Real-Time Quantitative Polymerase Chain Reaction Detection of Minimal Residual Disease by Standardized WT1 Assay to Enhance Risk Stratification in Acute Myeloid Leukemia: A European LeukemiaNet Study
Risk stratification in acute myeloid leukemia (AML) is currently based on pretreatment characteristics. It remains to be established whether relapse risk can be better predicted through assessment of minimal residual disease (MRD). One proposed marker is the Wilms tumor gene WT1, which is overexpressed in most patients with AML, thus providing a putative target for immunotherapy, although in the absence of a standardized assay, its utility for MRD monitoring remains controversial.
Nine published and in-house real-time quantitative polymerase chain reaction WT1 assays were systematically evaluated within the European LeukemiaNet; the best-performing assay was applied to diagnostic AML samples (n = 620), follow-up samples from 129 patients treated with intensive combination chemotherapy, and 204 normal peripheral blood (PB) and bone marrow (BM) controls.
Considering relative levels of expression detected in normal PB and BM, WT1 was sufficiently overexpressed to discriminate ≥ 2-log reduction in transcripts in 46% and 13% of AML patients, according to the respective follow-up sample source. In this informative group, greater WT1 transcript reduction after induction predicted reduced relapse risk (hazard ratio, 0.54 per log reduction; 95% CI, 0.36 to 0.83; P = .004) that remained significant when adjusted for age, WBC count, and cytogenetics. Failure to reduce WT1 transcripts below the threshold limits defined in normal controls by the end of consolidation also predicted increased relapse risk (P = .004).
Application of a standardized WT1 assay provides independent prognostic information in AML, lending support to incorporation of early assessment of MRD to develop more robust risk scores, to enhance risk stratification, and to identify patients who may benefit from allogeneic transplantation.
Current treatment protocols for acute myeloid leukemia (AML) are based on prognostic factors that contribute to therapy stratification.1,2 Key prognostic factors so far identified include pretreatment characteristics such as age, WBC count, karyotype, mutation status of the FLT3, NPM1, and CEBPA genes and relative levels of expression of BAALC, EVI1, and ERG.3–8 Morphologic response to induction chemotherapy provides an additional predictive factor, which has been incorporated into current risk stratification schemes used to inform decisions regarding consolidation therapy, particularly allogeneic transplantation.9 Although such parameters distinguish groups of patients at broadly differing risk of relapse, there is a pressing need to refine risk stratification to more reliably identify those patients most (and least) likely to benefit from transplantation. A number of studies have highlighted the potential of minimal residual disease (MRD) monitoring by real-time quantitative polymerase chain reaction (RQ-PCR) to detect leukemia-specific targets (ie, fusion gene transcripts such as PML-RARA or mutations such as that in NPM1) that would reveal those patients at highest risk of relapse and provide an opportunity for early treatment intervention (10). However, approximately half of AML patients lack a suitable leukemia-specific target; thus there has been considerable interest in developing alternative approaches to allow MRD detection to be extended to a much greater proportion of patients. One strategy involves use of flow cytometry to identify and monitor leukemia-associated aberrant phenotypes; it has the advantage of wide applicability, but it is technically demanding.10 Another approach involves using RQ-PCR assays to detect transcripts that are highly overexpressed in AML blasts relative to normal PB and BM, with most of the attention being focused on the Wilms tumor (WT1) gene.10
WT1 was originally identified for its involvement in the pathogenesis of Wilms tumor (reviewed11) and has been shown to be highly expressed in several hematopoietic tumors, including AML.11–25 Although the mechanisms leading to WT1 overexpression remain poorly understood, this phenomenon can be exploited as a marker to establish the presence, persistence, or reappearance of leukemic hematopoiesis. However, the clinical utility of WT1 monitoring has been somewhat controversial, which may in part reflect differences in performance of the assays evaluated to date. It has become apparent that a number of RQ-PCR assays are designed to amplify 3′ regions of the gene, where mutations that occur in approximately 10% of AML patients tend to cluster,26–30 potentially giving rise to false-negative results.31 Therefore, we established a network of 11 laboratories within the MRD Workpackage of the European LeukemiaNet (ELN) to systematically evaluate nine published and in-house WT1 RQ-PCR assays, leading to the selection of a standardized assay with optimal performance that was shown to provide an independent prognostic factor in AML.
