Bone Marrow Transplantation, (1998) 21, 159–166 1998 Stockton Press All rights reserved 0268–3369/98 $12.00 Detection of CBFb/MYH11 fusion transcripts in patients with inv(16) acute myeloid leukemia after allogeneic bone marrow or peripheral blood progenitor cell transplantation AH Elmaagacli1, DW Beelen1, M Kroll1, S Trzensky1, C Stein2 and UW Schaefer1 Departments of 1Bone Marrow Transplantation, and 2Department of Forensic Medicine, University Hospital of Essen, Essen, Germany Summary: Keywords: AML; inv(16); CBFb/MYH11; MRD; BMT We evaluated the occurrence of the CBFb/MYH11 fusion transcripts by PCR analysis in 10 patients with inv(16)(p13;q22) acute myeloid leukemia (AML) who Acute myelomonocytic leukemia with bone marrow eosino- underwent allogeneic bone marrow transplantation philia (AML-M4Eo), according to the French–American– (BMT) (n = 5), peripheral blood progenitor cell trans- British (FAB) classification, is a distinct subtype of AML. plantation (PBPCT) (n = 3), or autologous transplan- AML-M4Eo is often associated with rearrangements of tation (n = 2). In addition to the analysis of minimal chromosome 16, mostly involving 16p13 and 16q22, lead- residual disease (MRD), the chimerism status of patients ing either to a pericentric inversion, inv(16)(p13q22) or, after allogeneic transplant was studied by PCR. The less commonly, to a translocation between the homologous CBFb/MYH11 fusion trancript was not detectable in six chromosomes, t(16;16)(p13;q22). The pericentric inversion of seven patients who remained in remission after allo- inv(16)(p13q22) is one of the most frequently occurring geneic BMT or PBPCT. Two of these patients in chromosomal rearrangements detected in this neoplasm, remission were monitored for 50 months and 64 months which has been reported to account for approximately 16% post-BMT. One patient in remission was PCR-positive of all AML.1–3 for CBFb 3 months post-BMT in a single BM sample, The breakpoints involved in the inv(16) have recently but not in a simultaneously examined blood sample, been cloned and shown to involve the core binding factor suggesting that analyses from BM samples are more b-gene (CBFb) on 16q22 and the smooth muscle heavy sensitive than those from blood samples. Sequential chain (MYH11) on 16p13. The formation of the chimeric PCR assays performed 6 and 12 months post-BMT CBFb/MYH11 fusion gene results in the disruption of the obtained from the same patient were negative. Another normal interaction of the transcription factor complex a, b, patient with a positive PCR assay 3 months post-allo- and ETS.3 geneic PBPCT, remained PCR positive for the Patients with AML-M4Eo with inv(16)(p13q22) appear CBFb/MYH11 fusion transcript when tested 6 months to have a high response rate to induction chemotherapy and post-PBPCT. A chimerism analysis by PCR revealed a a favorable prognosis, but relapse of AML after chemo- mixed chimerism status in this patient. He relapsed 7 therapy remains a major cause of treatment failure. Allo- months post-transplant. Before transplant, in all nine geneic bone marrow transplantation (BMT) has become an patients who were in complete remission of AML (eight effective treatment modality for patients with hematological patients in 1CR, one patient in 2CR), the CBFb/MYH11 malignancies. Patients with AML in remission who transcript was detectable. In one patient in relapse, the undergo allogeneic BMT have a significantly lower risk of fusion transcript was not only detectable in blood and relapse than their counterparts treated with chemotherapy bone marrow, but also in a cerebrospinal fluid sample alone. On the other hand allogeneic BMT is associated with prior to transplant. Two patients who received autolog- more treatment-related mortality than autologous BMT or ous BMT were monitored for CBFb/MYH11 transcripts chemotherapy alone. 3 months after BMT. The CBFb/MYH11 was detected Several reports suggest that the CBFb/MYH11 fusion in these patients. Both patients subsequently relapsed 3 mRNA can often be detected in patients in long-term months and 23 months post-autologous BMT. The remission after chemotherapy.4–7 To date, other than single results study show that analysis of the CBFb/MYH11 case reports, studies of the detection of the CBFb/MYH11 fusion transcript by PCR seems to be a suitable method fusion transcripts after allogeneic BMT do not exist.7,8 for monitoring minimal residual disease in AML The aim of the present study was to determine if patients with inv (16). CBFb/MYH11 fusion transcripts can be detected in patients treated with allogeneic BMT (n = 5), allogeneic peripheral blood progenitor cell transplantation (PBPCT) (n = 3), or Correspondence: Dr AH Elmaagacli, Department of Bone Marrow Trans- = plantation, University Hospital of Essen, Hufelandstr. 