Autologous del(20q)-positive erythroid progenitor cells, re-emerging after DLI treatment of an MDS patient relapsing after allo-SCT, can provide a normal peripheral red blood cell count


A 54-year-old RhD-negative male with del(20q)-positive myelodysplastic syndrome was transplanted with bone marrow from an HLA-identical RhD-positive sibling donor. Cytogenetic relapse was detected 21 months after stem cell transplantation (SCT), with reappearance of the original del(20q)-positive clone and reversion to recipient RhD-negative blood group. The patient received sequential donor lymphocyte infusions (DLIs), resulting in mild graft-versus-host disease and pure red cell aplasia. At 2 years post DLI, the patient remains in a stable condition, despite a dominance of recipient-derived erythro- and granulopoiesis originating in del(20q)-carrying progenitor cells. We conclude that reappearance of autologous erythropoiesis, upon relapse after allogeneic SCT, may be predictive of erythropenia after DLI and that re-emerging autologous del(20q)-positive erythropoiesis post DLI can provide a normal peripheral red blood cell count. Furthermore, in patients relapsing after blood-group-mismatched transplantation, a possible reversion to recipient blood group should be considered prior to blood transfusion or DLI.


Investigating erythrocyte chimerism by red blood cell (RBC) phenotyping after allogeneic stem cell transplantation (SCT) has been a long-standing method used for monitoring of donor marrow engraftment and disease relapse,1,2 and several studies indicate that reappearance of recipient erythrocytes may herald relapse in haematological malignancies.1,2,3,4,5 Residual host cells may, however, represent normal haematopoiesis without any evidence of recurrent relapse.2,6 A possible source of detectable autologous erythrocytes post SCT would be a progenitor cell in common with the original malignant clone. Alternatively, the recipient RBCs may emerge from residual nonmalignant host stem cells. In the present study, we investigate the possible presence of cytogenetic abnormalities, initially observed at the time of diagnosis, in subsets of reappearing host cells, hereby elucidating the matter of recipient cell origin.

Case report

A 54-year-old previously healthy male was diagnosed with myelodysplastic syndrome (MDS RAEB-2), according to the WHO classification.7 The patient presented with pancytopenia (haemoglobin 122 g/l, white blood cells (WBCs) 3.0 × 109/l and platelets 72 × 109/l). A bone marrow examination revealed mild dysplastic features, most prominent in megakaryocytes, and the presence of 12% myeloblasts, with frequent occurrence of Auer rods. The karyotype by G-binding analysis was 46,XY,del(20)(q11)[24]/46,XY[1] (Table 1). Thus, according to the International Prognostic Scoring System for MDS,8 the patient was assigned to the Intermediate-2 group, necessitating more intensive treatment. The patient's RBCs typed as A RhD-negative.

Table 1 Cytogenetic findings in bone marrow at diagnosis and during follow-up

Following induction chemotherapy, morphological remission was achieved, although extensive megakaryocytic dysplasia was found in the marrow. The morphological remission was stable, and the fraction of dysplastic megakaryocytes decreased following two courses of post remission chemotherapy. At 4 months after diagnosis, the patient underwent allogeneic SCT, with bone marrow from an HLA-identical A RhD-positive sibling donor. The conditioning regimen consisted of cyclophosphamide 60 mg/kg once daily i.v. on days 1 and 2 (total dose 120 mg/kg) and fractionated total body irradiation (6 × 20 Gy). Conversion to donor RhD blood group was observed 90 days after SCT. At 1 year following SCT, the patient was in cytogenetic remission with a stable RhD-positive phenotype (Table 2).

Table 2 Monitoring of RhD phenotypic, STR-PCR, and cytogenetic findings

Cytogenetic relapse was detected 21 months after SCT. Apart from a clone with del(20q) as the sole change, several subclones with structural abnormalities, mainly balanced translocations, were also identified (Table 1). Clinically, the patient was well, but a slight thrombocytopenia was observed. Megakaryocytic and granulocytic dysplasia, but no increase in blast cells, was found upon bone marrow examination. RhD phenotyping revealed the reappearance of autologous RhD-negative RBCs (Table 2), without any detectable RhD-positive donor erythrocytes using standard serological typing techniques.

