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2048
Prediction of relapse of acute myeloid leukemia in allogeneic transplant recipients by marrow CD34 þ donor cell chimerism analysis
Leukemia (2004) 18, 2048–2050. doi:10.1038/sj.leu.2403507 Published online 7 October 2004 TO THE EDITOR
Chimerism analysis after allogeneic hematopoietic stem cell transplantation (AHSCT) unveils the conversion of recipient’s hematopoiesis to donor type and is useful for detection of graftfailure states. However, the prognostic significance of persistence or recurrence of host-derived cells in relation to relapse and long-term survival of acute leukemias has, as yet, been controversial.1–3 Moreover, little is known with regard to whether the analysis of donor cell chimerism (DCC) can be used to detect MRD in otherwise marker-negative hematological malignancies, and whether it adds to the prediction of relapse after AHSCT in leukemias. PCR-based amplification of highly polymorphic di-, tri- and tetranucleotide repeat sequences (short tandem repeat (STR) markers) interspersed throughout the genome allow for quantitative discrimination between host and donor hematopoiesis, and are now being evaluated for their clinical relevance in MRD monitoring and relapse prediction.4,5 In this report, we prospectively analyzed lineage-specific chimerism using the STR amplification technique of serial samples of peripheral blood (PB) and bone marrow (BM) aspirates from 20 AML patients following AHSCT, to investigate whether post-transplant chimerism is able to predict host-derived leukemic hematopoiesis early on and whether it can be used as a MRD marker. From July 1999 to July 2002, 20 patients with primary or secondary AML were enrolled in a prospective reduced-intensity AHSCT protocol at the University of Muenster. Patients were eligible for the study if excluded from conventional AHSCT because of age (450 years), prior severe infections and/or history of invasive fungal infection, heavy pretreatment, and/or prior severe organ toxicity. Disease and treatment characteristics for the 20 patients who were studied prospectively are summarized in Table 1. All patients were conditioned with a radiation-based preparative regimen consisting of 4 Gy TBI (n ¼ 1), 6 Gy TBI (n ¼ 3) or 8 Gy TBI (n ¼ 16) in combination with fludarabine. A total dose of 120 mg/m2 fludarabine was given on four consecutive days. Median patient age was 49.5 (range 21–65 years). Acute graft-versus-host disease prophylaxis consisted of antithymocyte globuline at a total dose of 40 mg/kg body weight and cyclosporine A, starting with a dose of 3 mg/kg/day. In addition, a short course of methotrexate was given at days þ 1 (15 mg/m2), þ 3, þ 6, þ 11 (10 mg/m2). Treatment of relapse consisted of DLTs at doses escalating from 5 106 to 1 107 cells/kg body weight and in four patients, followed by a second stem cell transfusion (DSCT) of the original donor (max. 2 106 CD34 þ cells/kg body weight). Prior to DSCT, patients received low-dose cytosine arabinoside (ARA-C) subcutaneously at a dose of 2 10 mg/m2 over 14 consecutive days. After DSCT, patients were treated with Correspondence: Dr J Kienast, Department of Medicine/Hematology and Oncology, University of Muenster, Albert-Schweitzer-Str. 33, D-48129 Muenster, Germany; Fax: þ 49 251 83 5 28 04; E-mail:
[email protected] Received 4 August 2004; accepted 5 August 2004; Published online 7 October 2004 Leukemia
granulocyte–macrophage colony-stimulating factor (GM-CSF) subcutaneously at a dose of 75 mg/m2/day for 4 consecutive weeks. Genomic DNA for multiplex PCR of microsatellite markers was extracted directly from total mononuclear cells or from purified subsets (PB: CD3 þ , CD14 þ , CD19 þ ; BM CD3 þ , CD14 þ , CD19 þ , CD34 þ ) using the QIAamp DNA blood kit (Quiagen, Hilden, Germany) according to the manufacturer’s recommendations. Subset purification was performed by magnetic cell separation technique using the autoMACS system (MACS; Miltenyi Biotech, Bergisch Gladbach, Germany) and by flow sorting using a FACS Vantage SE (Becton Dickinson, Mountain