Prognostic significance of quantitative analysis of WT1 gene transcripts by competitive reverse transcription polymerase chain reaction in acute leukaemia

Prognostic significance of quantitative analysis of WT1 gene

transcripts by competitive reverse transcription polymerase

chain reaction in acute leukaemia

Mamta Garg, Helen Moore, Khalid Tobal and John A. Liu Yin University Department of Haematology,

Manchester Royal Infirmary, Manchester, UK

Received 14 February 2003; accepted for publication 23 May 2003

Summary. We have developed a sensitive, competitive,nested reverse transcription polymerase chain reaction(RT-PCR) titration assay that quantifies the number ofWilm’s tumour (WT1) gene transcripts in bone marrow(BM) and peripheral blood (PB), coupled with a competitiveRT-PCR protocol for the ABL gene as control. We studiedBM/PB samples from 107 acute myeloid leukaemia (AML)patients and 22 acute lymphoblastic leukaemia (ALL)patients at presentation and detected the WT1 gene in> 90% of patients by a qualitative assay. Quantitativeanalysis of WT1 transcript at presentation in 66 patients(52 AML, 14 ALL) correlated significantly with remissionrate, disease-free survival (DFS) and overall survival (OS)(P ¼ 0Æ003). WT1 levels were normalized to 105 ABLtranscripts. Within good and standard cytogenetic riskgroups, high WT1 levels correlated with poorer outcome.Serial quantification was performed in 35 patients (28 AML,

seven ALL); those with less than 103 copies of WT1 afterinduction and second consolidation chemotherapy hadsignificantly better DFS and OS. Fourteen patients haverelapsed with a median complete remission duration of 12(range 4–49) months. We detected a rise in WT1 levels innine out of 14 patients, 2–4 months before the onset ofhaematological relapse, whereas in the remaining fivepatients, WT1 levels remained persistently high during thedisease course. WT1 levels were lower in PB than in BM, butmirrored changes in the BM samples and were equallyinformative. We suggest that WT1 is a useful moleculartarget to monitor minimal residual disease in acute leuk-aemia, especially in cases without a specific fusion gene.

Keywords: WT1 gene transcript, reverse transcriptionpolymerase chain reaction, acute leukaemia, prognosis,minimal residual disease.

Relapse remains the main cause of treatment failure inacute leukaemia (AL). There is now cumulative evidencethat monitoring of minimal residual disease (MRD) is usefulin predicting relapse in patients with AL (San Miguel et al,1997; Cave et al, 1998; Grimwade, 1999; Liu Yin & Tobal,1999). Detection of MRD is typically based on eithermolecular or immunological markers, which are presentin leukaemic cells but not in normal cells, thus allowing fortheir specific identification. Polymerase chain reaction(PCR) techniques can detect one leukaemic cell in105)106 normal cells, but are applicable only to leukaemiasthat bear specific DNA markers including fused genes suchas BCR-ABL, PML-RARA, AML1-MTG8 and CBFb-MYH11.Hence, they are suitable for only about 30% of cases with

acute myeloid leukaemia (AML) where specific transloca-tions such as t(8:21), t(15:17) and inv(16) have beencharacterized (Liu Yin & Tobal, 1999). Moreover detectionof MRD in acute lymphoblastic leukaemia (ALL) by PCRamplification of junctional regions of rearranged immuno-globulin and TCR genes requires sequencing and cloning ofthe junctional regions in the presentation sample (Caveet al, 1998). There is therefore a critical need to identifyalternative gene targets that are expressed in the majority ofAL patients. The behaviour or level of these molecularmarkers needs to be altered in the leukaemic clone eventhough they may not be specific to a particular type ofleukaemia. One such example is the Wilm’s tumour gene(WT1).

