, with studies first introduced by Monaco et al.6 – 8 To date, unless radical maneuvers exist, including whole body x-irradiation, or other exceptional circumstances, the results from other centers have been equivocal.9 –11 However, more recently, over the long term we have been following a group of 62 cadaver kidney transplant recipients given donor bone marrow compared with 215* noninfused controls treated with equivalent
immunosuppression, using OKT3 induction and maintenance tacrolimus, mycophenolate, and methylprednisolone,12–14 with results that have appeared more encouraging. In the present study we report the most recent update (ie, a 6-year follow-up of actuarial patient and graft survival). In addition, we have described in several publications the technique of quantitative cellular chimerism measurement by PCR-Flow.15,16 This has enabled us to analyze which DBMC subsets have persisted or expanded in our characterization of multi-lineage DBMC chimerism in the recipient.
* In previous reports, these included one patient in the DBMC group who had a second transplant, as well as two patients in the control group who had multiorgan transplants and two who were living-related donor kidney recipients, mistakenly consented, entered, and followed in the study as intent-to-treat. (There was no influence on the results by removing them and cleaning up the data for this update.)
From the Department of Surgery, Division of Transplantation, the Diabetes Research Institute, University of Miami School of Medicine; and the Miami Veterans Affairs Medical Center, Miami, Florida. Address reprint requests to Joshua Miller, MD, Department of Surgery, Division of Transplantation, University of Miami School of Medicine, PO Box 012440, Miami, FL 33101.
Fig 1. Six-year actuarial patient survival of DBMC-infused versus noninfused (control) first cadaver renal transplant recipients. 0041-1345/01/$–see front matter PII S0041-1345(01)02626-4
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3840
Transplantation Proceedings, 33, 3840–3843 (2001)
PERIOPERATIVE DONOR BONE MARROW
3841
Fig 2. Six-year actuarial graft survival of DBMC-infused versus noninfused (control) first cadaver renal transplant recipients.
METHODS Patients All sequential first cadaver kidney recipients transplanted between September 1994 and May 1998 were entered into the study. Informed consent was obtained as per our established institutional guidelines. When vertebral body bone marrow was available, it was infused in two aliquots on day 4 and day 11 after transplantation, averaging 7.1 ⫻ 108 ⫾ 1.9 ⫻ 108 cells/kg per body weight in total per patient. Most received two infusions. However, in a few patients, there was only enough marrow available for the first infusion (⬃half the total dose). Control recipients had the same
Fig 3. Six-year actuarial graft survival with death censored that was unrelated to rejection in DBMC-infused versus noninfused (control) first cadaver renal transplant recipients.
demographics in a retrospective analysis,14 had signed informed consent, but did not have marrow available from their respective donors.
Laboratory Methods and Biopsies The PCR-Flow assay has been described in several previous reports.12,14 Other laboratory assays consisted of routine renal function assessment, hematology, and chemistry. Kidney biopsies were performed when indicated by a rising serum creatinine concentration. Standardized Banff criteria were used to assess whether acute or chronic rejection was present.
3842
GARCIA-MORALES, CIANCIO, MATHEW ET AL
Table 1. Peripheral Blood DBMC Chimerism in Bone Marrow-Infused Recipients by PCR-Flow (cells/million counted; and %) ⴞ SD Year Posttransplant
1 (n ⫽ 42) 2 (n ⫽ 44) 3 (n ⫽ 38) 4 (n ⫽ 19) 5 (n ⫽ 22) 6 (n ⫽ 4)
Total
2263 ⫾ 1868 (0.2%) 1225 ⫾ 1208 (0.1%) 872 ⫾ 239 (0.08%) 727 ⫾ 89 (0.07%) 739 ⫾ 91 (0.07%) 813 ⫾ 25 (0.08%)
Statistics Patient and graft survival were analyzed by Kaplan-Meier graphs, and routine statistical analyses were performed on other laboratory values.