View, CA, USA). Nine tetra-nucleotide microsatellite regions were co-amplified with dye-labeled primers using the AmpFLSTR Profiler PCR amplification kit (Applied Biosystems, Weiterstadt, Germany). PCR reaction and fluorescent fragment analysis were performed as described by Thiede et al.6 The lowest starting cell number for chimerism analysis with a clear readout was 500. In addition, all serial samples underwent immunophenotyping using a three-color panel with 13 antibody combinations for identification and quantification of leukemic blasts by inappropriate cell surface antigen expression with a lower limit of detection of 0.5%. Furthermore, cytogenetic analysis of each bone marrow aspirate was performed. In order to be able to detect impending relapses, DCC of total mononuclear cells and subsets from PB and BM was determined in short intervals within the first 2 years: 4 week intervals during the first 12 months after engraftment, and in 2–3-month intervals thereafter. The median number of informative alleles for related donor–recipient pairs was 4 (range, 2–6) and for unrelated pairs 7 (range, 4–9). The median follow-up for the patients on study was 546 days (range, 93–1483), with a median number of analyses per patient of 10 (range, 2–26). DCC analysis of BM specimens at engraftment revealed that only CD3 þ T cells were of partial host origin (mean, 82.7%; s.d., 722.7%), whereas monocytes (mean, 99.3%; s.d., 71.5%), B cells (mean, 98.9; s.d., 71.6%) and CD34 þ progenitors (mean, 95.9; s.d., 76.4%) were of almost complete (495%) donor origin. Comparable results were obtained from peripheral blood (data not presented). By sequentially monitoring marrow CD34 þ DCC, we were able to detect relapse only in those patients with CD34 þ expression on leukemic blasts (UPN 9904, 9918, 0004, 0010, 0023, 0105, 0136, 0202, 0204), suggesting that its decrease was caused by reappearance of the leukemic clone. Notably, in some patients, marrow CD34 þ DCC decreased 21–91 days prior to the diagnosis of relapse by morphology, cytogenetics or immunophenotyping. Furthermore, a marrow CD34 þ DCC decreasing to below 75% was highly predictive of relapse in AML patients expressing CD34 antigen on the leukemic clone (Po0.001; Table 2). In contrast, patient UPN 0104 with CD34-negative AML at diagnosis showed nearly complete DCC (94%) in the CD34 þ flow-sorted cell fraction even at the time relapse was diagnosed by morphology. Of note, DCC analysis of total mononuclear cells or subsets (CD3 þ , CD14 þ , CD19 þ ) from PB and BM were not predictive of relapse (data not presented). The clinical potential of sequential analysis of marrow CD34 þ DCC for guidance of post-transplant treatment interventions is demonstrated in Figure 1. UPN 0004, a male patient with secondary AML M2 in CR after HLA-identical AHSCT, developed decreasing marrow CD34 þ DCC beyond day þ 315. At that time,
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2049 Table 1
Patient characteristics
UPN
FAB type
Leukemic phenotype CD34+
Donor
Status before transplant
Status post-transplant
9904 9918 0004 0010 0023 0105 0111 0112 0114 0136 0202 0204 0216 9921 0009 0020 0104 0106 0207 0225
M2 M5 M2 M1 M5 M5 M4eo M2 M2 M2 M5 M4 M6 M3 M1 M6 M5 M5 M2 M5
+ + + + + + + + + + + + +
MRD MRD MRD MRD MRD MUD MUD MRD MUD MUD MUD MUD MRD MRD MUD MRD MUD MRD MRD MUD
CR1 RD REL1 RD RD CR1 CR1 RD CR2 RD CR2 RD CR1 CR2 RD RD RD REL1 CR1 CR3
CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR CR
aGvHD
Relapse
2 2 2 2 2 4 1
+ + + cyR + + + + + + +
Treatment post-transplant
Days follow-up (cause of death)
DSCT DLT, DSCT DLT DLT, AHSCT DLT, DSCT DLT DLT DLT, DSCT DLT
390a (PD) 161a (PD) 1474b (rel.) 384a (c.d.) 629a (PD) 1138b (PD) 1048b (CR) 146a (TR, inf.) 1021b (CR) 728a (PD) 134a (PD) 414a (PD) 664b (CR) 1545b (CR) 102a (TR, GvHD) 1286b (CR) 162a (PD) 93a (PD) 738b (CR) 594b (CR)
Marrow CD34+ donor cell chimerism and relapse
Leukemic phenotype CD34+ AML
CD34 AML
DCC 475% o75% 475% o75%
Patients (n) Relapses (n) 3 10 5 2
0 9 1 1
w2 test Po0.001% NS
DCC, donor cell chimerism. NS, not significant. Pearson w2-test.