Total RNA was extracted and reverse transcribed using the standardized protocol developed within the Europe Against Cancer (EAC) program.32 RQ-PCR assays were performed using EAC conditions, with primers and probes at final concentrations of 300 nmol/L and 200 nmol/L, respectively.32 Primers and probes for WT1 assays and the published EAC ABL control gene assay33 were centrally distributed along with respective plasmid standards (a kind gift from Ipsogen, Marseille, France). RQ-PCR assays were predominantly performed on Applied Biosystems platforms (ABI7000/7700/7900; Applied Biosystems, Foster City, CA); one laboratory used the Lightcycler 1.5 (Roche Applied Science, Penzberg, Germany), one used a Rotor-Gene instrument (3000/3000A; Corbett Life Science, Sydney, Australia), and one used the Mx3000P (Stratagene, La Jolla, CA). Data were reported using a common threshold of 0.1, where the Ct (cycle threshold) value denotes the PCR cycle at which fluorescence is detected above the background level (reviewed34). WT1 transcripts were normalized to ABL by using the respective plasmid standards to generate normalized copy numbers in addition to using the ΔCt method.35 All assays were run in triplicate wells with appropriate water controls. To define sensitivities for the WT1 assays under evaluation, the detection limit was designated as the lowest dilution of a WT1-positive cell line (HL60) giving rise to amplification above the background level (ie, normalized WT1 ΔCt ≥ 1) detected in peripheral blood lymphocytes (PBLs) in at least two of three replicates with Ct ≤ 40. For analysis of AML samples and normal controls, those with detectable WT1 copy numbers (ie, with specific amplification in at least two of three replicates with Ct ≤ 40) were expressed per 104 ABL copies. Follow-up samples with ABL Ct more than 29 were excluded from analysis, according to EAC criteria.32 Samples were studied after institutional review board approval, and informed patient consent was provided in accordance with the Declaration of Helsinki.
The selection and evaluation process involved analyses conducted in 11 leukemia diagnostic laboratories spread across eight countries within the MRD Workpackage (WP12) of the European LeukemiaNet (www.leukemia-net.org). In phase I, nine published and in-house WT1 RQ-PCR assays (designated assays 1 to 9) were systematically evaluated (Fig 1, Table 1; Appendix Table A1, online only, for primer and probe sequences). These were tested on cDNA obtained from serial dilutions of HL60 AML cells (in which WT1 is highly expressed) into normal PBLs. All assays were also tested on cDNAs derived from undiluted HL60 cells and PBLs. RNA specificity of each assay was evaluated by including analysis of “no reverse transcriptase” controls and genomic DNA as described.34 On the basis of the performance profile, particularly in terms of sensitivity and RNA specificity, three assays were excluded at this stage (Fig 1; Table 1). In phase II, the remaining six assays were tested in parallel in the reference laboratory in Turin on a full-length WT1 plasmid standard (Ipsogen). Primary data were reviewed by members of all 11 participating laboratories; the three assays with the best performance (ie, efficiency and sensitivity) were taken forward to phase III, which involved parallel analysis by the network of laboratories on centrally distributed plasmid standards and cDNAs derived from serial dilutions of HL60 cells in PBLs. Assay 9, published previously by Van Dijk et al,37 (forward primer, 5′-CGCTATTCGCAATCAGGGTTA-3′; reverse primer, 5′-GGGCGTGTGACCGTAGCT-3′; probe, 5′-FAM-AGCACGGTCACCTTCGACGGGA-TAMRA-3′) was selected as the ELN WT1 assay and evaluated in normal control and AML patient samples (phase IV).
|WT1 Assay||Assay Location||Assay Reference||Sensitivity to Detect WT1 in HL60 Cell Line Dilutions||Phase of Exclusion||Reason for Exclusion|
|1||Exons 6-7||Ogawa et al22||10−3||I||Lack of RNA specificity*|
|2||Exons 7-8||Osborne et al36||10−2||II||Reduced efficiency and sensitivity|
|3||Exons 6-7||Østergaard et al23||10−4||III||Location of primers and probe|
|4||Exons 7-8||Unpublished||10−3||I||Reduced efficiency and sensitivity|
|5||Exons 7-8||Unpublished||10−3||II||Reduced efficiency and sensitivity|
|6||Exons 7-8||Unpublished||10−2||I||Low sensitivity|
|7||Exons 7-8||Unpublished||10−4||II||Lower level of efficiency|
|8||Exons 7-8||Cilloni et al15||10−3-10−4||III||Inferior level of efficiency|
|9||Exons 1-2||Van Dijk et al37||10−4||Not excluded||Not applicable|
*All other assays were confirmed to be RNA-specific.