WT1 maps to the chromosome 11p13 and encodes atumour suppressor gene associated with the development ofWilm’s tumour, a paediatric kidney neoplasm (Call et al,1990). WT1 has been shown to be overexpressed in> 90% of leukaemia cells, especially those with myeloid

Correspondence: John A. Liu Yin, University Department of Hae-

matology, Manchester Royal Infirmary, Oxford Road, Manchester

M13 9WL, UK. E-mail: [email protected]

British Journal of Haematology, 2003, 123, 49–59

� 2003 Blackwell Publishing Ltd 49

characteristics (Miwa et al, 1992; Miyagi et al, 1993; Inoueet al, 1994). Thus, the use of WT1 as a molecular target forMRD detection is potentially attractive, as it can be appliedto the majority of AL patients, particularly those who lack aspecific fusion gene. Several preliminary studies in the use ofWT1, both as a prognostic marker and for monitoring MRDin AL, have been published (Inoue et al, 1994, 1996;Bergmann et al, 1997). Data on the prognostic significanceof WT1 expression at diagnosis and during remission in ALare somewhat conflicting (Inoue et al, 1996; Schmid et al,1997; Gaiger et al, 1998, 1999). This could be due to thevariable levels of sensitivity of the protocols used forquantification of WT1 transcripts, with some of thesestudies relying on either qualitative or semi-quantitativereverse transcription (RT)-PCR protocols. Moreover, thequantification of WT1 gene transcript appears to be usefulin predicting early relapse in AL patients (Cilloni et al,2002); these results therefore emphasize the need foradditional studies using sensitive methods to quantify andexamine the kinetics of the WT1 transcript during differentphases of the disease.

We have developed a sensitive, quantitative, nestedRT-PCR assay to estimate levels of WT1 transcript and, byinference, levels of MRD that is coupled with a competitiveRT-PCR for the ABL transcript as a control. We report hereour evaluation of this method in the assessment ofpresentation and serial bone marrow (BM) and peripheralblood (PB) samples from a large group of patients with AL.

MATERIALS AND METHODS

Patients. Patients with AL who presented between June1994 and May 2000 were studied and followed untilDecember 2001. The diagnosis of AL was made accordingto the French–American–British (FAB) classification. Acuteleukaemia patients were treated according to United King-dom (UK) Medical Research Council AML 10/12 or UK ALLXII protocols.

AML 10/12 protocols included remission induction withdaunorubicin, cytarabine and 6-thioguanine (DAT 3 + 10)followed by consolidation with DAT 3 + 8, amsacrine,cytarabine and etoposide (MACE), and mitoxantrone andcytarabine (MiDAC). Patients aged < 50 years either had anallogeneic stem cell transplant if they had a matched siblingdonor or an autograft, except for patients with good riskcytogenetics (Grimwade et al, 1998).

The UK ALL XII protocol comprises remission inductionin two phases with daunorubicin, vincristine, prednisolone,cyclophosphamide, 6-thioguanine, l-asparaginase andcytarabine followed by consolidation with three infusionsof high-dose methotrexate. Patients aged < 50 years had anallogeneic stem cell transplant if they had a matched siblingdonor; otherwise, they received maintenance chemotherapyfor 2 years, which also included two intensification courses.

BM and/or PB samples at presentation from 129 patients(107 AML, 22 ALL) and BM/PBPC from 24 normal donorswere analysed qualitatively for the presence of WT1transcript. BM and PB paired samples were available in19 patients and seven normal donors. The median age of

these patients was 51 years (range 16–78 years). Quanti-tative analysis of the presentation BM and/or PB sampleswas performed in 66 patients (52 AML, 14 ALL) and 24normal samples. Cytogenetic data at presentation wereavailable in 50/52 patients with AML and 11/14 patientswith ALL.

Serial samples were analysed at presentation, afterinduction chemotherapy, second consolidation course, dur-ing remission at 3-monthly intervals, after bone marrowtransplantation and at relapse in 28 patients with AML. Theequivalent time points for patients with ALL (seven patients)were at presentation, after phase 1 of induction chemo-therapy, after consolidation with methotrexate, duringremission at 3-monthly intervals, after bone marrowtransplantation and at relapse.

RNA preparation. Mononuclear cells (MNC) were isolatedfrom BM and PB using the Ficoll-Hypaque density gradientcentrifugation method and stored at )80�C. RNA wasextracted from 1–2 · 106 cells of the NB4 cell line andpatients’ samples according to the guanidinium–phenol–chloroform method of Chomzynski & Sacchi (1987), withminor modifications as described previously. Extracted RNAwas quantified by spectrophotometry at 260 and 280 nm.

