Department of Bone Marrow Transplantation, University Hospital Essen, Essen, Germany

Correspondence to: Dr A H Elmaagacli, Department of Bone Marrow Transplantation, University Hospital Essen, Hufelandstr.55, 45122 Essen, Germany

We studied the role of wt-1 as a minimal residual disease (MRD) marker in 46 patients with acute leukemia (AL) (1st CR n = 24; 2nd CR n = 9, in relapse n = 13) after allogeneic bone marrow or peripheral blood stem cell transplantation. Prior to allogeneic transplant, wt-1 transcripts were detected by PCR in 38 of 46 patients (83%) with AL. After transplant, in 14 of 38 patients (37%) wt-1 transcripts were detected in at least one PCR assay at a median of 12 months post transplant (range 1-89 months). Twelve of the 38 patients relapsed after transplant, but only seven of the 12 were wt-1 positive after transplant. In five relapsing patients the wt-1 test remained negative 0 to 3 months prior to relapse. On the other hand, only seven of 14 patients with a positive test for wt-1 after transplant, relapsed consecutively. In 17 of the 46 study patients chromosomal abnormalities had been found prior to transplant (AML-M4eo with inv16 n = 7, AML-M2 with t(8;21) n = 3, AML-M3 with t(15;17) n = 1, AML-M5 with t(4;11) n = 1, ALL with t(9;22) n = 5). In these 17 patients, we analyzed the wt-1 transcript simultaneously with a specific chimeric transcript characteristic for the corresponding chromosomal abnormality. In 32 of 45 samples (71%) the results for the MRD marker and wt-1 transcript were concordant, but differed in 13 patients. We conclude that detection of wt-1 transcripts does not predict leukemic relapse reliably and is therefore not a suitable MRD marker in patients with acute leukemia after allogeneic BM or PBSC transplantation. Bone Marrow Transplantation (2000) 25, 91-96.

BMT; wt-1; PCR; AML; ALL; MRD

Wilms' tumor is a pediatric neoplasm of the kidneys that is thought to arise as a result of inactivation of both alleles of the Wilms' tumor gene (wt-1) located at chromosome 11p13.¹ The wt-1 gene encodes a zinc finger DNA-binding protein with a complex pattern of alternative splicing with different binding specificities and probably different targets.² The wt-1 protein is a transcription factor with mostly repressing activity when bound to the early growth response-1 (EGR-1) DNA consensus sequence which is present in growth factor gene promoters such as the platelet-derived growth factor A chain (PDGF-A) promoter and insulin-like growth factor II (IGF-II) promoter.^3,4,5 Other target genes of the wt-1 protein are macrophage colony-stimulating factor (M-CSF), transforming growth factor beta-1 (TGF-beta 1) and, wt-1 gene itself. Unlike the tumor suppressor gene p53, which is expressed ubiquitously, the expression of the wt-1 gene is not only restricted to a limited set of organs as in the developing kidney, testis, ovary, spleen and cells of hematopoietic origin, but is also expressed in a time-specific manner.^6,7

Recently, the expression of wt-1 in the majority of patients with acute leukemia and chronic myelogenous leukemia has been reported. In about 80% of all patients with acute leukemia the wt-1 gene transcript was detectable by PCR analysis.^8,9 In several studies the association between the detection of wt-1 gene product and leukemic relapse in patients in complete remission after chemotherapy has been investigated in order to use wt-1 as a 'panleukemic' minimal residual disease (MRD) marker. In the majority of these studies the authors suggested that a positive PCR result for wt-1 after chemotherapy may be associated with an increased risk of leukemic relapse, whereas only a few authors could not confirm such an association.^8,9,10 Moreover, it has been reported that there is a relationship between a positive PCR result for wt-1 at diagnosis and prognostic features of acute leukemia such as FAB classification, cell counts, LDH, karyotype, response to chemotherapy and survival.⁸

Although many studies on wt-1 detection after chemotherapy exist, studies on the association of wt-1 and the occurrence of leukemic relapse after allogeneic bone marrow (BM) or peripheral blood stem cell (PBSC) transplantation are rare. Therefore, the aim of this study was to evaluate the prognostic value of wt-1 detection with regard to leukemic relapse in patients with acute leukemia after allogeneic transplants. Further, we wanted to determine the value of the detection of wt-1 as a minimal residual disease marker (MRD) by correlating the detection of wt-1 transcripts with the detection of various transcripts or chromosomal abnormalities in acute leukemia.