The patient was treated with sequential donor lymphocyte infusions (DLIs) at 1 month (1 × 107 CD3+ cells/kg), 3 months (7.4 × 107 CD3+ cells/kg) and 6 months (1.3 × 108 CD3+ cells/kg) after cytogenetic relapse. Chimerism in peripheral blood subsets was monitored monthly by short tandem repeat-polymerase chain reaction (STR-PCR) analysis during the course of DLI, showing 66–76% myeloid autologous cells and <1% autologous cells in the CD3+ and CD19+ subsets (Table 2). At 2 months after the last DLI, the patient developed mild graft-versus-host disease (GvHD). In addition, the patient developed a pure red cell aplasia (PRCA) with erythroblastopenia, requiring repeated blood transfusions (of A RhD-negative RBCs). Erythropoietin was administered during 136 days, with no apparent effect. After steroid treatment, however, erythropoiesis was normalized. WBC and platelet counts were continuously normal. At 6 months after DLI, the bone marrow morphology was unchanged compared to the time of relapse, and cytogenetic remission has never been achieved (Tables 1 and 2).

At 2 years after initiation of DLI therapy, that is, 48 months post SCT, the patient is in a stable condition, requiring no treatment. He has a normal peripheral blood cell count (haemoglobin 130 g/l, WBC 10.3 × 109/l and platelets 144 × 109/l), no change in bone marrow morphology and no overt GvHD. In chimerism analysis by STR-PCR, 84% of the bone marrow cells in the myeloid subset are of recipient origin. The RhD phenotyping is negative, indicating a dominance of autologous erythropoiesis (Table 2).

In order to determine the origin of the re-emerging autologous erythrocytes post DLI, erythroid bone marrow cells of different maturities were sorted and examined by interphase fluorescence in situ hybridisation (FISH) analysis, using a probe mapping to 20q12 (LSID20S108; Vysis Inc., Downers Grove, IL, USA). Mononuclear cells (MNCs) were obtained from a bone marrow aspirate and CD34+ cells were isolated using magnetic microbeads (CD34 isolation kit, Milteny Biotec, Bergisch Gladbach, Germany). The isolated CD34+ cells (1.6% of MNCs) and the remaining CD34-negative cells were subsequently sorted on a FACSVantage Cell Sorter (Becton Dickinson, San Jose, CA, USA) into subpopulations. The CD34+ cells were sorted for erythroid progenitor cells (expressing blood group A antigen) and for nonerythroid progenitors (Figure 1). The CD34-negative cells were sorted for maturing erythroid cells, expressing both CD71 (the transferrin receptor) and glycophorin A (GPA), for T-cells (CD3+), B-cells (CD19+), and for more (CD15high/CD33low) and less (CD15low/CD33high) mature granulocytic cells (Figure 1). The interphase FISH analysis (Figure 1) revealed that the absolute majority of the erythroid (Ahigh/CD34high, Alow/CD34low) and nonerythroid progenitor cells (A-/CD34+) harboured the del(20q), as did the maturing erythroid cells (CD71+/GPA+) and the different granulocytic cell populations (CD15high/CD33low and CD15low/CD33high), whereas the T-cells (CD3+) and B-cells (CD19+) were del(20q)-negative (Table 3).

Figure 1

Bone marrow cells sorted on a FACSVantage Cell Sorter and further analysed by FISH, using a probe for 20q12 (D20S108). Unsorted cells, forward scatter (FSC)/side scatter (SSC): (a) CD34+ cells, (b) CD34− cells. Sorting of CD34+ cells: (c) nonerythroid progenitors (A-/CD34+) and erythroid progenitors (Alow/CD34low and Ahigh/CD34high). Sorting of CD34− cells: (d) maturing erythroid cells (CD71+/GPA+), (e) B-cells (CD19+) and T-cells (CD3+), (f) less mature (CD15low/CD33high) and more mature (CD15high/CD33low) granulocytic cells. In FISH analysis, only one signal, indicating the presence of del(20q), is seen in (g) most maturing erythroid cells (CD71+/GPA+) and (h) erythroid progenitors (Ahigh/CD34high).

