Ruri Kato

Risk and prevention of graft failure in patients with preexisting donor-specific HLA antibodies undergoing unmanipulated haploidentical SCT.

A role of donor-specific HLA antibodies (DSA) in graft failure after SCT has been suggested, but the relevance of DSA in unmanipulated haploidentical SCT (haplo-SCT) remains unknown. We prospectively examined HLA antibodies using the Luminex-based single Ag assay for 79 adult patients undergoing unmanipulated haplo-SCT. Among them, 16 (20.2%) were HLA Ab-positive, including five patients with antibodies not corresponding to donor HLA Ags and 11 DSA-positive patients. Of the 11 DSA-positive patients, five received treatments to decrease DSA levels, including two, who received plasma exchange and rituximab, two who received platelet transfusions from healthy-related donors having DSA-corresponding HLA Ags and one who received bortezomib. Platelet transfusion was the most simple and effective treatment option for class I DSA. The cumulative incidence of neutrophil recovery was significantly lower in pretransplant (post-treatment) DSA-positive patients than in DSA-negative patients (61.9 vs 94.4%, P=0.026). Notably, three of five patients with high levels of DSA had graft failure. Donors should be selected on the basis of an evaluation of HLA antibodies. If haplo-SCT from donors with HLA Ags that correspond to high levels of DSA must be performed, then recipients should be treated for DSA to improve the chances of successful donor engraftment.

Frequency of CD4(+)FOXP3(+) regulatory T-cells at early stages after HLA-mismatched allogeneic hematopoietic SCT predicts the incidence of acute GVHD.

Acute GVHD (aGVHD) is a major obstacle to allogeneic hematopoietic SCT (alloHSCT). Although it is thought that aGVHD is initiated in secondary lymphoid organs at a very early stage of alloHSCT, whether CD4+FOXP3+ regulatory T-cells (Tregs) have an impact on aGVHD development during this period remains unclear. Here, we measured Tregs in peripheral blood as early as possible after HLA-mismatched alloHSCT, and assessed the incidence of aGVHD. Flow cytometric analyses revealed that at the second week after HSCT, patients with aGVHD had significantly (P=0.018) lower Treg:CD4+T-cell ratios than those without aGVHD. As these differences were seen before the development of aGVHD, these ratios can predict the incidence of aGVHD. The cumulative incidence of aGVHD in patients with ratios of <9% was significantly higher than that in patients with ratios of ⩾9% (P=0.0082, log-rank test). Additionally, the specific ratio of Tregs:CD4+T-cells was the most significant value among all other possible lymphocyte-associated ratios and absolute cell counts. These findings suggest that the ratio of Tregs:CD4+T-cells at the second week post HLA-mismatched alloHSCT might be a potent predictor of aGVHD in these patients. The practical efficacy of this finding should be verified in further interventional studies.

Salvage haploidentical transplantation for graft failure using reduced-intensity conditioning

Graft failure is a major concern after cord blood transplantation (CBT) or HLA-haploidentical transplantation (haplo-SCT). As patients who undergo CBT or haplo-SCT almost always lack both matched-related and -unrelated donors, salvage transplantation would also be limited to either CBT or haplo-SCT. In this study, we assessed eight patients who received haplo-SCT as salvage therapy for graft failure. Five and three patients had received haplo-SCT and CBT, respectively, which resulted in graft failure. The median interval from the failed transplantation to salvage transplantation in six patients with primary graft failure was 33.5 days. The reduced-intensity conditioning regimen consisted of fludarabine, thiotepa, rabbit antithymocyte globulin and low-dose TBI. All eight patients achieved neutrophil engraftment, and seven patients achieved platelet recovery. The median times to neutrophil recovery and platelet recovery were 10 and 20 days, respectively. Three patients died from treatment-related causes: two from GVHD and one from rupture of carotid artery aneurysm. Five patients are alive, at a median follow-up of 946 days. The probability of overall survival at 5 years was 75%. These findings may serve as a rationale for giving precedence to haplo-SCT over CBT in salvage SCT after graft failure.

Intrabone Marrow Transplantation of Unwashed Cord Blood Using Reduced-Intensity Conditioning Treatment: A Phase I Study

The outcome of cord blood transplantation following reduced-intensity conditioning is suboptimal because of fatal infection triggered by prolonged neutropenia and graft-versus-host disease (GVHD) in addition to graft rejection. Intrabone marrow injection (IBMI) may improve the outcome by providing better hematopoietic engraftment and less GVHD. We therefore evaluated IBMI safety in reduced-intensity stem cell transplantation. Furthermore, we used unwashed cord blood to avoid stem cell loss. Ten patients (median age = 61 years old) were enrolled. Cord blood cells were thawed at the bedside and injected into 4 iliac bone sites (2 at each hemipelvis). The procedure was well tolerated with no injection-related complications. Nine patients achieved donor engraftment. The median time to neutrophil recovery (>0.5 × 10(9)/L) was 17 days, and platelet recovery was achieved in 8 patients. Early full donor chimerism was achieved (median of 15 and 20 days in T cells and myeloid cells, respectively). Three of 9 evaluable patients developed grade II to III GVHD, and 5 of 10 patients died of treatment-related toxicities. The probability of survival at 1 year was 46.7%. IBMI of unwashed cord blood following reduced-intensity conditioning is safe, well tolerated, and may lead to an increased donor engraftment rate.