morphologic relapse was diagnosed, showing 20% blasts in BM cytology. Treatment interventions consisted of dose-escalating DLTs (days þ 326, þ 341, þ 355), low-dose ARA-C (days þ 368 through þ 395), followed by DSCT (day þ 399) and GM-CSF (days þ 400 through þ 427). This treatment led to a complete conversion of CD34 þ chimerism to donor origin with restoration of complete hematological remission. However, another significant decrease of CD34 þ DCC occurred at day þ 683 (66%). This time, diagnosis of relapse by BM cytology and immunophenotyping was delayed by 91 days. With the aim of preventing impending relapse, early interventions with dose-escalating DLTs (days þ 733, þ 747, þ 762), low-dose ARA-C (days þ 762 through day þ 824), DSCT (day þ 826) and GM-CSF (days þ 827 through day þ 855) were initiated. With this approach of early interventions based on marrow CD34 þ DCC, CRs could be re-established in this patient twice during a period of 3.5 years. These results demonstrate that a decrease of marrow CD34 þ DCC is highly predictive for relapse in AML patients with a CD34 þ leukemic phenotype (Po0.001). In contrast, chimerism analyses of T cells, B cells and monocytes from PB or BM were not or far less sensitive for diagnosis of imminent relapse. In our observations, post-transplant chimerism analysis by STR-PCR had advantages over other methods of MRD detection. In
DCC CD34 + BM cells (%)
Table 2 incidence
100 90 80 70 60 50 40 30 20 10 0
UPN 0004 BM blasts >5% day+315 day+774
Relapse treatment #1 : day+326-427 #2 : day+733-855
0
150
300 450 600 Days after AHSCT
750
20 18 16 14 12 10 8 6 4 2 0 900
CD33 + CD117 + BM cells (%)
AHSCT, allogeneic hematopoietic stem cell transplantation from second donor; c.d., cardiac death; CR, complete remission; cyR, cytogenetic relapse; DLT, donor lymphocyte transfusion; DSCT, donor stem cell transfusion; aGvHD, acute graft-versus-host disease; inf., infection; MRD, matched related donor; MUD, matched unrelated donor; PD, progressive disease; RD, refractory disease; REL, untreated relapse; TR, treatment related. a Death. b Alive.
Figure 1 Sequential monitoring of CD34 þ DCC as a predictive marker and guidance for therapeutic interventions in high-risk AML. Percentage of CD34 þ DCC compared to the percentage of immature myeloid blasts (CD33 þ , CD117 þ ) in the bone marrow of patient UPN 0004 after AHSCT. A decreasing marrow CD34 þ DCC was observed 91 days prior to diagnosis of second relapse by cytology (day þ 683 vs day þ 774). Therapeutic interventions (DLTs, low-dose ARA-C, DSCT, GM-CSF) starting at days þ 326 and þ 733 were able to restore complete marrow CD34 þ DCC and induce complete hematological remissions.
several instances, molecular chimerism analysis revealed disease recurrence significantly earlier than both multiparameter flow cytometry and cytogenetics. However, some limitations of the data presented require consideration. First, AML patients commonly express CD34 antigen (70–75%) on malignant cells, but the leukemic immunophenotype can alter from diagnosis to relapse.7 Nonetheless, shifts in CD34 phenotype should not be of particular concern since gain of CD34 expression on leukemic cells is more common than its loss.8 Second, the Leukemia
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2050 number of patients studied is limited and all transplant recipients received a dose-reduced conditioning. Thus, in order to more broadly establish the role of MRD detection using chimerism analysis in marrow CD34 þ progenitors, confirmative studies in AML and potentially other leukemias, and particularly in transplant recipients treated with conventional conditioning regimens, will be required. Furthermore, the cutoff of 75% DCC was arbitrarily set and marrow CD34 þ DCC-guided treatment post-transplant, as suggested by the present data, will need to be studied prospectively in larger cohorts of patients. In conclusion, CD34 þ chimerism analyses of bone marrow samples collected in monthly intervals during the first year following AHSCT appears to be an appropriate method of monitoring MRD in high-risk AML with CD34 expression. It may provide guidance for clinical interventions, thereby allowing for earlier treatment and, in some cases, prevention of overt relapses. The data shown here might also be a basis for further studying the principle of choosing leukemia/lymphoma-specific cell surface markers and chimerism analysis of the cell population sorted for this marker for early detection of MRD or relapse.
Acknowledgements We thank Christina Burhoi, Simone Niehues, Annegret Rosemann and Colette Huenefeld for their excellent technical assistance and Dr Andreas Grote for statistical analysis.
