Successful non-T-cell-depleted nonmyeloablative hematopoietic stem cell transplantation (NST) from an HLA-haploidentical 2-loci-mismatched sibling in a heavily transfused patient with severe aplastic anemia based on the fetomaternal microchimerism

Summary:

A 37-year-old Japanese man with systemic hemochromatosis due to multiple transfusions was referred to us for the treatment of severe aplastic anemia (SAA), from which he had been suffering for 24 years. The patient had diabetes arising from the hemochromatosis, chronic anal fissures, and a kidney abscess due to neutropenia. He was treated with a nonmyeloablative preconditioning regimen followed by non-T-cell-depleted (non-TCD) allogeneic peripheral blood stem cell transplantation (PBSCT) from his human leukocyte antigen (HLA)-haploidentical 2-loci-mismatched sibling. Prompt engraftment of granulocytes and platelets was observed, and graft-versus-host disease was easy to control. Noninherited maternal antigens in the donor were confirmed prior to PBSCT, and they were also detected in small quantities in the recipient. This report describes the first successful nonmyeloablative hematopoietic stem cell transplant in a heavily transfused SAA patient from an HLA-haploidentical 2-loci-mismatched sibling donor. The result suggests that a long-term fetomaternal microchimerism-positive sibling can be a second-line donor if an alternative HLA-identical donor is not available.

Main

Allogeneic stem cell transplantation (allo-SCT) is one of the curative treatments for patients with severe aplastic anemia (SAA). Unfortunately, the majority of patients lack a sibling donor who is human leukocyte antigen (HLA) compatible. Allo-SCT using a mismatched related or unrelated donor is associated with a higher incidence of treatment failure than is deemed acceptable for practical procedures because of graft rejection, especially in patients who have been previously heavily transfused.1, 2, 3, 4 There have not yet been any successful reports of nonmyeloablative hematopoietic SCT (NST) from a haploidentical sibling donor for a very high-risk SAA patient with a history of frequent transfusions. Recently, progress has been made in clarifying the processes of the immunological interactions between the fetus and mother. The fetus is exposed to noninherited maternal antigens (NIMA), while the mother is exposed to paternal antigens of the fetus, and it is likely that immunological tolerance is established between the mother and fetus.5, 6, 7 Based on this hypothesis, it is feasible to perform non-T-cell-depleted (non-TCD) SCT between haploidentical family members and patients with refractory, high-risk hematological malignancies.8 However, for nonmalignant diseases, no successful cases of NST from a haploidentical sibling donor have been reported. This patient was refractory to anabolic steroids, some immunosuppressive drugs, and antithymocyte globulin (ATG). He had received routine blood and platelet transfusions from 13 years of age. HLA-matched platelet infusions were thus required. The patient also suffered from diabetes arising from hemochromatosis and chronic or recurrent infections. Consequently, we carried out a non-TCD NST from the 2-loci-mismatched sibling in whom long-term fetomaternal microchimerism had been confirmed. The patient is now quite healthy and was confirmed to be hematologically normal 8 months after transplantation.

Case report

A 37-year-old Japanese man had suffered from SAA for 24 years. He had received cyclosporin A, ATG, prednisolone (PSL), granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor, and erythropoietin. However, none of these drugs had any significant effect on his disease. Consequently, he was forced to undergo repeated transfusions due to severe anemia and bleeding.

He was diabetic because of the hemochromatosis secondary to the numerous red blood cell transfusions, and HLA-matched platelets were needed because of anti-HLA antibodies resulting from repeated platelet transfusions. He frequently had fever and sepsis due to a chronic anal fissure and suffered from a kidney abscess because of neutropenia.

On admission, his hemoglobin was 3.5 g/dl, leukocyte count 0.9 × 109/l (stab and segmented neutrophils: 46%; eosinophils: 1%; monocytes: 8%; and lymphocytes: 45%), platelets 1 × 1010/l, lactate dehydrogenase level of 94 IU/l, and C-reactive protein 0.7 mg/dl (normal range below 0.3 mg/dl). Bone marrow aspirate showed severely fatty marrow. He not only had HLA complete matched siblings but was also much older and too heavily transfused for us to expect a reasonable result in an SCT from an unrelated donor. Although his pancytopenia was progressive and immediate transplantation was necessary, the coordination of an unrelated donor would have taken at least 3 months. We therefore decided to perform an HLA-haploidentical stem cell transplant from a sibling. There was a two-locus mismatch in both the graft-versus-host and host-versus-graft directions between patient and donor. (Patient: HLA-A 26,31; -B 61,56; -C 3,4, DR 8,9. Donor: HLA-A2, 31; -B 35,56; -C 3,4 DR 8,9. Their mother: HLA-A 2,26; -B 35,61; -C 3,-; DR 9,-). The result of the HLA-nested polymerase chain reaction with sequence-specific primer typing (PCR-SSP) for detecting the NIMA is shown in Figure 1. The donor tested positive for the donor NIMA that were positive in the recipient (Figure 1a). The recipient tested slightly positive for the recipient NIMA, which were positive in the donor (Figure 1b). Frequent transfusions were the cause of the weak recipient NIMA. These results suggest immunological tolerance between donor and patient. The patient was treated with a nonmyeloablative preconditioning regimen that included fludarabine (Flu, 25 mg/m2 for 5 days), ATG (5 mg/kg for 3 days), and total body irradiation (TBI, 6 Gy in three fractions over 2 days) followed by a non-TCD allogeneic peripheral blood stem cell transplantation (PBSCT) from his HLA-haploidentical 2-loci-mismatched sibling. The patient received 5.09 × 106/kg of G-CSF mobilized CD34-positive cells. Graft-versus-host disease (GVHD) prophylaxis was tacrolimus 0.02 mg/kg by continuous intravenous infusion and methotrexate, 10 mg/m2 on day 1, and 7 mg/m2 on days 3 and 5, for GVHD prophylaxis. The clinical course is shown in Figure 2. Neutrophil engraftment occurred on day 16 and platelet engraftment on day 26. A short tandem repeat analysis of peripheral blood (PB) was performed. CD3-, CD14-, 15-, and CD56-positive cells were almost 100% of donor origin after neutrophil engraftment. Mild chronic GVHD, that is, dry skin and slight stomatitis, developed on day 86 after SCT, which resolved within 1 week after administration of 25 mg of PSL. PSL was then tapered by 5 mg every 2 weeks. Thereafter, no other complications occurred.