Donor-derived HLA antibody production in patients undergoing SCT from HLA antibody-positive donors

Pre-existing donor-specific HLA antibodies in patients undergoing HLA-mismatched SCT have increasingly been recognized as a risk factor for primary graft failure. However, the clinical implications of the presence of HLA antibodies in donors remain unknown. We prospectively examined 123 related donors for the presence of HLA antibodies by using a Luminex-based single antigen assay. Of these, 1/57 (1.8%) male, 6/27 (22%) parous female and 0/39 (0%) nonparous female donors were HLA antibody-positive. Then, we determined the presence of HLA antibodies in seven patients who received SCT from antibody-positive donors. Of these, four became HLA antibody-positive after SCT. The specificities of the antibodies that emerged in the patients closely resembled those of the antibodies found in the donors, indicating their production by donor-derived plasma cells. Moreover, the kinetics of the HLA antibody levels were similar in all four patients: levels started increasing within 1 week after SCT and peaked at days 10–21, followed by a gradual decrease. These results suggest that donor-derived HLA antibody production frequently occurs in patients undergoing SCT from antibody-positive donors. Further studies are warranted for clarifying the clinical significance of donor-derived HLA antibodies, including the role of these antibodies in post transplant platelet transfusion refractoriness.

Incidence of extramedullary relapse after haploidentical SCT for advanced AML/myelodysplastic syndrome

Extramedullary (EM) relapse of leukemia after allo-SCT in patients with AML/myelodysplastic syndrome has been increasingly reported. The reduced effectiveness of the GVL effect in EM sites, as compared with BM, has been suggested to underlie this problem. We retrospectively analyzed the pattern of relapse after haploidentical SCT (haplo-SCT), performed as the first or second SCT. Among 38 patients who received haplo-SCT as their first SCT, the cumulative incidences of BM and EM relapse at 3 years were 40.5 and 10.9%, respectively. Among 19 patients who received haplo-SCT as their second SCT, the cumulative incidences of BM and EM relapse were 30.9 and 31.9%, respectively. Moreover, most of the patients who underwent repeat haplo-SCT for the treatment of EM relapse had further EM relapse at other sites. Post-relapse survival did not differ significantly with different patterns of relapse. The frequent occurrence of EM relapse after haplo-SCT, particularly when performed as a second SCT, suggests that the potent GVL effect elicited by an HLA disparity also occurs preferentially in BM. Our findings emphasize the need for a treatment strategy for EM relapse that recognizes the reduced susceptibility of EM relapse to the GVL effect.

Different mechanisms causing loss of mismatched human leukocyte antigens in relapsing t(6;11)(q27;q23) acute myeloid leukemia after haploidentical transplantation

Mismatched human leukocyte antigens (HLAs) on leukemic cells can be targeted by donor T cells in HLA‐mismatched/haploidentical stem cell transplantation. In two cases of acute myeloid leukemia with t(6;11)(q27;q23) abnormality presented here, flow cytometry analysis showed a lack of HLA‐A unshared between recipients and donors in relapsing leukemic cells after HLA‐haploidentical transplantation. However, high‐resolution HLA genotyping showed that one case lacked a corresponding HLA haplotype, whereas the other preserved it. These cases suggest that leukemic cells, which lacked mismatched HLA expression, might have an advantage in selective expansion under donor T‐cell immune surveillance after HLA‐haploidentical transplantation. Most importantly, down‐regulation of unshared HLA expression potentially occurs by genetic alterations other than loss of HLA alleles.

Haploidentical hematopoietic stem cell transplantation for lymphoma with monosomy of chromosome 6 (loss of heterozygosity in the HLA region)--who should be a donor?

Hematopoietic stem cell transplantation (HSCT) from an HLA haploidentical family donor is an option for patients who do not have a full HLA matched donor and lack the time to find an unrelated one [1,2]. Furthermore, it may facilitate a powerful graft versus leukemia/lymphoma (GVL) effect to help combat hematological malignancies by directly targeting the mismatched HLA expressed on leukemia/lymphoma cells [3]. On the contrary, leukemia/lymphoma cells escape from the surveillance of the donor-derived GVL effect by losing the target HLA (mismatched HLA in GVH direction). This mechanism has been called loss of heterozygosity (LOH) in the HLA gene region on chromosome 6 [4,5]. Taking the above into account, in this case report, we present a case of lymphoma with monosomy 6, which means natural LOH of HLA, and suggest that selection of a haploidentical family donor matched with the missing HLA haplotype seems to be very effective.