Reference values of normalized WT1 transcript levels in normal peripheral blood (PB), bone marrow (BM), and PB stem cells (PBSCs) were established using the selected ELN WT1 assay by analyzing a total of 118 PB, 61 BM, and 25 granulocyte colony-stimulating factor mobilized PBSC samples collected from healthy volunteers. To investigate the range of WT1 expression observed in AML, 620 pretreatment samples (238 PB, 382 BM) derived from 504 patients (median age, 45 years; range, 0 to 85 years) were studied. The predictive value of MRD assessment using the ELN WT1 assay was determined in a cohort of 129 patients (median age, 45 years; range, 2 to 76 years) treated with intensive anthracycline and cytarabine–based combination chemotherapy.38–42 To establish whether hematopoietic regeneration following chemotherapy modulates WT1 expression, follow-up samples were analyzed from a cohort of 16 patients with AML in which pretreatment WT1 transcript levels were shown to be low, comparable to those observed in normal BM (< 250 copies/104 ABL copies).
Given that the ELN assay spans exons 1 and 2 of the WT1 gene, in 32 patients with AML found to have low WT1 expression at diagnosis (drawn from the cohort of 504 patients), the 5′ region of the gene was PCR-amplified using primers ATGGGCTCCGACGTGCGGGAC and TCAAAGCGCCAGCTGGAGTTTGG. The 5′ region was then sequenced to investigate for possible mutations disrupting the primer or probe binding sites by using standard methods and the ABI PRISM 3100 genetic analyzer (Applied Biosystems).35 To determine whether the presence of mutations in the 3′ region (exons 7 and 9) of WT1 was correlated with differences in WT1 expression level (as determined by the ELN assay with respect to patients with wild-type WT1), cDNAs from AML patients of known mutation status28 (gift from Jude Fitzgibbon, St Bartholemew's Hospital, London, United Kingdom) were received for analysis.
Correlations between continuous variables were calculated using Spearman's correlation coefficient. Comparison between two groups of continuous variables was performed using the nonparametric Mann-Whitney U test; 2 × 2 contingency tables were analyzed using the Mantel-Haenszel test. Survival analyses were performed using either the log-rank test for comparison of two groups or Cox regression for continuous covariate or multivariable analysis. Cytogenetic risk groups were defined according to the original Medical Research Council classification.43 Significance was set at P < .05.
As a first step to selecting the optimal assay for MRD assessment in AML, nine published and in-house WT1 RQ-PCR assays were evaluated in parallel on serial dilutions of HL60 cells in normal PBLs that exhibit high and low levels of WT1 expression, respectively (phase I, Fig 1). Comparison of WT1 expression in HL60 cells relative to ABL expression using the ΔCt method suggested significant differences in efficiency between the assays, which had an impact on the maximal achievable sensitivities to detect WT1 transcripts (Appendix Table A2, online only). In phase II, six assays with better performance profiles were taken forward for parallel evaluation on WT1 plasmid standards, which revealed variation in assay efficiency and sensitivity (Appendix Table A3, online only). The three best-performing assays (assays 3, 8, and 9) were analyzed in parallel on centrally distributed plasmid standards and cDNA dilution series of HL60 in PBLs in phase III (Fig 1). Assay 8 was found to be less efficient on standard curve analysis (Appendix Fig A1, online only). Assays 3 and 9 were comparable in their performance profile (Appendix Figs A1 and A2, online only) and given that the region amplified by the former is more prone to mutation in AML,26–30 assay 9 (hereinafter ELN WT1 assay) published previously by Van Dijk et al37 was selected for validation.