RESULTS Patient and Graft Survival, Chimerism Analysis, and Chronic Rejection
An early tendency in the postoperative course toward infections in the DBMC group, primarily CMV, has been previously reported,14 and this resulted in deaths that were not associated with rejection. There has, therefore, been equivalence in both groups in patient survival up to the present time (Fig 1). Until now there has been a statistically significant difference because of this between the DBMC and control groups only by excluding nongraft-loss-related deaths (Fig 3) (P ⫽ .04). There now also appears to be a trend, even if one does not exclude deaths (Fig 2), in which the graft survival curves begin to separate in that an inflection point is reached ⬃42 months, due to the fact that the later incidence of chronic rejection on biopsy has been greater in the control nonmarrow-infused group.14 This difference in graft survival has continued to be amplified with time (P ⫽ .06). Tables 1, 2, and 3 demonstrate that chimerism in iliac crest bone marrow aspirates of the
CD3⫹
CD34⫹
1032 ⫾ 779 (0.1%) 547 ⫾ 473 (0.05%) 392 ⫾ 112 (0.03%) 339 ⫾ 73 (0.03%) 329 ⫾ 58 (0.03%) 395 ⫾ 31 (0.04%)
663 ⫾ 564 (0.06%) 332 ⫾ 333 (0.03%) 222 ⫾ 108 (0.02%) 155 ⫾ 39 (0.02%) 152 ⫾ 54 (0.02%) 135 ⫾ 31 (0.01%)
DBMC-infused patients, performed yearly, has now increased to greater than 1.5% of the total donor bone marrow cells/million marrow cells counted. This trend toward increasing bone marrow chimerism in the recipients has more than quadrupled in the 6-year interval of followup. However, no attempt has been made to totally wean patients from immunosuppressive medications, and both groups still show no statistical differences in dosaging of either maintenance tacrolimus, mycophenolate, or corticosteroids. DISCUSSION AND CONCLUSIONS
Although no attempt has been made in this protocol to wean immunosuppressive therapy from DBMC-infused recipients, there has been statistically significant and increasing differences in graft survival caused by DBMC infusion, with fewer patients in this group experiencing chronic rejection on biopsy. Several mechanisms have been proposed for the improved graft acceptance associated with bone marrow infusion as a result of a number of in vitro studies from this laboratory by Mathew et al17–19 and Jin et al.20,21 As a result, the suppressor cell functions of DBMC have been much more clearly defined and may well be the effective mechanisms for explaining the decreased incidence of chronic rejection in this group.
Table 2. Iliac Crest DBMC Chimerism in Bone Marrow-Infused Recipients by PCR-Flow (cells/million counted; and %) ⴞ SD Year Posttransplant
1 (n ⫽ 42) 2 (n ⫽ 44) 3 (n ⫽ 39) 4 (n ⫽ 22) 5 (n ⫽ 17) 6 (n ⫽ 4)
Total
CD3⫹
CD34⫹
4210 ⫾ 3294 (0.4%) 7492 ⫾ 4966 (0.7%) 10,200 ⫾ 6763 (1.0%) 11,783 ⫾ 8221 (1.2%) 13,186 ⫾ 8383 (1.3%) 17,778 ⫾ 7036 (1.8%)
1749 ⫾ 1643 (0.2%) 3236 ⫾ 2415 (0.3%) 4408 ⫾ 3183 (0.4%) 5136 ⫾ 3805 (0.5%) 6008 ⫾ 4253 (0.6%) 9360 ⫾ 4505 (0.9%)
1616 ⫾ 1494 (0.2%) 2503 ⫾ 1805 (0.3%) 3191 ⫾ 2297 (0.3%) 3849 ⫾ 2745 (0.4%) 3958 ⫾ 2810 (0.4%) 5540 ⫾ 2929 (0.5%)
Table 3. Iliac Crest DBMC Chimerism in Noninfused Recipients by PCR-Flow (cells/million counted; and %) ⴞ SD Year Posttransplant
1 (n ⫽ 30) 2 (n ⫽ 22) 3 (n ⫽ 19) 4 (n ⫽ 12) 5 (n ⫽ 4) 6 (n ⫽ 5)
Total
717 ⫾ 510 (0.07%) 1116 ⫾ 474 (0.1%) 1491 ⫾ 462 (0.1%) 1475 ⫾ 538 (0.1%) 1663 ⫾ 1064 (0.2%) 2080 ⫾ 1085 (0.2%)
CD3⫹
CD34⫹
347 ⫾ 319 (0.03%) 574 ⫾ 375 (0.06%) 814 ⫾ 390 (0.08%) 683 ⫾ 476 (0.07%) 793 ⫾ 829 (0.08%) 1250 ⫾ 863 (0.1%)
113 ⫾ 126 (0.01%) 173 ⫾ 122 (0.02%) 288 ⫾ 75 (0.03%) 168 ⫾ 131 (0.02%) 160 ⫾ 188 (0.02%) 186 ⫾ 140 (0.02%)
PERIOPERATIVE DONOR BONE MARROW