Materials and methods

Patients

Forty-six patients who were transplanted for acute leukemia between November 1991 and July 1998 with BM (n = 30) or PBSC (n = 16) at our institution were studied (Table 1). Thirty-five patients were diagnosed with acute myeloid leukemia (AML), and 11 patients were diagnosed with acute lymphoblastic leukemia (ALL). Approval for this study was obtained from the Institutional Review Board on Medical Ethics at the Essen University Hospital. All patients gave informed consent before material was obtained.

Patients were transplanted in first remission (1st CR, 24 patients), second or third remission (2nd CR or 3rd CR, nine patients), and in relapse (REL, 13 patients).

Twenty-nine donor/recipient pairs were matched at the HLA-A, B, C and DR loci and were nonreactive in mixed lymphocyte culture (MLC), and 17 patients received a graft from a HLA partially matched donor. Ten patients received grafts from unrelated donors, 36 patients were transplanted with grafts from family donors.

Conditioning regimens and GVHD prophylaxis

The conditioning regimen consisted in 39 patients of cyclophosphamide (60 mg/kg/day ´ 2) and fractioned total body irradiation (TBI) delivered by a cobalt source in 4 daily fractions of 2.5 Gy to a total dose of 10 Gy. Two patients received busulfan (4.0 mg/kg/day for 4 days) followed by cyclophosphamide (60 mg/kg/day ´ 2), and five, busulfan (4.0 mg/kg/day for 4 days), thiotepa (5 mg/kg/day for 2 days) and cyclophosphamide (60 mg/kg/day ´ 2).

Two patients who were retransplanted with PBSCs received a conditioning regimen consisting of busulfan (4.0 mg/kg/day for 4 days), fludarabine (30 mg/m² for 6 days) and anti-T lymphocyte globuline (10.0 mg/kg/day for 4 days). With the exception of one, all transplants were performed without prior ex vivo removal of donor lymphocytes from the graft. Irradiated leukocyte-depleted blood products were exclusively used for blood component substitution throughout the post-transplant course.

Prophylaxis of acute GVHD of patients treated by allogeneic transplantation consisted of short-course methotrexate and cyclosporin A (CsA) in 35 patients,¹¹ of CsA alone in 10 patients, and one patient received a T cell-depleted peripheral blood stem cell graft (Table 1).

Morphology and cytogenetics

AML was diagnosed based on standard FAB morphological and cytochemical criteria. Chromosome analyses were performed on bone marrow cells (24 and/or 48 h in vitro culture with GTG (G bands obtained by trypsin and stained with Giemsa)).

Isolation of RNA and cDNA synthesis and reverse transcription (RT)

RNA was prepared from peripheral blood cells, Kasumi-1 and K562 cells as positive control for RT-PCR. RNA was extracted by the acid guanidium/phenol/chloroform method.¹² For each PCR, cDNA was synthesized from 2 g of total RNA with 200 U Moloney murine leukemia virus (Mo-MuLV) reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA) in PCR buffer (50 mmol/l KCl, 10 mmol/l Tris-HCl pH 8.3, 1.5 mmol/l MgCl₂, 0.001% gelatin (Perkin-Elmer-Cetus, Weiterstadt, Germany), using random hexamers (10 mmol/l) (Boehringer, Mannheim, Germany), deoxynucleotide triphosphates dNTP; 1 mmol/l each), 20 U RNAsin at a final volume of 20 l at 37°C for 1 h.

Detection of wt-1 gene product

Ten l of cDNA was used in a total volume of 50 l containing 1´ PCR buffer 50 mmol/l KCl, 10 mmol/l Tris-HCl pH 8.3, 1.5 mmol/l MgCl₂ 0.001% gelatin (Perkin-Elmer-Cetus), 0.2 mmol/l each of NTP, 2.5 U Taq polymerase (Perkin-Elmer-Cetus) and 0.3 mol/l of each of the primers WT1, 5'-GGCATCTGAGACCAGTGAGAA-3' and WT2, 5'-GAGAGTCAGACTTGAAAGCAGT-3'.¹³ After an initial denaturation step of 3 min at 94°C, 30 s cycles consisting of 15 s at 94°C, 30 at 60°C and 45 s at 72°C were performed followed by a final extension of 10 min at 72°C.

In the second round of PCR 2 l of the first PCR products were added in a 50 l total volume containing 1 ´ PCR buffer, 50 mmol/l KCl, 10 mmol/l Tris-HCl pH 8.3, 1.5 mmol/l MgCl₂, 0.001% gelatin, 200 mol/l of each NTP, 0.3 l of inner primers WT3, 5'-GCTGTC- CCACTTACAGATGCA-3', and WT4, 5'-TCAAAGCGC- CAGCTGGAG TTT-3',¹³ and 2.5 U Taq DNA polymerase. Amplification was performed with an initial denaturation step of 3 min at 94°C, followed by 30 cycles of denaturation at 94°C for 15 s, annealing at 60°C for 30 s and 45 s extension at 72°C with a final extension of 10 min at 72°C.