Table 3 Interphase FISH detection of del(20q) in sorted bone marrow cell populations


This study reveals the presence of a donor-derived lymphopoiesis and a recipient-derived myelopoiesis after DLI treatment following relapse, with the vast majority of erythrocytic and granulocytic cells being del(20q)-positive. The erythrocytic and granulocytic cells that were negative for the del(20q) may be donor-derived, as indicated by the 16% donor cells found in the myeloid subset by STR-PCR. However, the possibility of co-existing host cells lacking the del(20q) cannot be excluded. Such host cells could be non-neoplastic or neoplastic, with the latter harbouring cytogenetically cryptic mutations occurring prior to the del(20q).9,10 Detection of the del(20q) in erythroid cells after relapse suggests an origin common to the original myeloid malignant cell clone and the reappearing RhD-negative autologous RBCs. This notwithstanding, the 20q deletion apparently does not compromise erythroid differentiation, considering that the erythroid del(20q)-positive progenitor cells produce functional RBCs and provide a normal RBC count. Also, the granulocytic differentiation seems undisturbed, with the majority of the maturing granulocytes carrying the del(20q), but without any tendency for granulocytopenia. Furthermore, although the bone marrow displayed signs of dysplastic megakaryocytopoiesis, the platelet count is normal. Hence, the del(20q), as well as the presence of several cytogenetically complex subclones, which is a characteristic karyotypic feature of haematologic malignancies relapsing after SCT,11 may be a relatively ‘benign’ genetic change in myeloid progenitor cells, still being capable of normal differentiation in vivo. The fact that the del(20q) as a sole anomaly in MDS is considered a good-risk abnormality agrees well with this,8 as does the reported study on a patient, in whom a del(20q)-containing bone marrow had been used successfully for an autotransplant, showing that such cells may yield normal blood values.12 Furthermore, a report on a series of 55 patients who had a morphologically normal bone marrow but a cytogenetically abnormal bone marrow karyotype was recently presented, suggesting that clones with 20q- are associated with minimal morphological aberrations, at least during the early stages of disease evolution.13 Thus, it is tempting to conclude that further genetic, and perhaps epigenetic, events are required for transformation of ‘preleukaemic’ del(20q)-positive progenitors into leukaemic cells. The additional post remission chemotherapy given to the patient prior to SCT may have increased the risk of such genetic events. Possibly, the course of DLI upon relapse has eradicated or provided control of more malignant cells, explaining the patient's currently stable clinical condition, while ‘preleukaemic’ del(20q)-positive progenitors have escaped the graft-versus-leukaemia effect.

In the present patient, bone marrow aplasia post DLI was restricted to the erythroid lineage, resulting in transfusion-requiring anaemia. The development of PRCA was interpreted to be an effect of GvHD, subsequently overcome by steroid treatment. Why the GvHD effect was restricted to erythroid progenitors is not clear.

In this case of RhD-mismatched SCT, reversion to recipient RhD-negative blood group was detected upon relapse. It is still not clear which haematological diagnoses or subgroups of diagnoses are prone to blood group reversion upon relapse. Therefore we suggest that a possible return to the recipient blood group should be considered prior to blood transfusion or DLI in patients who relapse after blood-group-mismatched SCT.


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We thank Bodil Strömbeck (Department of Clinical Genetics in Lund) for her technical assistance in FISH analyses, as well as Anna Fossum and Dr Zhi Ma (Department of Stem Cell Biology in Lund) for their assistance in cell sorting. We also thank the Swedish Cancer Society, the Swedish Research Council, the Medical Faculty at Lund University and the Lund University Hospital Donation Funds for financial support of this study.

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Correspondence to J H Dykes.

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Dykes, J., Lindmark, A., Lenhoff, S. et al. Autologous del(20q)-positive erythroid progenitor cells, re-emerging after DLI treatment of an MDS patient relapsing after allo-SCT, can provide a normal peripheral red blood cell count. Bone Marrow Transplant 33, 559–563 (2004) doi:10.1038/sj.bmt.1704383

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  • red blood cells
  • blood group
  • 20q deletion
  • myelodysplastic syndrome
  • allogeneic stem cell transplantation
  • donor lymphocyte infusion

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