REFERENCES 1. Barber WH, Mankin JA, Laskow DA, et al: Transplantation 51:70, 1991 2. Corry RJ, Chakrabarti PK, Shapiro R, et al: Ann Surg 230:372, 1999 3. Fontes P, Rao AS, Demetris AJ, et al: Lancet 344:151, 1994 4. Barber WH, Mankin JA, Laskow DA, et al: Transplantation 51:70, 1991 5. Rao AS, Phil D, Fontes P, et al: Adv Exp Med Biol 417:269, 1997 6. Monaco AP, Clark AW, Wood ML, et al: Surgery 79:384, 1976 7. Okazaki H, Maki T, Wood ML, et al: Transplantation 32:111, 1981 8. Monaco AP, Wood ML: Transplant Proc 2:489, 1970 9. Shapiro R, Starzl TE: Transplant Proc 30:1371, 1998 10. Rao AS, Shapiro R, Corry R, et al: Transplant Proc 30:1367, 1998 11. McDaniel DO, Naftilan J, Hulvey K, et al: Transplantation 57:852, 1994
3843 12. Garcia-Morales R, Carreno M, Mathew J, et al: Transplantation 65:956, 1998 13. Garcia-Morales R, Carreno M, Mathew J, et al: J Clin Invest 99:1118, 1997 14. Ciancio G, Miller J, Garcia-Morales RO, et al: Transplantation 71:827, 2001 15. Garcia-Morales R, Esquenazi V, Zucker K, et al: Transplantation 62:1149, 1996 16. Garcia-Morales R, Esquenazi V, Zucker K, et al: Transplant Proc 29:1219, 1997 17. Mathew JM, Carreno M, Fuller L, et al: Transplantation 68:1172, 1999 18. Mathew JM, Fuller L, Carreno M, et al: Transplantation 70:1752, 2000 19. Mathew JM, Fuller L, Carreno M, et al: Transplant Proc 33:108, 2001 20. Jin Y, Fuller L, Esquenazi V, et al: Hum Immunol 61:538, 2000 21. Jin Y, Fuller L, Wei Y, et al: Hum Immunol 61:1233, 2000
P
ERIOPERATIVE infusion of donor-specific bone marrow cells (DBMC) at the time of organ transplantation has had a somewhat renewed interest using clinical trials with novel immunosuppressive strategies.1–5 These have evolved from animal work, with studies first introduced by Monaco et al.6 – 8 To date, unless radical maneuvers exist, including whole body x-irradiation, or other exceptional circumstances, the results from other centers have been equivocal.9 –11 However, more recently, over the long term we have been following a group of 62 cadaver kidney transplant recipients given donor bone marrow compared with 215* noninfused controls treated with equivalent
immunosuppression, using OKT3 induction and maintenance tacrolimus, mycophenolate, and methylprednisolone,12–14 with results that have appeared more encouraging. In the present study we report the most recent update (ie, a 6-year follow-up of actuarial patient and graft survival). In addition, we have described in several publications the technique of quantitative cellular chimerism measurement by PCR-Flow.15,16 This has enabled us to analyze which DBMC subsets have persisted or expanded in our characterization of multi-lineage DBMC chimerism in the recipient.
* In previous reports, these included one patient in the DBMC group who had a second transplant, as well as two patients in the control group who had multiorgan transplants and two who were living-related donor kidney recipients, mistakenly consented, entered, and followed in the study as intent-to-treat. (There was no influence on the results by removing them and cleaning up the data for this update.)
From the Department of Surgery, Division of Transplantation, the Diabetes Research Institute, University of Miami School of Medicine; and the Miami Veterans Affairs Medical Center, Miami, Florida. Address reprint requests to Joshua Miller, MD, Department of Surgery, Division of Transplantation, University of Miami School of Medicine, PO Box 012440, Miami, FL 33101.
Fig 1. Six-year actuarial patient survival of DBMC-infused versus noninfused (control) first cadaver renal transplant recipients. 0041-1345/01/$–see front matter PII S0041-1345(01)02626-4
© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
3840
Transplantation Proceedings, 33, 3840–3843 (2001)
PERIOPERATIVE DONOR BONE MARROW
3841
Fig 2. Six-year actuarial graft survival of DBMC-infused versus noninfused (control) first cadaver renal transplant recipients.