The PCR product was amplified as a band at 481 bp (WT1WT2) or 343 bp (WT3WT4) on a 1.5% agarose gel stained with ethidium bromide (WT1WT2 product not shown).

Detection of pml-rar, aml1-eto, cbf-myh11, mll/af4, bcr-abl gene products

RT-PCR of pml-rar, aml1-eto, cbf-myh11, mll/af4, bcr-abl gene products were performed with specific nested primers as previously described.^{14,15,16,17,18}

RNA quality control

The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) messenger RNA was coamplified as a control in all RT-PCR.^14,19 From all samples, the cDNA was divided into two tubes. One tube was used as a RNA quality control containing a final volume of 25 ml with 1´ PCR buffer with 50 mmol/l KCl, 10 mmol/l Tris-HCl pH 8.3, 1.5 mmol/l MgCl₂, 0.001% gelatin (Perkin-Elmer-Cetus), 0.2 mmol/l each of NTP, 2.5 U Taq polymerase (Perkin-Elmer-Cetus) and 12 pmol/l of each GAPDH specific primers, which results in a positive 200 bp band from all human RNA and therefore controls for the quality of RNA and successful PCR amplification.^14,19

PCR controls

Elaborate measures were taken to minimize contamination following the recommendations of Kwok and Higuchi.²⁰ All PCR products were kept in separate laboratory rooms from patient samples, RNA and PCR reagents.

Blank controls were included in all RNA extraction procedures as negative controls to assess quality and cross-contamination between samples. Cells from NB-4, Kasumi-1, SD-1 and MV4-11 cell lines, and cells of a cbf/myh11-positive patient were included as a positive control.

For a patient sample to be considered negative for any PCR amplification, it had to: (1) be amplified in two independent experiments using 2.0 g of total cellular RNA per reaction; (2) be successfully coamplified for the GAPDH transcript in each reaction; (3) be successfully coamplified for a positive control in both reactions.

Oligonucleotide primers were synthesized on an Applied Biosystems 380 B DNA synthesizer (Foster City, CA, USA).

Sensitivity of PCR assays

A sensitivity of 1:10⁵ PCR was obtained for PCR of mll-af4, cbf-myh11, aml1-eto, pml-rar gene transcripts as previously published.^{14,15,16,17,18}

To assess the sensitivity of the RT-PCR amplification of wt-1 gene transcripts, 1 g of total RNA isolated from cells of K562 cell line was serially diluted in RNA isolated from mononuclear peripheral blood cells of a healthy donor. The resulting cDNA was then submitted to 30 cycles of amplification with primers WT1 and WT2, followed by 30 cycles of amplification with nested primers WT3 and WT4. A sensitivity of 1:10⁵ was measured for wt-1.

Definition of relapse

Hematologic relapse was diagnosed on the basis of standard hematological criteria.

Clinical evaluation and statistical analysis

The diagnosis of acute and chronic GVHD was based on the characteristic clinical appearance of the symptoms of organ involvement. Grading of acute or chronic GVHD followed commonly accepted criteria.^21,22

Differences between frequencies were compared by the two-tailed Fisher exact test.

Results

Detection of wt-1 transcripts in patients before transplant

In 32 of 35 patients with AML and six of 11 patients with ALL wt-1 transcripts were detected by PCR prior to transplant. Twenty of 38 patients (53%) with a positive wt-1 result prior to transplant were diagnosed to be in 1st CR at time of transplant. Eight patients were in 2nd CR or 3rd CR and 10 patients were in relapse at time of transplant.

Detection of wt-1 transcripts in patients after transplant

Thirty-eight patients who were positive for wt-1 before transplant were further monitored for wt-1 after transplant. PCR tests for wt-1 were performed between 1 and 89 months post transplant. In 14 of 38 patients (37%) wt-1 transcripts were detectable in at least one PCR assay at a median of 3 months post transplant (range 1-28 months).

Relapse occurred in 12 of 38 patients in a median of 6 months (range 1-13 months) after transplant. Seven of these 12 patients were positive in at least one PCR assay for wt-1 prior to relapse (a median of 3 months before relapse, range 0-5). Five of 12 patients (42%) who were wt-1 positive before transplant remained negative after transplant up to the time of relapse.