C Scheffold1 M Kroeger1 M Zuehlsdorf1 J Tchinda1 G Silling1 G Bisping1 M Stelljes1 T Buechner1 WE Berdel1 J Kienast1
1 Department of Medicine/Hematology and Oncology, University of Muenster, Muenster, Germany
References 1 Bader P, Beck J, Frey A, Schlegel PG, Hebarth H, Handgretinger R et al. Serial and quantitative analysis of mixed hematopoietic chimerism by PCR in patients with acute leukemias allows the prediction of relapse after allogeneic BMT. Bone Marrow Transplant 1998; 21: 487–495. 2 Mattsson J, Uzunel M, Tammik L, Aschan J, Ringden O. Leukemia lineage-specific chimerism analysis is a sensitive predictor of relapse in patients with acute myeloid leukemia and myelodysplastic syndrome after allogeneic stem cell transplantation. Leukemia 2001; 15: 1976–1985. 3 Lawler M, Humphries P, McCann SR. Evaluation of mixed chimerism by in vitro amplification of dinucleotide repeat sequences using the polymerase chain reaction. Blood 1991; 77: 2504–2514. 4 Bader P, Kreyenberg H, Hoelle W, Dueckers G, Handgretinger R, Lang P et al. Increasing mixed chimerism is an important prognostic factor for unfavorable outcome in children with acute lymphoblastic leukemia after allogeneic stem-cell transplantation: possible role for pre-emptive immunotherapy? J Clin Oncol 2004; 22: 1696–1705. 5 Thiede C, Bornhauser M, Oelschlagel U, Brendel C, Leo R, Daxberger H et al. Sequential monitoring of chimerism and detection of minimal residual disease after allogeneic blood stem cell transplantation (BSCT) using multiplex PCR amplification of short tandem repeat-markers. Leukemia 2001; 15: 293–302. 6 Thiede C, Florek M, Bornhauser M, Ritter M, Mohr B, Brendel C et al. Rapid quantification of mixed chimerism using multiplex amplification of short tandem repeat markers and fluorescence detection. Bone Marrow Transplant 1999; 23: 1055–1060. 7 Baer MR, Stewart CC, Dodge RK, Leget G, Sule N, Mrozek K et al. High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361). Blood 2001; 97: 3574–3580. 8 Thomas X, Campos L, Archimbaud E, Shi ZH, Treille-Ritouet D, Anglaret B et al. Surface marker expression in acute myeloid leukaemia at first relapse. Br J Haematol 1992; 81: 40–44.
Extramedullary manifestation of a donor-derived acute myeloid leukemia in a liver transplant patient
Leukemia (2004) 18, 2050–2053. doi:10.1038/sj.leu.2403498 Published online 7 October 2004 TO THE EDITOR
Improved patient care and new immunosuppressive therapy regimens result in better post-transplant survival and contribute to a growing number of transplantations. Hence, the frequency of post-transplant malignancies increases.1 Here, we describe a clinical case with unusual extramedullary presentation of an allograft-derived acute myeloid leukemia (AML) occurring
Correspondence: Dr CA Schmidt, Department of Hematology/ Oncology, Universita¨t Greifswald, KIM C, Sauerbruchstrae, 17487 Greifswald, Germany; Fax: 49 3834 86 6716; E-mail:
[email protected] Received 26 March 2004; accepted 24 June 2004; Published online 7 October 2004 Leukemia
3 years after orthotopic liver transplantation (oLTX). HLA-typing, STR, cytogenetic and FICTION analyses proved this leukemia to be donor-derived.2 To our knowledge, this is the first report demonstrating allograft-localized donor-derived leukemia. The patient, a 43-year-old male, presented with mild fever, jaundice and abdominal pain. He had received an oLTX 3 years ago for nutritive-toxic liver cirrhosis (child C). The clinical course after oLTX was uncomplicated, including a routine check-up 1 month prior to admission. Laboratory findings on admission: leukocytes 9.0/ml (3.1–9.6/nl), Hb 8.2 g/dl (14–18 g/dl), platelets 280/nl (130–340/nl), hyperbilirubinemia 7.3 mg/dl, conjugated bilirubin 5.6 mg/dl (up to 1.0 mg/dl), alkaline phosphatase 470 U/l (60–180 U/l), g-glutamyl-transferase 67 U/l (up to 28 U/l), creatinine 1.7 mg/dl (up to 1.2 mg/ml), urea nitrogen 52 mg/dl (14–46 mg/dl) and uric acid 7.2 mg/dl (3.0–6.9 mg/dl). Other laboratory findings were normal. An abdominal ultrasound revealed a heterogeneous parenchymal structure in the liver, with a mass (2 cm) at the liver hilus,