Given that WT1 is expressed in normal hematopoietic cells, it is critical to establish levels of expression observed in normal control samples so that thresholds can be defined that distinguish residual leukemia from background amplification. Analysis of 204 control samples derived from healthy volunteers using the ELN WT1 assay confirmed that WT1 expression in PB, BM, and PBSC samples is low, with a median value of 19.8 WT1 copies/104 ABL copies (range, 0 to 213) in BM, 0.01 (range, 0.01 to 47.6) in PB, and 6.1 (range, 0 to 39) in PBSC (Fig 2). WT1 expression in PB was significantly lower than in BM (P < .0001). Based on these results, the upper limit of normal was defined as 250 copies for BM and 50 copies for PB.
To evaluate the applicability of the ELN WT1 RQ-PCR assay to detect MRD, 620 pretreatment samples (238 PB and 382 BM) from 504 patients were analyzed. WT1 was overexpressed above background (defined as > 250 and > 50 WT1 copies/104 ABL copies in BM and PB, respectively) in 86% and 91% of BM and PB diagnostic AML samples, respectively (Fig 2). The median value of WT1 copies/104 ABL copies was 2,505 (range, 0 to 7.5 × 105) in BM (P < .0001 v normal BM) and 3,107 (range, 0 to 1.13 × 106) in PB (P < .0001 v normal PB). There was no significant difference in expression between PB and BM across the whole cohort, as confirmed by results obtained in patients with paired diagnostic PB and BM samples (Appendix Fig A3, online only). Considering the upper threshold limits defined in normal control samples, WT1 expression was sufficiently high at diagnosis to allow detection of at least a 2-log reduction in transcripts in 46% and 13% of AML patients according to whether PB or BM samples are used to assess MRD status. Variation in normalized WT1 expression level was observed according to cytogenetics (Fig 3; P < .001), with particularly high levels in patients with inv16 (median, 2.31 × 104; range, 12 to 3.14 × 105). Significantly higher WT1 levels were also detected in AML with NPM1 mutations (NPM1 mutant: median, 1.44 × 104; range, 0 to 1.13 × 106; NPM1 wild type: median, 6,566; range, 0 to 7.5 × 105; P = .005).
The levels of WT1 expression (as defined by ELN WT1 assay) in 15 patients harboring mutations in exons 7 and 9 of the WT1 gene were comparable to patients with wild type WT1 (P = .2). However, sequence analysis of a series of 32 patients in which the ELN WT1 assay suggested a low level of WT1 transcript expression (< 250 copies/104 ABL copies), showed that in three patients (9.4%), this was associated with mutations that disrupted the forward primer binding site (Appendix Fig A4, online only).
To establish the potential utility of any overexpressed gene as a putative MRD target, it is critical to exclude the possibility that the transcript in question is significantly modulated on regeneration after chemotherapy. Therefore, serial follow-up PB and BM samples taken from 16 patients in whom diagnostic samples revealed low levels of WT1 transcripts (< 250 copies/104 ABL copies) were analyzed. No significant impact of hematopoietic recovery on normalized WT1 levels was observed (P = .1); these levels remained below the upper limits defined in normal PB and BM samples (Appendix Fig A5, online only).
To investigate the predictive value of MRD assessment using the ELN WT1 assay, follow-up samples were analyzed from a cohort of 129 adults treated with conventional anthracycline and cytarabine-based chemotherapy in which diagnostic WT1 levels exceeded 2 × 104 copies/104 ABL copies, allowing discrimination of at least a 2-log reduction compared with the pretreatment level. Greater reduction in WT1 after the first cycle of chemotherapy predicted a decreased risk of subsequent relapse (hazard ratio [HR] 0.54; range, 0.36 to 0.83 per log reduction; P = .004; Fig 4), with no evidence of heterogeneity according to treatment protocol (P = .4). Regression analysis demonstrated that the magnitude of WT1 log reduction after induction chemotherapy provided an independent predictor of relapse, remaining significant when adjusting either for age (HR, 0.54; range, 0.35 to 0.83; P = .005), WBCs (HR, 0.54; range, 0.35 to 0.81; P = .003), or cytogenetic risk group (HR, 0.63; range, 0.41 to 0.98; P = .04). Magnitude of log reduction in WT1 transcripts after induction remained highly significant when censoring at time of transplantation undertaken in first remission (HR, 0.42; range, 0.25 to 0.69; P = .0007). The prognostic impact of normalized WT1 transcript level was also determined immediately after completion of consolidation; at this time point, detection of WT1 transcripts at a level exceeding the upper normal values predicted a significantly increased risk of relapse (67% v 42% at 5 years; HR, 0.23; range, 0.09 to 0.63; P = .004).