METHODS Patients All sequential first cadaver kidney recipients transplanted between September 1994 and May 1998 were entered into the study. Informed consent was obtained as per our established institutional guidelines. When vertebral body bone marrow was available, it was infused in two aliquots on day 4 and day 11 after transplantation, averaging 7.1 ⫻ 108 ⫾ 1.9 ⫻ 108 cells/kg per body weight in total per patient. Most received two infusions. However, in a few patients, there was only enough marrow available for the first infusion (⬃half the total dose). Control recipients had the same
Fig 3. Six-year actuarial graft survival with death censored that was unrelated to rejection in DBMC-infused versus noninfused (control) first cadaver renal transplant recipients.
demographics in a retrospective analysis,14 had signed informed consent, but did not have marrow available from their respective donors.
Laboratory Methods and Biopsies The PCR-Flow assay has been described in several previous reports.12,14 Other laboratory assays consisted of routine renal function assessment, hematology, and chemistry. Kidney biopsies were performed when indicated by a rising serum creatinine concentration. Standardized Banff criteria were used to assess whether acute or chronic rejection was present.
3842
GARCIA-MORALES, CIANCIO, MATHEW ET AL
Table 1. Peripheral Blood DBMC Chimerism in Bone Marrow-Infused Recipients by PCR-Flow (cells/million counted; and %) ⴞ SD Year Posttransplant
1 (n ⫽ 42) 2 (n ⫽ 44) 3 (n ⫽ 38) 4 (n ⫽ 19) 5 (n ⫽ 22) 6 (n ⫽ 4)
Total
2263 ⫾ 1868 (0.2%) 1225 ⫾ 1208 (0.1%) 872 ⫾ 239 (0.08%) 727 ⫾ 89 (0.07%) 739 ⫾ 91 (0.07%) 813 ⫾ 25 (0.08%)
Statistics Patient and graft survival were analyzed by Kaplan-Meier graphs, and routine statistical analyses were performed on other laboratory values.
RESULTS Patient and Graft Survival, Chimerism Analysis, and Chronic Rejection
An early tendency in the postoperative course toward infections in the DBMC group, primarily CMV, has been previously reported,14 and this resulted in deaths that were not associated with rejection. There has, therefore, been equivalence in both groups in patient survival up to the present time (Fig 1). Until now there has been a statistically significant difference because of this between the DBMC and control groups only by excluding nongraft-loss-related deaths (Fig 3) (P ⫽ .04). There now also appears to be a trend, even if one does not exclude deaths (Fig 2), in which the graft survival curves begin to separate in that an inflection point is reached ⬃42 months, due to the fact that the later incidence of chronic rejection on biopsy has been greater in the control nonmarrow-infused group.14 This difference in graft survival has continued to be amplified with time (P ⫽ .06). Tables 1, 2, and 3 demonstrate that chimerism in iliac crest bone marrow aspirates of the
CD3⫹
CD34⫹
1032 ⫾ 779 (0.1%) 547 ⫾ 473 (0.05%) 392 ⫾ 112 (0.03%) 339 ⫾ 73 (0.03%) 329 ⫾ 58 (0.03%) 395 ⫾ 31 (0.04%)
663 ⫾ 564 (0.06%) 332 ⫾ 333 (0.03%) 222 ⫾ 108 (0.02%) 155 ⫾ 39 (0.02%) 152 ⫾ 54 (0.02%) 135 ⫾ 31 (0.01%)
DBMC-infused patients, performed yearly, has now increased to greater than 1.5% of the total donor bone marrow cells/million marrow cells counted. This trend toward increasing bone marrow chimerism in the recipients has more than quadrupled in the 6-year interval of followup. However, no attempt has been made to totally wean patients from immunosuppressive medications, and both groups still show no statistical differences in dosaging of either maintenance tacrolimus, mycophenolate, or corticosteroids. DISCUSSION AND CONCLUSIONS
Although no attempt has been made in this protocol to wean immunosuppressive therapy from DBMC-infused recipients, there has been statistically significant and increasing differences in graft survival caused by DBMC infusion, with fewer patients in this group experiencing chronic rejection on biopsy. Several mechanisms have been proposed for the improved graft acceptance associated with bone marrow infusion as a result of a number of in vitro studies from this laboratory by Mathew et al17–19 and Jin et al.20,21 As a result, the suppressor cell functions of DBMC have been much more clearly defined and may well be the effective mechanisms for explaining the decreased incidence of chronic rejection in this group.