Reverse transcription (RT). Total RNA (1 lg) was dena-tured at 72�C for 5 min and snap cooled on ice. RT wasperformed by adding RT reaction mixture (final concentra-tion in a 20-ll reaction volume; 1· first-strand buffer,10 mmol/l dithiothreitol, 0Æ25 lg of pd(N)6, 0Æ5 mmol/ldNTP, 200 U of Moloney murine leukaemia virus reversetranscriptase, 40 U of RNAsin) to the RNA and incubationat room temperature for 10 min. RT reaction was per-formed at 37�C for 60 min, then at 45�C for 30 min and at72�C for 5 min.

ABL RT-PCR (qualitative). The ABL gene transcript waschosen as a control gene to assess the quality and quantityof amplifiable mRNA used in the RT-PCR assays. PCRamplification was performed in a 25-ll reaction containing1· PCR buffer, 0Æ5% W-1, 1Æ5 mmol/l MgCl2, 0Æ25 mmol/ldNTP, 15 pmol of primers (A2 and CA3), 1 U of Taq (Gibco)with 2 ll of cDNA. ABL PCR was performed at 97�C for1 min 30 s, 64�C for 50 s, 72�C for 1 min (one cycle); 97�Cfor 30 s, 64�C for 50 s, 72�C for 1 min (40 cycles); 72�C for5 min (one cycle). PCR products were electrophoresed on a2% agarose gel. Any sample demonstrating the presence ofgenomic DNA was subjected to repeat RNA extraction.However, contamination with genomic DNA was encoun-tered in only a minority of samples. Expected band size forABL transcript was 276 bp.

WT1 RT-PCR (qualitative). Two microlitres of cDNA wassubjected to two rounds of PCR amplification for the WT1transcript. First-round PCR was performed in a 50-llreaction containing 1· PCR buffer, 0Æ5% W-1, 1Æ5 mmol/lMgCl2, 0Æ25 mmol/l dNTP, 15 pmol of primers (WT1 andWT4) and 1 U of Taq (Gibco). The first-round PCR wasperformed in a 50-ll reaction containing primers WT1 andWT4 at 93�C for 2 min (one cycle); 93�C for 30 s, 55�C for50 s, 72�C for 1 min (40 cycles); 72�C for 5 min (onecycle). Two microlitres of first-round products were used ina 50-ll second-round PCR containing nested primers WT3

50 M. Garg et al

� 2003 Blackwell Publishing Ltd, British Journal of Haematology 123: 49–59

and WT11 under the same PCR conditions. PCR productswere electrophoresed on a 2% agarose gel. The expectedband size for WT1 transcript is 412 bp in the first round and242 bp in the second round.

ABL competitor construction (Fig 1). The ABL competitorwas constructed using splicing by the overlap extensiontechnique as described before (Clarkson et al, 1991; Tobal &Liu Yin, 1996). The size of the constructed ABL competitorwas 285 bp.

ABL competitive RT-PCR. This is based on co-amplifica-tion of two fragments (gene and competitor) with the sameset of primers, but produces two different-sized products. If aPCR contains the same amount of both transcript andcompetitor, then equal amounts of product will be seen onthe agarose gel (same intensity of the bands) (Cross et al,1993). Competitive RT-PCR was performed using 2 ll ofsample cDNA and 2 ll of competitor DNA in a 25-llreaction subjected to the same PCR conditions as describedpreviously (Tobal & Liu Yin, 1996). The expected band sizefor the ABL transcript was 276 bp and ABL competitor was232 bp. We estimated that there were 105 copies of ABLtranscript in 1 lg of RNA.

WT1 competitor construction (Fig 2). The WT1 competitorwas prepared by PCR amplification of WT1 transcript fromNB4 cDNA using primers WT5 and WT12. Primer WT12 isthe reverse primer and has a tail that corresponds to reverseprimers WT4 and WT11 that were used in two rounds of

PCR amplification. The expected band size of the WTcompetitor was 323 bp.