Detection of donor-derived CMV-specific T cells in cerebrospinal fluid in a case of CMV meningoencephalitis after cord blood stem cell transplantation.

Cytomegalovirus (CMV) meningoencephalitis is a rather rare complication after allogeneic stem cell transplantation. We describe here the case of a 59-year-old man with acute myeloid leukemia who developed CMV meningoencephalitis after cord blood transplantation. The patient presented with a sudden onset of neurological symptoms, such as convulsion, on day 37. The analysis of cerebrospinal fluid (CSF) sample revealed an increase in the number of cells, which were of donor (cord blood) origin, consisting mainly of T cells. No bacteria were detected in the CSF sample. Real-time PCR analysis revealed that the CSF sample was positive for CMV, but was negative for HHV-6, adenovirus, or BK virus. The patient was diagnosed with CMV meningoencephalitis and received cidofovir. His neurological symptoms were gradually improved and completely disappeared by day 60. CMV-specific dextramer-positive CD8+ T cells were detected in the peripheral blood and CSF samples, with the frequency being much higher in the CSF. To our knowledge, this is the first report on the appearance of CMV-specific T cells in CSF samples from a patient with CMV meningoencephalitis. Cord blood-derived CMV-specific T cells may develop early after transplantation, enter the intrathecal compartment, and likely contribute to the regulation of CMV-meningoencephalitis.

Direct antiglobulin test-negative autoimmune hemolytic anemia associated with HLA-haploidentical stem cell transplantation

Alloimmune hemolysis mainly associated with incompatibility in ABO blood groups between donors and recipients is well recognized in immune-mediated hemolysis after allogeneic hematopoietic stem cell transplantation (SCT) [1]. In contrast, autoimmune hemolytic anemia (AIHA) due to antibodies produced by the donor’s immune system against donor red cell antigens is rare. This occurs at a median time of 147 days after SCT, and shows no association with ABO incompatibility between recipient and donor pairs [2]. Here, we present a direct antiglobulin test (DAT)-negative AIHA on a patient who had undergone reduced-intensity SCT from a human leukocyte antigen (HLA)-mismatched ABO-incompatible related donor, although a DAT showing the binding of antibodies or complement system factors to red blood cell (RBC) surface antigens in vivo is usually helpful for AIHA diagnosis. A 54-year-old woman with acute megakaryoblastic leukemia with trilineage dysplasia achieved complete remission after a single course of induction chemotherapy. Because the megakaryoblastic subtypes and dysplasia indicated high risk of relapse, we proceeded with reducedintensity SCT from her HLA-2-antigen-mismatched son. Reduced-intensity conditioning consisted of fludarabine, busulfan, high-dose cytarabine, and rabbit antiT-lymphocyte globulin, with intravenous tacrolimus and methylprednisolone (1 mg kg day) as prophylaxis for acute graft-versus-host disease (GVHD), as previously reported [3]. Donor stem cells successfully engrafted and chimerism analysis on day 9 showed 100% donor type. Ganciclovir was administered at 5 mg kg day after the cytomegalovirus pp65 antigenemia assay turned positive on day 28. Grade 2 skin GVHD developed on day 73 at a prednisolone dose of 35 mg/day, but disappeared immediately on increasing the dose. There were no transplant complications thereafter, until Escherichia coli sepsis developed simultaneously with hemolytic anemia refractory to RBC transfusions on day 225. The hemolytic laboratory findings gradually resolved as the infection improved following the administration of biapenem. On day 298, hemolytic anemia reappeared after tapering the prednisolone dose to 18 mg/day; no symptoms of bacterial infection or chronic GVHD were present (Fig. 1). Complete blood counts showed normocytic anemia with reticulocytosis, whereas white blood cell and platelet counts were within normal ranges. Laboratory findings revealed elevated lactate dehydrogenase and undetectable haptoglobin levels. The ABO group was determined to be AB, as forward and reverse groupings were positive for both A and B antigens on RBC surfaces and negative for both anti-A and anti-B antibodies in the sera. Bone marrow aspirates showed normal trilineage hematopoiesis without abnormal cells. Moreover, fluorescence in situ hybridization with sex chromosome probes revealed complete donor chimerism in bone marrow cells. Thrombotic microangiopathy was excluded as neither neurological symptoms nor red cell fragments on peripheral blood films were observed. These findings suggested AIHA, but a routine DAT was nonetheless negative. Eventually, we diagnosed DATnegative AIHA on the basis of RBC-bound immunoglobulin G (RBC-IgG) using an immunoradiometric assay, J. Nakata H. Tamaki (&) K. Ikegame R. Kato S. Yoshihara K. Kaida T. Inoue M. Okada H. Ogawa Division of Hematology, Department of Internal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan e-mail: tamakhi@hyo-med.ac.jp