Table 2. Iliac Crest DBMC Chimerism in Bone Marrow-Infused Recipients by PCR-Flow (cells/million counted; and %) ⴞ SD Year Posttransplant
1 (n ⫽ 42) 2 (n ⫽ 44) 3 (n ⫽ 39) 4 (n ⫽ 22) 5 (n ⫽ 17) 6 (n ⫽ 4)
Total
CD3⫹
CD34⫹
4210 ⫾ 3294 (0.4%) 7492 ⫾ 4966 (0.7%) 10,200 ⫾ 6763 (1.0%) 11,783 ⫾ 8221 (1.2%) 13,186 ⫾ 8383 (1.3%) 17,778 ⫾ 7036 (1.8%)
1749 ⫾ 1643 (0.2%) 3236 ⫾ 2415 (0.3%) 4408 ⫾ 3183 (0.4%) 5136 ⫾ 3805 (0.5%) 6008 ⫾ 4253 (0.6%) 9360 ⫾ 4505 (0.9%)
1616 ⫾ 1494 (0.2%) 2503 ⫾ 1805 (0.3%) 3191 ⫾ 2297 (0.3%) 3849 ⫾ 2745 (0.4%) 3958 ⫾ 2810 (0.4%) 5540 ⫾ 2929 (0.5%)
Table 3. Iliac Crest DBMC Chimerism in Noninfused Recipients by PCR-Flow (cells/million counted; and %) ⴞ SD Year Posttransplant
1 (n ⫽ 30) 2 (n ⫽ 22) 3 (n ⫽ 19) 4 (n ⫽ 12) 5 (n ⫽ 4) 6 (n ⫽ 5)
Total
717 ⫾ 510 (0.07%) 1116 ⫾ 474 (0.1%) 1491 ⫾ 462 (0.1%) 1475 ⫾ 538 (0.1%) 1663 ⫾ 1064 (0.2%) 2080 ⫾ 1085 (0.2%)
CD3⫹
CD34⫹
347 ⫾ 319 (0.03%) 574 ⫾ 375 (0.06%) 814 ⫾ 390 (0.08%) 683 ⫾ 476 (0.07%) 793 ⫾ 829 (0.08%) 1250 ⫾ 863 (0.1%)
113 ⫾ 126 (0.01%) 173 ⫾ 122 (0.02%) 288 ⫾ 75 (0.03%) 168 ⫾ 131 (0.02%) 160 ⫾ 188 (0.02%) 186 ⫾ 140 (0.02%)
PERIOPERATIVE DONOR BONE MARROW
REFERENCES 1. Barber WH, Mankin JA, Laskow DA, et al: Transplantation 51:70, 1991 2. Corry RJ, Chakrabarti PK, Shapiro R, et al: Ann Surg 230:372, 1999 3. Fontes P, Rao AS, Demetris AJ, et al: Lancet 344:151, 1994 4. Barber WH, Mankin JA, Laskow DA, et al: Transplantation 51:70, 1991 5. Rao AS, Phil D, Fontes P, et al: Adv Exp Med Biol 417:269, 1997 6. Monaco AP, Clark AW, Wood ML, et al: Surgery 79:384, 1976 7. Okazaki H, Maki T, Wood ML, et al: Transplantation 32:111, 1981 8. Monaco AP, Wood ML: Transplant Proc 2:489, 1970 9. Shapiro R, Starzl TE: Transplant Proc 30:1371, 1998 10. Rao AS, Shapiro R, Corry R, et al: Transplant Proc 30:1367, 1998 11. McDaniel DO, Naftilan J, Hulvey K, et al: Transplantation 57:852, 1994
3843 12. Garcia-Morales R, Carreno M, Mathew J, et al: Transplantation 65:956, 1998 13. Garcia-Morales R, Carreno M, Mathew J, et al: J Clin Invest 99:1118, 1997 14. Ciancio G, Miller J, Garcia-Morales RO, et al: Transplantation 71:827, 2001 15. Garcia-Morales R, Esquenazi V, Zucker K, et al: Transplantation 62:1149, 1996 16. Garcia-Morales R, Esquenazi V, Zucker K, et al: Transplant Proc 29:1219, 1997 17. Mathew JM, Carreno M, Fuller L, et al: Transplantation 68:1172, 1999 18. Mathew JM, Fuller L, Carreno M, et al: Transplantation 70:1752, 2000 19. Mathew JM, Fuller L, Carreno M, et al: Transplant Proc 33:108, 2001 20. Jin Y, Fuller L, Esquenazi V, et al: Hum Immunol 61:538, 2000 21. Jin Y, Fuller L, Wei Y, et al: Hum Immunol 61:1233, 2000