A novel real-time RT-PCR assay for quantification of OTT-MAL fusion transcript reliable for diagnosis of t(1;22) and minimal residual disease (MRD) detection

TO THE EDITOR Acute megakaryoblastic leukemia (AMKL), M7 in the FAB nomenclature, is a heterogeneous subtype of acute myeloid leukemia (AML) whose exact frequency ranges from 3 to 14% of AMLs, depending on the different institutions,1 and is higher in children than in adults. It is the most frequent type of AML occurring in young patients with Down's syndrome (DS),2 in which it is associated with acquired GATA1 mutations.3 AMKL may also occur as a de novo leukemia in the absence of DS or as a secondary malignancy, following myelodysplasia or therapy for a prior malignancy.1 Accordingly, a large variety of chromosomal abnormalities can be observed in AMKL. The most specific and recurring chromosomal abnormality in pediatric AMKL is the t(1;22)(p13;q13) translocation, which is present in one-third of de novo AMKL, mainly in infants.4 The t(1;22) translocation is routinely detected by standard cytogenetic analysis but could remain undetected, in part because of the difficulty to obtain accurate bone marrow (BM) aspirates of these patients because of an associated myelofibrosis or the low number of blasts present in the samples. Translocation t(1;22) has been shown to result in the in-frame fusion of the OTT(RBM15) on 1p13 and MAL(MKL1) on chromosome 22q13.5,6 Since the OTT-MAL fusion transcripts can be detected in the cells that underwent the translocation, we developed a real-time quantitative RT-PCR (RQ-RT-PCR) in order to assess and quantify the OTT-MAL fusion transcript associated with the t(1;22). Using this approach we analyzed BM samples taken at diagnosis and during the treatment, of seven children with AMKL referred to our hospital since May 1996. The diagnosis was established on the basis of the FAB criteria by studies of cell morphology and cytochemistry, and was validated according to the immunological criteria recommended by the European Group for the Immunological Classification of Acute Leukemias (EGIL). Five patients (median age: 38 months) presented with a 'de novo' AMKL and two patients were diagnosed with AMKL following another hematopoietic disorder: a recurrent and severe neutropenia in one case and an hypoplastic anemia in the other. BM cells were obtained at the time of the diagnosis and during cytological remission and cryopreserved before use for molecular analysis. Cytogenetic studies were carried out on BM cells at the diagnosis of each patient and the classical t(1;22)(p13;q13) was found in only one case. For the molecular studies we performed total RNA extraction and cDNA preparations as previously reported.7 The primers and probe used for real-time quantitative PCR amplification (RQ-PCR) of OTT-MAL fusion tran-scripts, aimed to detect both common and variant fusion transcripts (Figure 1) were: forward primer, OTT-127 Fw (nucleotides 2819–2838 of OTT transcript, AJ297259): -AGC AGT TCC TGG ATT CCC CT-; reverse primer, MAL- 227 Rv (nucleotides 220–240 of MAL transcript, AJ297257): - ATG AAA TGC GGC TGG ACT TTT-; TaqMan probe: p-OTT-148: -CCA AGG CAC TGG CCA AAT CTG AAG AA-. Detection of the ABL transcript was performed as reported.7 In order to quantify the expression of OTT-MAL transcripts, we first validated a standard curve constructed with serial dilution of known starting copy number (108–102) of the appropriate cDNA. Figure 2a indicates the change in reporter fluorescence, which is measured continuously during the PCR so that the kinetics of each reaction can be assessed. The standard curve is then obtained by correlation of standard concentration vs the Cp value (crossing point) as shown in Figure 2b. A strong linear relation between the Cp and the OTT-MAL transcripts copy number (r>0.99) was found over a range of at least seven orders of magnitude with a PCR efficiency value of 90%. Similar results were obtained with the standard curve of the endogenous control ABL (data not shown). For each patient and control sample the quality and quantity of RNA was assessed by the amplification of ABL gene transcripts in independent QR-RT-PCR runs. Samples were considered to be eligible for testing only when the Cp of the internal reference ABL was lower than 30. Quantitative results were thus expressed as normalized copy number of the target gene against the copy number of the endogenous control, ABL gene, 1000. PCR reactions were performed using the Light Cycler System (Hoffman-Roche). For each reaction, 100 ng of reverse-transcribed RNA sample was added to 15 l volume of PCR mix containing 1 LC master mix, 5 mM MgCl2, 300 M of each primer and 200 M of probe. Thermal cycling conditions consisted of an initial denaturation step at 95°C for 10 min followed by 50 cycles at 95°C for 15 s and 60°C for 1 min Experiments were performed in duplicate for each data point. Each PCR run included the standard curve, a control without reverse transcriptase and a control without template. The assay allows accurate detection down to 10 copies of OTT-MAL target cDNA copies with a within-run coefficient of variation of Ct values less than 2.2%. Fusion OTT-MAL transcripts were detected in two patients, although