A number of studies have highlighted the opportunity afforded by the overexpression of the WT1 gene in the majority of AML patients to provide a target for novel immunotherapeutic approaches and to advocate it as a universal marker for MRD assessment.10,44,45 The value of WT1 monitoring in AML has been a matter of some debate; however, optimized assays are becoming increasingly important as tools for enhancing risk stratification in AML and assessing response to WT1-targeted therapies. To address this issue we undertook a systematic evaluation of a range of published WT1 assays; this evaluation revealed marked variation in performance that is likely to have an impact on their clinical utility. Our assessment led to the selection of an assay with superior performance that afforded a sensitivity of 1 in 104 in cell-line dilutions. Moreover, analysis of a large number of control samples has allowed us to establish reference ranges for WT1 expression in normal PB, BM, and PBSCs, which enables transcript levels indicative of residual leukemia to be distinguished from background levels. The location of the selected ELN WT1 assay spanning exons 1 and 2 is also advantageous, since this region is less prone to mutations than exons 7 and 9, where the majority of WT1 assays are situated and thus could give rise to false-negative results.26–31 Indeed, in a focused analysis of AML patients in whom WT1 expression fell within the normal range, as determined by the ELN WT1 assay, 9.4% were found to harbor mutations in the amplified region.
While some studies have reported that pretreatment WT1 expression level is predictive of outcome,12,16,46 it was not confirmed in this study (P = .8). However, in accordance with previous studies,16,19,21,23 we found that determining the kinetics of WT1 transcript reduction following induction chemotherapy provides a key prognostic factor, distinguishing patients at differing risk of relapse within cytogenetically defined risk groups. We also found that failure to normalize WT1 transcript level by the postconsolidation time point distinguishes a group of patients at significantly increased risk of relapse. Together, these results highlight the potential of early assessment of WT1 transcript reduction to identify residual leukemic cells and enhance risk stratification schemes in AML.
There has also been interest in the potential for WT1 to provide a target for sequential MRD monitoring, particularly as a tool to direct the need for treatment modification following allogeneic transplantation, including donor leukocyte infusion.22 WT1 appears not to be modulated on engraftment following transplantation22; through the analysis of sequential follow-up samples taken from patients with AML in which WT1 was not overexpressed, we have been able to show that WT1 expression is not significantly modulated during hematopoietic recovery following intensive chemotherapy. In addition, when background levels of expression observed in normal PB and BM are taken into account, our data show that the degree of WT1 overexpression in most patients with AML is too modest to afford a highly sensitive universal marker for sequential MRD monitoring but nevertheless show that early assessment of decline in WT1 transcripts following therapy in informative patients is highly predictive of risk of subsequent relapse.
Importantly, the establishment of a standardized WT1 assay with independent prognostic value in AML provides a firm basis for determining prospectively in large-scale multicenter clinical trials whether early MRD assessment using this target to define kinetics of disease response can serve to further refine risk stratification to dictate consolidation therapy including transplantation and whether this approach will lead to meaningful improvements in survival. Such studies should evaluate paired PB and BM samples to establish which is most informative, considering background levels of WT1 expression in the respective normal tissues. While the majority of follow-up samples analyzed were BM aspirates, our study suggests that use of PB may increase the proportion of patients in whom WT1 can be used to assess treatment response. Finally, this study has established a standardized assay to assess patients who are potential candidates for WT1-directed therapies and to monitor response to such treatment approaches at the molecular level.
Supported by Leukaemia Research of Great Britain and the European LeukemiaNet (J.V.J. and D.G.).
Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a “U” are those for which no compensation was received; those relationships marked with a “C” were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors.