WT1 competitive RT-PCR. cDNA (2 ll) positive for WT1and 2 ll of competitor DNA were mixed together in a 50-llreaction and subjected to PCR amplification as describedabove. The expected band size of the competitor (WTC)produced was 275 bp in first round and 185 bp in thesecond round. Each sample was quantified at every order ofmagnitude and then at every half order of magnitude. Thepoint of equivalence was assessed by gel densitometry.Because of the size difference between the WT1 transcript(242 bp) and the competitor band (185 bp), the number ofcompetitor molecules at the point of equivalence wasmultiplied by 0Æ76 (the ratio of the size of competitor andthe transcript). The level of WT1 transcript was normalizedto 105 copies of ABL transcript present, thus eliminatingerrors resulting from sample variation and handling.

Sequences of PCR primers. Outer forward primer WT15¢-GGCATCTGAGACCAGTGAGAA-3¢

Inner forward primer WT3 5¢-GCTGTCCCACTTACAGATGCA-3¢

Inner reverse primer WT11 5¢-GACAGCTGAAGGGCTTTTCA-3¢

Outer reverse primer WT4 5¢-TCAAAGCGCCAGCTGGAGTTT-3¢

Forward WT5 5¢-TCAGGATGTGCGACGTGTGC-3¢Reverse WT12 5¢-TCAAAGCGCCAGCTGGAGTTTGAC

AGCTGAAGGGCTTTTCATTTCGCTGACAAGTTTTA-3¢CA3 5¢-TGTTGACTGGCGTGATGTAGTTGCTTGG-3¢A2 5¢-TTCAGCGGCCAGTAGCATCTGACTT-3¢ABL and WT1 degradation rates. Rates of degradation

studies were carried out for both transcripts at 24 h andFig 1. Diagrammatic description of the preparation of ABL gene

competitor using the ‘splicing by overlap extension’ technique.

Fig 2. Diagrammatic description of the preparation of WT1 gene

competitor.

Monitoring of WT1 Transcript Levels in Acute Leukaemia 51


48 h using the NB4 cell line. The NB4 cell line sample wasdivided into three equal parts. One part was subjected toFicoll-Hypaque density gradient centrifugation as describedabove, then frozen directly at )80�C; the other two partswere incubated at room temperature for 24 and 48 h,respectively, before centrifugation. The levels of ABL andWT1 transcripts were estimated in all three parts todetermine the rates of degradation.

Accuracy and reproducibility of assays. Positive and neg-ative controls were used in each assay. The NB4 cell linewas used as positive control. Negative controls includedsterile H2O as a replacement for RNA or cDNA. Contam-ination was avoided through the use of an ultravioletflow cabinet, designated PCR pipettes and filtered tips.Each assay was repeated at least twice to confirm theresults.

Statistical analysis. Overall survival (OS) was calculatedfrom presentation until death, and the disease-free survival(DFS) from the achievement of complete remission (CR)until relapse. Kaplan and Meier life tables were constructedfor the survival data, i.e. OS and DFS, and were comparedby means of the log rank test.

RESULTS

Degradation rates for ABL and WT1 transcriptsThe degradation rates of the ABL and WT1 transcripts werefound to be equal. After incubation of samples at roomtemperature, the levels of both transcripts decreasedequally, by 0Æ5 log after 24 h and 1 log after 48 h. Theseresults indicated that ABL is a suitable control transcript forquantification of WT1 transcript. RNA and cDNA samplesstored at )80�C for up to 2 months showed no degradationof either transcript.

Linearity and sensitivity analysis (Fig 3)Serial dilutions of NB4 cells were made in sterile water. TheRT-PCR method was found to be linear over a wide range ofWT1 transcript levels as shown. The method has asensitivity level of one NB4 cell in 104 dilution and, insome experiments, we were able to detect 1 in105. With thismethod, we were able to detect as few as eight copies ofWT1 transcripts per 105 ABL transcripts.

Qualitative assay (Table I)We performed this assay in 94 BM and 32 PB samples in107 patients with AML. Twenty-one BM and eight PBsamples were analysed in 22 patients with ALL. Altogether,120/129 patients with AL (> 90%) had detectable levels ofWT1 at presentation. It was not detected in five BM and twoPB samples in seven patients with AML (patient FAB typeswere: one M2, one M4Eo, four M5, one not specified), and2/22 patients with ALL. In the normal donors, WT1transcript was not detected in 20 BM/PBSC samples,whereas a positive result was obtained in four. A positivequalitative ABL RT-PCR was sufficient to confirm thequality of the extracted RNA and, hence, the samples thatwere negative for WT1 transcript were not subjected toquantitative RT-PCR for ABL.