the level of expression varied widely (1590 and 9 OTT-MAL transcripts copy number, respectively). These results are consistent with cytogenetic data, since a t(1;22)(p13;q13) was observed in the patient expressing the high copy number of OTT-MAL transcripts. However, no detectable chromosomal abnormalities were observed in the BM sample from the patient with a low OTT-MAL transcripts copy number. Although the positive result in this patient was confirmed in an independent quantitative RT-PCR run, the paucity of blast cells recovered from the BM aspirate prevented us from repeating the RT-PCR reaction starting from another frozen sample. Since OTT-MAL fusion transcripts are specifically associated with the t(1;22) translocation, they represented a suitable marker for monitoring leukemic cells during treatment. Follow-up samples from the t(1;22)- positive patient were available for RQ-RT-PCR analysis (see Figure 3). The OTT-MAL transcripts were quantified at the time of evaluation of the complete remission (CR), day 35, within 3 and 7 months from the diagnosis and at the time of the autologous bone marrow transplant (ABMT). Three additional samples were analyzed following the ABMT at 12, 18 and 30 months from the initial diagnosis. As shown in Figure 3, we observed a steady decrease of the estimated number of OTT-MAL transcripts in the samples obtained during the first 7 months of treatment. The fusion was not detected in the samples collected after the ABMT. This result appears to be coherent with the clinical status of the patient, who remains in persistent CR 54 months after the initial diagnosis. These data illustrate the potential use of OTT-MAL transcripts expression as a marker to monitor residual leukemia and assess the response to treatment. Our RT-PCR assay is a reliable and sensitive method to search for the presence of t(1;22) translocation and to evaluate the level of the fusion OTT-MAL transcripts expression in diagnostic and follow-up samples from patients with a t(1;22) AMKL. The assay enabled us to detect the OTT-MAL fusion in one case with normal leukemic karyotype and to estimate the expression of the fusion gene during the different phases of the treatment in the BM cells from the patient with the t(1;22)(p13;q13)- positive leukemia at the diagnosis. The finding of OTT-MAL transcripts in a patient with a normal karyotype was unexpected. The discrepancy observed between molecular and cytogenetic data may result from various artefacts. One may reside in the known difficulties to obtain abnormal metaphases of leukemic cells in patients with AMKL because of the overwhelming number of nonleukemic cell mitosis. Furthermore, the expression level of OTT-MAL transcripts in the diagnostic sample of patient, without a cytogenetic evidence of t(1;22) translocation was low, in a range similar to that estimated in the follow-up samples analyzed (Figure 3). Thus, the t(1;22) could have been undetected by cytogenetic means because of the very low number of malignant cells carrying the translocation in that BM sample. In addition, it cannot be excluded that the blasts frequency in the BM cells recovered after the Ficoll separation for molecular studies differed from that of BM cells used for karyotype analysis. Finally, the paucity of the material available from the diagnosis prevents us from further investigating for the presence of t(1;22) by means of FISH analysis, in the hypothesis of a cryptic OTT-MAL gene fusion event. A similar result was observed in another case of AMKL with a normal karyotype, published in the study of the Groupe Français de Cytogénétique Hématologique on Megakaryoblastic Leukemia by Dastugue and colleagues. These findings suggest that the real frequency of OTT-MAL recombination events may be underestimated at least in the childhood group of AMKL. Recent analysis of treatment results obtained in pediatric AMKL indicates that the outcome is particularly poor in patients with de novo AMKL compared to other forms of AML and to the general good outcome of AMKL DS patients1. However, more intensive treatments, including high doses of aracytine and BM transplantation during complete remission, may significantly improve the survival rate of patients with de novo AMKL. None of the current biological features – that is, WBC, platelet counts or Hb level at with diagnosis, age, etc. - seem associated with the fraction of 'good responders' to intensive treatments. It would be of interest to investigate the role of the recurrent t(1;22) translocation in response to therapy. It is worth noting that long-term survivors could be found among patients with t(1;22)- positive AMKL, even those treated with 'standard' AML therapy.8 The presently described reliable assay for the detection and monitoring of the OTT-MAL fusion transcripts is of value in assessing the therapeutic response, the presence of residual cells in the autologous BM prior to BM transplantation and the choice of the best drugs combinations for the treatment of t(1;22) AMKL. It is also a suitable complement of cytogenetic search of the t(1;22)(p13;q13) translocation in AMKL. This work was supported by INSERM and the Ligue Nationale Contre le Cancer (Comité National and Comité de Paris).