Two patients who were wt-1 positive after transplant and relapsed subsequently were retransplanted. One of these two patients, who had an AML-M3 with translocation t(15;17), relapsed again after the second transplant. Although this patient was wt-1 positive before, five examinations at monthly intervals remained negative after the second transplant, whereas four of five tests for pml-rar transcripts were positive in simultaneous PCR analysis.

Results of the wt-1 PCR had a specificity of 73%, a sensitivity of 58%, and a negative predictive value of 82% with respect to the development of relapse in this study.

Detection of wt-1 and MRD in patients with chromosomal abnormalities after transplant

Seventeen patients with chromosomal abnormalities were wt-1 positive by PCR prior to transplant. In all patients the specific chimeric transcript was also detectable prior to transplant. Seven patients were diagnosed with an AML-M4eo with inversion 16, three patients had an AML-M2 with a t(8;21), one patient had an AML-M3 with a t(15;17), and one patient had an AML-M5 with t(4;11). Five patients with ALL had a t(9;22) and were m-bcr-abl (n = 3) or M-bcr-abl positive (n = 2) before transplant.

Forty-five samples of 17 patients were analyzed after transplant for the specific chimeric transcript of the MRD marker and the wt-1 transcript simultaneously. In 32 of 45 samples (71%) the results for the MRD marker and the wt-1 transcript were concordant. All data of patients who were examined for wt-1 and a specific MRD are shown in Figure 1.

Discussion

We studied the role of wt-1 as a MRD marker in 46 patients with acute leukemia after allogeneic bone marrow or peripheral blood stem cell transplantation.

Although wt-1 transcripts can also be detected in normal subjects, wt-1 transcripts are aberrantly overexpressed in patients with leukemia.²³ This may imply the involvement of this gene in human leukemogenesis. Therefore, Inoue et al concluded that wt-1 is not only suitable as a MRD marker but also qualified as a prognostic factor for acute leukemia.^23,24

In this study, the wt-1 transcript was found in 38 of 46 patients (83%) with acute leukemia prior to transplant. There was no association between the stage of disease and the detection of wt-1 transcript prior to transplant, since 20 of 38 patients with a positive wt-1 result prior to transplant were diagnosed to be in first complete remission at time of transplant. On the other hand, one patient who was in relapse at time of transplant had a negative PCR result for wt-1. However, these results do not support those studies which found that wt-1 transcripts could no longer be detected in patients who achieved a complete remission of leukemia.

We detected wt-1 transcripts in 14 of 38 patients (37%) in at least one PCR assay after transplant in a median time interval of 12 months post transplant (range 1-89 months). Seven of these 14 patients relapsed consecutively resulting in a poor sensitivity of 58%, a specificity of 73%, and positive predictive value of 82% for the detection of wt-1 transcripts by PCR when compared to the sensitivity (91%) and specificity (85%) of our PCR assay for bcr-abl transcript (data not published).

Only seven of the 12 relapsing patients who had positive PCR results for wt-1 prior to transplant were also wt-1 positive after transplant, and in five patients the wt-1 test remained negative 0-3 months prior to the date of relapse.

In 17 patients with various chromosomal abnormalities (AML-M4eo with inv 16 n = 7, AML-M2 with t(8;21) n = 3, AML-M3 with t(15;17) n = 1, AML-M5 with t(4;11) n = 1, ALL with t(9;22) n = 5); we analyzed wt-1 transcripts simultaneously with a specific chimeric transcript characteristic for the chromosomal abnormality and correlated these results. Only in 32 of 45 samples (71%) were the results for the MRD marker and wt-1 transcripts concordant.

Further, the low reliability of the detection of wt-1 transcripts as a MRD marker was also demonstrable in an AML patient with translocation t(15;17) who was wt-1 positive prior to first relapse after first transplant, but was negative prior to the second relapse in five PCR analyses performed monthly after the second transplant, whereas simultaneously performed PCR analyses for the characteristic pml-rar transcripts were positive after the first and second transplant.

In summary, although we found that wt-1 is more often detectable in patients with a consecutive relapse after transplant than in patients in remission, it may not be regarded as a reliable MRD marker in patients after allogeneic BM or PBSC transplantation. The low specificity and sensitivity of this assay suggest that detection of wt-1 after transplant is not a reliable predictor of leukemic relapse after transplant.

Acknowledgements

This work was supported by a grant from 'Aktion Kampf dem Krebs' of the German Cancer Society and a grant from 'Deutsche Krebshilfe' No. 70-1669-EL-T. We thank Jitka Stockova and Melanie Kroll for their excellent technical performance of the PCR analyses.

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Figure 1 Simultaneous detection of different MRD markers and wt-1 transcript by PCR in patients after transplant.

Table 1  Patients' characteristics