Levels at presentation (Table II)Quantitative analysis was performed in 66 patients (52AML, 14 ALL). The median level of WT1 in AML patientswas 8Æ9 · 104 (range 2 · 103)3 · 106) and 3Æ2 · 104

(range 8 · 102)8 · 105) copies/105 ABL for BM and PBrespectively. Patients with ALL had a median level of6 · 104 (range 8 · 103)8 · 104) and 2Æ4 · 104 (range8 · 102)8 · 104) copies/105 ABL for BM and PB respect-ively. Eleven PBSC and 13 BM were analysed from normaldonors. The gene was not detected in 9/11 PBSC and 11/13BM, whereas in the remaining four normal samples, themedian level of WT1 transcript was 80 copies/105 ABL(range 8–2 · 102).

Critical presentation level (Fig 4)Patients in our cohort could be divided into three groupsaccording to the levels of WT1 in the BM samples atpresentation, namely < 104, 104)105 and > 105 copies.Sixteen patients (10 AML, six ALL) had between 103 and104 copies/105 ABL, and 100% achieved CR with inductionchemotherapy, whereas in 32 patients (24 AML, eight ALL)with levels between 104 and 105 copies at presentation, theCR rate was 68%. Eighteen patients (all AML) had more

Fig 3. Linearity and sensitivity studies of the competitive nested

RT-PCR titration assay for WT1. The figure above each track

indicates the number of WT1 competitor molecules added. Dilution

of the NB4 cell line in sterile H2O at factors of 100·, 1000· and

10 000· resulted in a reduction in the detectable number of com-

petitor molecules 10-, 100- and 1000-fold respectively.

52 M. Garg et al


than 105 copies, and only 50% achieved CR. The differencein the CR rates in the three groups was highly significant(P ¼ 0Æ001).

Probability of survival at 3 years was 81%, 37% and 22%for patients with levels < 104, between 104 and 105 and> 105 copies respectively. Patients with WT1 levels < 104

had significantly better OS (P ¼ 0Æ0003) and DFS(P ¼ 0Æ0004) compared with patients with levels between104 and 105 or > 105 copies (Fig 4). We did not find asignificant difference in DFS (P ¼ 0Æ1577) and OS(P ¼ 0Æ2394) when patients with levels between 104 and105 and > 105 copies were compared. Similar survivalresults were obtained when patients with AML wereanalysed separately.

Correlation between cytogenetics and WT1 level in the BMat presentation

Acute myeloid leukaemia. Patients were classified accordingto their karyotypic abnormalities (Grimwade et al, 1998).

Good risk cytogenetics (13 patients). A total of 8/13 hadWT1 levels > 104, only two patients have remained in CR,whereas six have succumbed to the disease. Five out of 13had < 104 copies; all remained in stable remission with amedian follow-up of 46 months (range 16–73 months).

Standard risk cytogenetics (23 patients). A total of 19/23patients had a WT1 level > 104, 15 of whom have relapsedafter a median CR duration of 14 months (range 6–42 months), two died during induction, one had refractoryleukaemia and only one patient is in CR for 19 months. Allfour of the 23 patients with < 104 copies of WT1 atpresentation remain in first CR at a median of 21 months(range 18–29 months).

Poor risk cytogenetics (14 patients). Thirteen patients hadWT1 levels > 104, and all either had refractory disease orhave relapsed, whereas the only patient with < 104 copiesremained in CR at 30 months.

Acute lymphoblastic leukaemia. Among six patients witht(9:22) at diagnosis, three patients with WT1 levels > 104 at

Table I. Disease characteristics and qualitative WT1 expression.

