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Donor choice in haploidentical stem cell transplantation: fetal microchimerism is associated with better outcome in pediatric leukemia patients

Bone Marrow Transplantation volume 50, pages 1367–1370 (2015)Cite this article

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Haploidentical hematopoietic stem cell transplantation (hHSCT) from a parent to a child has become well established for pediatric patients with high-risk leukemia in the absence of an HLA-matched donor.1, 2, 3, 4 In most cases, both parents are eligible and willing to donate stem cells for their child. Which parent is the better stem cell donor depends on transplant modality and in particular on graft manipulation: in T cell-depleted hHSCT (TCD-hHSCT) the mother is the better donor, whereas in non-depleted hHSCT the father is the better donor.5 In TCD-hHSCT, improved event-free survival and lower risk of relapse were reported for mother-to-child transplantations compared with father-to-child transplantations.6, 7 In the group of mother-to-child transplantations, transplant-related mortality and incidence of GvHD were markedly lower compared with the father-to-child transplantations. We assumed that pregnancy left an imprint on the mother’s immune system, which is beneficial in hHSCT from the child’s mother. During pregnancy, a bidirectional cell transfer, from mother to child and vice versa, may result in persisting fetal cells in the mother (fetal microchimerism), which are detectable as long as three decades after birth.8, 9 Hence, we hypothesized that (i) fetal microchimerism (FM) has a role in hHSCT and that (ii) FM may be influenced by the killer immunoglobulin-like receptors (KIRs) pattern on maternal natural killer (NK) cells and KIR ligands on the child’s cells. CD56bright NK cells make up ~70% of the lymphocytes in the decidua during the first trimester of pregnancy and represent the major cell subset interacting with cells of the developing fetus.10 Not surprisingly, certain maternal KIR/fetal KIR ligand combinations are associated with recurrent spontaneous abortions or preeclampsia.11

In compliance with the regulations of the Institutional Review Board (decision PV4296), we retrospectively analyzed 46 pediatric patients who underwent T cell-depleted hHSCT from a parent between 2003 and 2013 in six German transplant centers. Median age was 5 years (range 4 months–21 years), and indications included malignant as well as nonmalignant diseases (Table 1). Patients had ALL (n=10), AML (n=10), juvenile myelomonocytic leukemia (n=1), immune deficiencies (n=8) or myelodysplastic syndrome (n=4). All patients underwent conditioning with a combination of fludarabine, thiotepa and melphalan as well as serotherapy with OKT3 or antithymocyte globulin before receiving the T cell-depleted graft of granulocyte-CSF mobilized PBSC. None of the patients received cyclophosphamide post transplantation. Median stem cell dose was 10.5 × 106 (range 2.2–80.2 × 106) per kg body weight and median T-cell dose was 4.8 × 104 (range 0.6–15.3 × 104) per kg body weight. Excess DNA of the donor extracted immediately before transplant from peripheral blood was assayed for fetal DNA (in mother-to-child transplantations) and for the donor’s KIR pattern as well as the recipient’s HLA-C type. First, specific markers were selected for each recipient/mother pair using an extended panel of human biallelic short insertion/deletion polymorphisms,12 as well as Y-chromosome-specific sequences.13 Real-time quantitative PCR (qPCR) was performed using the TaqMan technology (Applied Biosystems, Foster City, CA, USA). To standardize DNA content, a second PCR reaction detecting the human hematopoietic cell kinase gene was carried out in the same well. A volume of 10 μL DNA (containing 200–400 ng DNA) was analyzed in 25 μL reaction mixes using the iTaq Universal Probes Supermix (Bio-Rad, Foster City, CA, USA). Primers and probes were obtained from Eurofins MWG GmbH (Ebersberg, Germany). Fetal cell content was quantified based on ct (= threshold cycle) values obtained after qPCR. When sufficient DNA was available, we performed 5–10 analyses of each sample at the detection limit of 5 × 10−4 to increase sensitivity. Each positive reaction was considered as one hit among the number of cells analyzed in that tube. The level of fetal microchimerism was defined as the number of maternal cells in which one hit was detected, for example, one hit in 300 000 maternal cells is reported as a FM level of 3 × 10−5. FM levels greater than one fetal cell in 250 000 maternal cells were considered as FM positive. KIR genotyping was performed by real-time PCR as described by Alves et al.14

Table 1 Patient characteristics
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Acknowledgements

This study was supported by Forschungs- und Wissenschaftsstiftung Hamburg, Hamburg, Germany, Fördergemeinschaft Kinderkrebs-Zentrum Hamburg eV, Hamburg, Germany, and Rüdiger Colditz Stiftung, Germany. Purchase of equipment for this work was supported by the Rudolf Bartling Stiftung (III/103).

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  1. A Kruchen and T Stahl: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Institute Children’s Cancer Center Hamburg, Hamburg, Germany

    A Kruchen, F Gieseke & I Müller

  2. Department of Pediatric Hematology and Oncology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    A Kruchen & I Müller

  3. Research Department Cell and Gene Therapy, Interdisciplinary Clinic of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    T Stahl & B Fehse

  4. Department for Transfusion Medicine, University Medical Center, Hamburg-Eppendorf, Hamburg, Germany

    T M C Binder

  5. Clinic for Pediatric Oncology, Hematology and Clinical Immunology, Center for Child and Adolescent Health, Medical Faculty, Heinrich Heine University, Düsseldorf, Germany

    Z Özcan & R Meisel

  6. Division for Stem Cell Transplantation and Immunology, Department for Children and Adolescents, University Hospital, Goethe University, Frankfurt/Main, Germany

    H Kreyenberg & P Bader

  7. Department of Pediatrics, Jena University Hospital, Jena, Germany

    B Gruhn

  8. Department of Pediatric Oncology, Hematology and Immunology, University Hospital, Ruprecht Karls University, Heidelberg, Germany

    J Greil

  9. Department of General Pediatrics, Hematology and Oncology, Children’s University Hospital, Eberhard Karls University, Tuebingen, Germany

    M Pfeiffer, M Döring & R Handgretinger

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Correspondence to A Kruchen.

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Kruchen, A., Stahl, T., Gieseke, F. et al. Donor choice in haploidentical stem cell transplantation: fetal microchimerism is associated with better outcome in pediatric leukemia patients. Bone Marrow Transplant 50, 1367–1370 (2015). https://doi.org/10.1038/bmt.2015.136

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