55, 45122 Essen, autologous BMT (n 2) for inv(16) positive AML at our Germany institution. In addition to the analysis of minimal residual Received 3 July 1997; accepted 13 August 1997 disease (MRD), the chimerism status of patients after allo- CBFb/MYH11 fusion transcripts in patients with AML AH Elmaagacli et al 160 geneic BMT or PBPCT was studied by PCR of minisatellite one busulfan (4 mg/kg/day for 4 days), thiotepa (5 gene regions (VNTR-PCR). mg/kg/day for 2 days) and cyclophosphamide (60 mg/kg/day × 2). All transplants were performed without prior ex vivo removal of donor lymphocytes from the graft. Materials and methods Irradiated leukocyte-depleted blood products were used exclusively for blood component substitution throughout Patients the post-transplant course. Prophylaxis of acute GVHD of patients treated by allo- Ten patients with inv(16) leukemia who underwent allo- geneic transplantation consisted of short-course methotrex- geneic transplantation with bone marrow (n = 5) or periph- ate and cyclosporin A (CsA) in five patients, of CsA alone eral blood progenitor cells (n = 3), or autologous BMT (n in three patients, as shown in Table 1. = 2) at our institution were studied (Table 1). Six patients were diagnosed with de novo FAB M4Eo, and four patients were diagnosed with AML M4. The median age at diag- nosis was 31 years (range 17–40). Seven patients were male Morphologic and cytogenetics and three female. Patients were transplanted in first remission (1CR, eight AML was diagnosed according to standard FAB morpho- 9 patients), second remission (2CR, one patient), and first logic and cytochemical criteria. Chromosome analyses relapse (1rel, one patient). Two patients were transplanted were performed on bone marrow cells (24 and/or 48 h in with autologous bone marrow, and eight patients received vitro culture with GTG (G-bands obtained by trypsin and allogeneic grafts. Seven donor/recipient pairs were matched stained with Giemsa)). In all patients a pericentric inversion at the HLA-A, B, C and DR loci and were nonreactive in 16 could be demonstrated prior to transplant. mixed lymphocyte culture (MLC), and one patient (UPN 936) received a graft from an antigen-mismatched unrelated donor (one mismatch of six). All recipients of allogeneic Isolation of RNA and cDNA synthesis and reverse transplantation except one received grafts from family transcription donors. BM or blood samples were studied 1 month before transplantation and 1 to 64 months after transplant. The RNA was prepared from fresh or cyropreserved peripheral median time to last follow-up for all patients was 6 months. blood cells or BM buffy coat cells, Kasumi-1 and K562 Approval for this study was obtained from the Institutional cells provided negative controls for RT-PCR. Blood from Review Board on Medical Ethics at the Essen University a patient (UPN 799) with a CBFb/MYH11 fusion transcript Hospital. All patients gave informed consent before was used as a positive control. RNA was extracted by the material was obtained. acid guanidium/phenol/chloroform method.10 For each PCR, cDNA was synthesized from 2 mg of total RNA with 200 U Moloney murine leukemia virus (MoMuLV) reverse Conditioning regimens and GVHD prophylaxis transcriptase (Gibco-BRL, Gaithersburg, MD, USA) in The conditioning regimen consisted in five patients of PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl pH 8.3, cyclophosphamide (60 mg/kg/day × 2) and fractioned total 1.5 mmol/l MgCl2, 0.001% gelatin (Perkin Elmer Cetus, body irradiation (TBI) delivered from a cobalt source in Weiterstadt, Germany), using random hexamers (10 four daily fractions of 2.5 Gy to a total dose of 10 Gy. mmol/l) (Boehringer, Mannheim, Germany), deoxynucleo- Four patients received busulfan (4 mg/kg/day for 4 days) tide triphosphates 8dNTP; 1 mmol/l each), 20 U RNAsin followed by cyclophosphamide (60 mg/kg/day × 2), and at a final volume of 20 mlat37°C for 1 h. Table 1 Clinical characteristics of the AML patients with inv(16) No. UPN Sex Age Trans Disease GVHD Condit GVHD HLA Present status R/D stage proph regimen ac/chr constel (months) 1 176 — 17 autoBMT 1CR — Bu + CY — — Relapse/death (3) 2 189 — 40 autoBMT 1CR — Bu + CY — — Relapse/death (23) 3 469 M/F 34 BMT 1CR CsA + MTX TBI + CY —/lim de novo si/id DSF (69) 4 554 M/F 34 BMT 1CR CsA + MTX Bu + CY II/lim si/id DFS (54) 5 799 M/F 32 PBPCT 1CR CsA + MTX TBI + CY II/lim si/id Relapse/death (7) 6 856 M/M 30 BMT 1CR CsA Bu,Th,CY II/lim bro/id Death through infection (12) 7 868 F/F 25 BMT 1CR CsA TBI + CY I/lim si/id DFS (12) 8 876 F/M 36 PBPCT 2CR CsA + MTX Bu,Th,CY II/lim bro/id DFS (10) 9 882 F/F 25 BMT 1CR CsA TBI + CY IV/ext si/id DFS (10) 10 936 M/M 35 PBPCT Relap CsA + MTX TBI + CY II/— unr/MM Death through infection (3) UPN = unique patient number; R = recipient; D = donor; GVHD = graft-versus-host disease; 1CR/2CR, first/second remission; PBPCT = peripheral blood progenitor cell transplantation; BMT = bone marrow transplantation; MTX = methotrexate; CsA = cyclosporin A; TBI = total body irradiation; CY = cyclophosphamide; Bu = busulfan; Th = thiotepa; bro = brother; id = identical; unr = unrelated; si = sister; lim = limited; ext = extensive; DFS = disease-free survivor.