Disease

(FAB type)

No. of

patients

WT1 expression

positive Percentage

Acute leukaemia 129 120 93

Acute myeloid leukaemia 107 100 93Æ5M0 2 2 100

M1 14 14 100

M2 25 24 96

M3 15 15 100

M4 6 5 83

M5 5 1 20

M6 5 5 100

M7 3 3 100

Not otherwise specified 14 13 93

RAEBt 18 18 100

Acute lymphoblastic leukaemia 22 20 91

Common ALL 12 12 100

T ALL 3 3 100

B ALL 1 1 100

Biphenotypic 1 1 100

Not otherwise specified 5 3 60

Normal controls 24 4 16

RAEBt, refractory anaemia with excess blasts in transformation.

Table II. WT1 expression level at presentation, disease relapse and in long-term remission.

Sample

type

No. of patients WT1

positive/total number

Normalized levels of

WT1 transcripts in BM

At presentation 66/66 > 103 copies/105 copies of ABL

Normal controls 4/24 < 2 · 102 copies/105 copies of ABL

Long-term remission 7/21 < 103 copies/105 copies of ABL

Follow-up > 36 months

At relapse 18/18 > 104 copies/105 copies of ABL



presentation were refractory to induction chemotherapy orsubsequently relapsed at a median duration of 8 months(range 4–15 months), whereas three patients who had< 104 copies of WT1 achieved remission with a medianduration of 30 months (range 27–34 months).

Critical remission level (Fig 5)WT1 levels were assessed at specific time points during thedisease course, namely after induction chemotherapy andsecond consolidation chemotherapy in 28 patients withAML. The equivalent time points for patients with ALL(seven patients) were after phase 1 of induction chemo-therapy and after consolidation with high-dose metho-trexate. Patients were divided into two groups accordingto the level of MRD detected by WT1 assay in the BM.Patients who were negative or had < 103 copies of WT1transcript after induction (Fig 5) and second consolidationchemotherapy (Figure not shown) had significantly betterDFS and OS (P < 0Æ0001).

Levels during remission (Table II and Fig 6)We studied serial BM and PB samples in 21 patients whocontinued to be in stable remission for a median follow-up of51 months (range 15–91 months). WT1 has not beendetected in 14 patients (10 AML, four ALL) since post-induction chemotherapy, whereas seven patients (six AML,one ALL) expressed WT1 at very low levels (< 103). Elevenpatients have remained in remission for more than 4 years.

Prerelapse levels (Figs 7 and 8)We studied serial samples in 14 patients (12 AML, two ALL)who subsequently relapsed at the median CR duration of12 months (range 4–49 months). In nine patients (eightAML, one ALL), relapse could be predicted 2–4 monthsbefore haematological relapse because of rising levels ofWT1 (1–5 log rise). The median level of WT1 transcript inthese samples was 6 · 104 (range 8 · 103)2 · 105) and8Æ9 · 103 (range: 2 · 103)3 · 104) copies/105 ABL in BMand PB respectively.

Fig 4. Kaplan–Meier plot showing the corre-

lation between the disease-free survival and

WT1 level in BM at presentation in patients

with acute leukaemia.

Fig 5. Kaplan–Meier plot showing the corre-

lation between the disease-free survival and

WT1 level in BM after induction chemother-

apy in patients with acute leukaemia.

54 M. Garg et al


Five patients (three AML, two ALL) had persistently highlevels, i.e. > 103 copies of WT1 throughout the treatmentcourse. These patients relapsed within 6 months of diagno-sis and succumbed to their disease (Fig 7).

Levels at relapseRelapsed disease was studied in 14 BM and 11 PB samplesfrom 18 patients. The median level of WT1 transcript was8Æ9 · 104 (range 2 · 104)3 · 106) and 8Æ3 · 104 (range:

Fig 6. Serial quantification of WT1 transcripts in four patients with AML in long-term CR (> 3 years). Levels of 1Æ00E)01 (¼ 1Æ0 · 10)1) are

RT-PCR negative.

Fig 7. Serial quantification of WT1 transcripts in three patients with AML and one patient with ALL who had persistently high levels of WT1

during the disease course. R, relapse. Levels of 1Æ00E)01 are RT-PCR negative.



8 · 103)3 · 105) copies/105 ABL in the BM and PBrespectively. Six patients (six BM and four PB samples)had > 105 WT1 copies at relapse.