Figure 1
figure1

Nested PCR-SSP revealed long-term fetomaternal microchimerism in the patient and his sister. (a) The HLA-A26 (NIMA)-specific band was detected in the PB of the donor in lanes 5 and 6 (lane 1: water; lane 2: donor PB, SSP of HLA-A26; lane 3: donor nail, nested PCR, 10−2 dilution of first PCR product; lane 4: donor nail, nested PCR, 10−3 dilution of first PCR product; lane 5: donor PB, nested PCR, 10−2 dilution of first PCR product; lane 6: donor PB, nested PCR, 10−3 dilution of first PCR product; lane 7: recipient PB, SSP of HLA-A26; lane 8: recipient, nested PCR, 10−6 dilution of first PCR product; lane 9: recipient, nested PCR, 10−8 dilution of first PCR product). (b) The HLA-A2 (NIMA)-specific band was barely detected in the PB of the recipient in lane 5 (lane 1: water; lane 2: recipient PB, SSP of HLA-A2; lane 3: recipient nail, nested PCR, 10−2 dilution of first PCR product; lane 4: recipient nail, nested PCR, 10−3 dilution of first PCR product; lane 5: recipient PB, nested PCR, 10−2 dilution of first PCR product; lane 6: recipient PB, nested PCR, 10−3 dilution of first PCR product; lane 7: donor PB, SSP of HLA-A2; lane 8: donor nested PCR, 10−6 dilution of first PCR product; lane 9: donor nested PCR, 10−8 dilution of first PCR product).

Figure 2
figure2

Clinical course of the patient. Normal engraftment was obtained and severe GVHD was not observed.

Discussion

AA is one of several life-threatening disorders of the bone marrow, but the introduction of immunosuppressive therapy has improved the prospects for severe AA patients. Although SCT from an HLA-matched sibling has shown favorable results, use of unrelated donors has resulted in a poor outcome, especially for high-risk patients.1 SAA patients sometimes experience organ failure after multiple transfusions and a long-disease course and develop severe infections due to the neutropenia. In these high-risk patients, conventional SCT has resulted in a poor outcome, with a high incidence of graft rejection. In this case, graft rejection was a concern because the patient had been sensitized with alloantigens after receiving multiple transfusions.

Intensive conditioning regimens, including low-dose (3–5 Gy) TBI, are required to ensure engraftment of donor stem cells in severe AA patients who have been repeatedly transfused.9 A conventional SCT, however, was risky for this patient because of the possibility of infection due to the kidney abscess and anal fissure and the diabetes; therefore, a reduced-intensity regimen was selected. The combination of Flu, ATG, and TBI was tolerated well and no infections were documented. Furthermore, graft rejection did not occur and only controllable GVHD was seen. The successful engraftment indicates that the Flu, ATG, and TBI combined regimen is sufficiently potent to suppress the host-versus-graft reaction, even in a multiply transfused, 2-locus-mismatched patient. Many transplant candidates lose the opportunity for SCT due to the lack of an HLA-matched donor. TCD or CD34-positive cell selection (CD34+) SCT has been previously performed for HLA-nonidentical SCT. In these cases, a high incidence of graft failure was reported.10 On the other hand, several investigators have reported non-TCD SCT from HLA-haploidentical donors based on the fetomaternal microchimerism theory.8, 11, 12 This patient was heavily transfused and was not eligible for transplantation from an unrelated donor. This report describes the first successful non-TCD NST for SAA from an HLA-haploidentical 2-loci-mismatched sibling donor. Judging from our experience, it may be possible to evaluate SCT from a haploidentical donor by using the fetomaternal microchimerism hypothesis, even for heavily transfused adult SAA patients. Furthermore, Flu, ATG, and 6 Gy of TBI may be sufficiently potent to suppress the immune reaction between a haploidentical 2-locus-mismatched sibling and a heavily transfused adult SAA patient.

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Correspondence to Y Tsutsumi.

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Tsutsumi, Y., Tanaka, J., Miura, T. et al. Successful non-T-cell-depleted nonmyeloablative hematopoietic stem cell transplantation (NST) from an HLA-haploidentical 2-loci-mismatched sibling in a heavily transfused patient with severe aplastic anemia based on the fetomaternal microchimerism. Bone Marrow Transplant 34, 267–269 (2004) doi:10.1038/sj.bmt.1704567

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Keywords

  • nonmyeloablative stem cell transplantation (NST)
  • HLA 2-loci-mismatched donor
  • noninherited maternal antigen (NIMA)
  • fetomaternal microchimerism
  • severe aplastic anemia (SAA)

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