Minimal residual disease quantification in patients with acute myeloid leukaemia and inv(16)/CBFB-MYH11 gene fusion Bert A. van der Reijden, 1 * Annet Simons, 2  Erna Luiten, 1 Sonja C. van der Poel, 1 Pauline E. Hogenbirk, 1 Evelyn To ¨nnissen, 3 Peter J. M. Valk, 1 Bob Lo ¨wenberg, 1 Georgine E. De Greef, 1 Martijn H. Breuning 2 and Joop H. Jansen 1 * 1 Institute of Haematology, Erasmus University Medical Centre, Rotterdam, 2 Department of Human Genetics, Leiden University Medical Centre, Leiden, and 3 Department of Haematology, University Medical Centre Nijmegen, Nijmegen, The Netherlands Received 14 January 2002; accepted for publication 26 April 2002 Summary. We have designed a real-time CBFB-MYH11 reverse transcription polymerase chain reaction (RT-PCR) assay to quantify minimal residual disease (MRD) in patients with inv(16)-positive acute myeloid leukaemia (AML). Six patients were followed for a median of 17Æ5 months after diagnosis during which 120 evaluable samples were ana- lysed. The CBFB-MYH11 expression at diagnosis varied only fourfold between the six patients and was virtually identical to that observed in the CBFB-MYH11-positive cell line ME-1. For two cases, a patient-specific real-time PCR for CBFB- MYH11 quantification at genomic DNA level was designed. Similar disease levels were found at the RNA and genomic DNA level during and after treatment, indicating that CBFB- MYH11 gene expression was unaltered during treatment and that the percentage of malignant cells can be accurately quantified at the RNA level. Following successive courses of chemotherapy, the reduction of malignant cells was found to be significantly more pronounced (80–250-fold greater) in peripheral blood compared with bone marrow in five out of six cases tested. Treatment with gemtuzumab ozogamicin as sole agent at relapse did not result in a selective decrease of tumour cells in three cases analysed. We conclude that real-time PCR is a powerful method of monitoring MRD levels and quantifying the antileukaemic effect of separate (experimental) courses of chemotherapy. Keywords: leukaemia, inv(16), CBFB-MYH11, MRD quan- tification, real-time PCR. An inv(16)(p13q22) or t(16;16)(p13;q22) can be found in approximately 10% of cases with acute myeloid leukaemia (AML) (Liu et al, 1995). Patients with these aberrations have a relatively good prognosis with a 5 year survival of approximately 50% (De la Chapelle & Lahtinen, 1983; Le Beau et al, 1983; Larson et al, 1986; Liu et al, 1993). Both the inv(16) and t(16;16) lead to the formation of a CBFB- MYH11 fusion gene that can be detected by reverse transcription polymerase chain reaction (RT-PCR) (Liu et al, 1993; Claxton et al, 1994; He ´bert et al, 1994; Poirel et al, 1995; van der Reijden et al, 1995). With qualitative PCR methods, CBFB-MYH11-positive cells have been detected in patients for prolonged periods of time without any signs of clinical relapse (He ´bert et al, 1994; Poirel et al, 1995; Tobal et al, 1995). The clinical value of qualitative CBFB-MYH11 PCR detection is therefore limited. Quantitative PCR assays could give more insight in the kinetics of the disease and may identify patients at risk of developing a clinical relapse (van der Reijden et al, 1997). When treatment affects the expression of tumour-specific target genes (as has been shown for interferon treatment of acute promyelocytic leukaemia cells inducing PML-RARA expression), minimal residual disease (MRD) levels meas- ured by RT-PCR at the RNA level do not accurately reflect the percentage of malignant cells (Chelbi-Alix et al, 1995; Lavau et al, 1995). In contrast, quantitative PCR at the genomic DNA level is independent of gene expression and directly reflects the percentage of malignant cells. For most mutated genes in AML, the breakpoints are scattered over large genomic regions, which severely hampers fusion gene detection by genomic DNA PCR. However, 90% of inv(16) breakpoints are clustered in two relatively small regions, allowing a feasible PCR amplification of genomic CBFB-MYH11 breakpoints (van der Reijden et al, 1999). We designed and validated a competitive and real-time Correspondence: Bert A. van der Reijden, PhD, Department of Haematology, University Medical Centre Nijmegen, Geert Grootep- lein zuid 8, PO Box 9101, 6500 HB, Nijmegen, The Netherlands. E-mail: b.vanderreijden@chl.azn.nl *Present address: Central Haematology Laboratory, University Medical Centre Nijmegen, Nijmegen, The Netherlands.  Present address: Department of Human Genetics, University Medical Centre Nijmegen, Nijmegen, The Netherlands. British Journal of Haematology, 2002, 118, 411–418 Ó 2002 Blackwell Science Ltd 411