Comparison of PB and BM analysis (Fig 9)BM and PB paired analysis was undertaken in 12 patients atpresentation. Sequential paired samples were tested for WT1levels in 12 patients who have relapsed and 10 patients whocontinued to be in CR for a median duration of 40 months(range 16–85 months). The levels in PB were lower than inBM by 1–2 log but mirrored changes in BM samples andwere therefore equally useful in predicting early relapse inAL patients.

DISCUSSION

The WT1 gene has been shown to be overexpressed inimmature leukaemia cells, especially those with myeloidcharacteristics (Miwa et al, 1992; Miyagi et al, 1993; Inoueet al, 1994). We confirmed that > 90% of AL cases expressthe WT1 gene as reported previously (Inoue et al, 1994;Menssen et al, 1995). It was not expressed in 80% of acutemonocytic and 33% of myelomonocytic (M4, M4Eo)leukaemia cases (Table I). Moreover, it was expressed at alower level in more differentiated leukaemias such as acutepromyelocytic leukaemia. However, unlike previouslyreported figures (Miwa et al, 1992; Menssen et al, 1995;

Patmasiriwat et al, 1999), we found WT1 to be expressed ina higher proportion of ALL (91%). This could be due to therelatively high sensitivity of the nested PCR technique wehave used to detect WT1 transcripts. These findings suggestthat WT1 gene expression is closely associated with acuteleukaemias of both myeloid and lymphoid lineages.

Data regarding the expression of WT1 on normalhaemopoietic progenitors are somewhat conflicting (Baird& Simmons, 1997; Inoue et al, 1997; Maurer et al, 1997;Patmasiriwat et al, 1999). This discrepancy appears toresult from the variable sensitivities of the different meth-odologies used to detect the WT1 transcript. Routinequalitative or semi-quantitative RT-PCR assays have notbeen able to detect the transcript in unsorted normal BMsamples (Brieger et al, 1994; Menssen et al, 1995), whereasa sensitive quantitative method has shown WT1 to beexpressed in normal CD34-selected progenitors (Inoue et al,1994, 1996; Baird & Simmons, 1997; Maurer et al, 1997).Using a very sensitive quantitative method, we were able todetect WT1 in four out of 24 normal BM or PBPC donors.The level in these samples was appreciably lower(8–2 · 102 copies) than the level we detected in leukaemicpatients at presentation (> 2 · 103). Thus, by quantitativeanalysis, we have shown WT1 expression to be at least 10times lower in some normal progenitors compared withleukaemic cells, similar to previously reported results.(Inoue et al, 1996, 1997).

Fig 8. Levels of WT1 transcripts in AL at different phases of disease. A, relapsed patients (n ¼ 14); B, remission patients (n ¼ 21). Levels of

1Æ00E)01 are RT-PCR negative. Post Con, post consolidation.

56 M. Garg et al


Several studies (Brieger et al, 1994; Schmid et al, 1997)using a qualitative assay for WT1 in AML found nocorrelation between WT1 expression at diagnosis andachievement of CR, whereas Bergmann et al (1997) haveshown a tendency towards a higher CR rate in patientswith low-level expression of WT1 mRNA using a semi-quantitative technique. However, Inoue et al (1996)showed a clear inverse correlation between high WT1levels in AL and prognosis in terms of CR, DFS and OS.Quantitative analysis, using either competitive RT-PCR(Bergmann et al, 1997) or visual densitometry (Inoue et al,1996), does not take into account the variation resultingfrom sample handling and quality of RNA and cDNA,whereas by performing double quantification for both WT1and ABL transcripts and adjusting the WT1 value to 105

copies of ABL transcripts, we attempted to eliminate suchpotential errors. In this study, we were able to divide thepatients into three groups according to the levels of WT1transcripts at presentation. We showed that the CR ratewas 100%, 66% and 50% in patients with WT1 levels of< 104, 104)105 and > 105 copies respectively. Further-more, the actuarial survival at 3 years was 81%, 37% and22%, respectively, in these three groups. Patients with< 104 copies in the BM at presentation had significantlybetter DFS and OS than patients with > 104 copies.Subgroup analysis showed similar results for AML patients.We confirm that WT1 levels at presentation in acuteleukaemia, as well as in AML, are prognostically importantfor CR achievement, DFS and OS.

We also attempted to correlate WT1 levels at presentationwith cytogenetic risk groups in AML. There appeared to be atrend for a higher proportion of patients with high WT1levels (> 104 copies) in the less favourable cytogenetic

groups (60% of good risk, 82% of standard risk and 93% ofpoor risk patients had > 104 copies). Within both good andstandard cytogenetic risk groups, high WT1 levels correla-ted with a poorer outcome. Thus, WT1 level at diagnosismay be another useful pretreatment characteristic, similarto Flt3 ligand mutation (Kottaridis et al, 2001), and mayenable the identification of high-risk patients in the cyto-genetically defined good and standard risk subsets of AML.However, this important observation remains to be con-firmed in a larger group of patients.

We carried out quantification of MRD in 35 patients (28AML, seven ALL) at two specific time points, i.e. afterinduction and after second consolidation chemotherapy,and serially during remission. We found that patients whowere negative or had < 103 copies at these time points hadsignificantly better DFS and OS than those with > 103 copiesof WT1. Similarly, during long-term remission, patients hadeither undetectable or < 103 copies of WT1. Furthermore,we were able to predict relapse in nine patients, in whomWT1 levels rose significantly, 2–4 months before the onsetof haematological relapse. In five patients, WT1 levelsremained persistently high during morphological and cyto-genetic remission, and they all relapsed within 6 months.Thus, our results confirm and extend the observations ofInoue et al (1996), who used semi-quantitative assays forthe WT1 transcript. Using a sensitive, quantitative method,we have also shown that PB is a reliable source of MRD inWT1-positive patients and could also be used successfully topredict early relapse. Clearly, a qualitative assay for WT1,because of its lack of sensitivity, is of no value in MRDmonitoring in AL. We also postulate that it may be possibleto establish critical MRD levels, based on WT1, above andbelow which patients are likely to relapse or remain in

Fig 9. Serial quantification of WT1 transcripts in four cases of relapsed AML and comparison of WT1 levels in BM and PB in two cases: FR (PB,

BM) and LL (PB, BM). R, relapse. Levels of 1Æ00E)01 are RT-PCR negative.



remission. These observations are in accordance with thosereported for quantitative MRD monitoring using AML1-ETOtranscripts in t(8:21)-positive patients (Tobal et al, 2000).More recent data also suggest that quantification of CBFb-MYH11 transcripts by real-time PCR in AML patients withinv(16) abnormality may allow the establishment of MRDthresholds for determining relapse risk (Buonamici et al,2002; Guerrasio et al, 2002). Real-time quantitative (RQ)-PCR has now been applied to quantify the WT1 transcript,and the results of early studies in AML also appearpromising (Kreuzer et al, 2001; Cilloni et al, 2002). WT1may thus be a valuable molecular target, and the results ofour study provide the basis for future MRD monitoring byRQ-PCR analysis in AML patients who lack a specific fusiongene and possibly also offer an alternative target in ALLpatients. Furthermore, RQ-PCR can produce a result in afew hours and lends itself to greater standardization andquality control.

In conclusion, we have shown that accurate quantifica-tion of WT1 transcripts in both AML and ALL patients atpresentation is an important prognostic pretreatment char-acteristic and is also a useful predictive marker of leukaemiarelapse. We also showed that, in at least 76% of patients inthis series (presentation WT1 level > 104 copies/105 ABL,i.e. at least 2 log higher than normal), WT1 is a suitablemarker for MRD monitoring. Our data suggest that it maynow be possible to offer MRD monitoring for most patientswith AML. This information on MRD may in future providethe basis for therapeutic intervention, for example pre-emptive treatment in cases of molecular relapse, includingstem cell transplantation or additional therapy for high-riskpatients, thus allowing specific treatment to be tailored toindividual patients. However, the clinical utility of WT1 as amarker of MRD in AML will need to be evaluated inprospective studies involving large numbers of patients.

ACKNOWLEDGMENT

Dr M. Garg was a Leukaemia Research Fund ClinicalTraining Fellow.

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Prognostic significance of quantitative analysis of WT1 gene transcripts by competitive reverse transcription polymerase chain reaction in acute leukaemia