Employment or Leadership Position: Fabienne Hermitte, Ipsogen (C); Susanne Schnittger, MLL Munich Leukemia Laboratory (C); Tamara Weiss, MLL Munich Leukemia Laboratory (C) Consultant or Advisory Role: David Grimwade, Ipsogen (U) Stock Ownership: Fabienne Hermitte, Ipsogen; Susanne Schnittger, MLL Munich Leukemia Laboratory Honoraria: None Research Funding: None Expert Testimony: None Other Remuneration: None
Conception and design: Daniela Cilloni, Giuseppe Saglio, David Grimwade
Financial support: Giuseppe Saglio, David Grimwade
Administrative support: Giuseppe Saglio, David Grimwade
Provision of study materials or patients: Daniela Cilloni, Aline Renneville, Fabienne Hermitte, Sarah Daly, Enrico Gottardi, Milena Fava, Susanne Schnittger, Tamara Weiss, Barbara Izzo, Josep Nomdedeu, Adrian van der Heijden, Bert A. van der Reijden, Joop H. Jansen, Vincent H.J. van der Velden, Hans Ommen, Claude Preudhomme, Giuseppe Saglio
Collection and assembly of data: Daniela Cilloni, Aline Renneville, Sarah Daly, Jelena V. Jovanovic, Enrico Gottardi, Milena Fava, Susanne Schnittger, Tamara Weiss, Barbara Izzo, Josep Nomdedeu, Adrian van der Heijden, Bert A. van der Reijden, Joop H. Jansen, Vincent H.J. van der Velden, Hans Ommen, Claude Preudhomme, David Grimwade
Data analysis and interpretation: Daniela Cilloni, Aline Renneville, Robert K. Hills, Sarah Daly, Jelena V. Jovanovic, Enrico Gottardi, Milena Fava, Tamara Weiss, Barbara Izzo, Josep Nomdedeu, Adrian van der Heijden, Joop H. Jansen, Vincent H.J. van der Velden, Hans Ommen, David Grimwade
Manuscript writing: Daniela Cilloni, Robert K. Hills, David Grimwade
Final approval of manuscript: Daniela Cilloni, Aline Renneville, Fabienne Hermitte, Robert K. Hills, Sarah Daly, Jelena V. Jovanovic, Enrico Gottardi, Milena Fava, Susanne Schnittger, Tamara Weiss, Barbara Izzo, Josep Nomdedeu, Adrian van der Heijden, Bert A. van der Reijden, Joop H. Jansen, Vincent H.J. van der Velden, Hans Ommen, Claude Preudhomme, Giuseppe Saglio, David Grimwade
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We thank Patricia Hoogeveen and Nicolas Maroc for technical assistance, Michelle Sale for data collection, Karin Summers for sample preparation, and Francesca Messa for assistance in the preparation of the figures; Peter Rohon, MD (Department of Hemato-oncology, University Hospital Olomouc, Czech Republic), Jaroslav Polák, and Cedrik Haskovec, PhD (DNA Diagnostics, Institute of Hematology, Prague, Czech Republic) also made significant contributions to this study.
|WT1 Assay||Primer and Probe Sequences||Location||Reference|
|1||5′-GATAACCACACAACGCCCATC-3′ (Fwd)||Exon 6||Ogawa et al|
|5′-ACACCGTGCGTGTGTATTCTGTATTGG-3′ (Probe)||Exon 6|
|5′-CACACGTCGCACATCCTGAAT-3′ (Rev)||Exons 6-7|
|2||5′-AGCTGTCCCACTTACAGATGCAC-3′ (Fwd)||Exon 7||Osborne et al|
|5′-CAGGAAGCACACTGGTGAGAAACCATACCA-3′ (Probe)||Exons 7-8|
|5′-TTCGTTCACAGTCCTTGAAGTCAC-3′ (Rev)||Exon 8|
|3||5′-AGAATACACACGCACGGTGTCT-3′ (Fwd)||Exon 6||Østergaard et al|
|5′-CTCCAGGCACACGTCGCACATCCTG-3′ (Probe)||Exons 6-7|
|5′-GATGCCGACCGTACAAGAGTC-3′ (Rev)||Exon 7|
|4||5′-TGCACAGCAGGAAGCACACT-3′ (Fwd)||Exon 7||Unpublished|
|5′-TGACTTCAAGGACTGTGAAC-3′ (Probe)||Exon 8|
|5′-TCTTTTGAGCTGGTCTGAACGA-3′ (Rev)||Exon 8|
|5||5′-TGCACAGCAGGAAGCACACT-3′ (Fwd)||Exon 7||Unpublished|
|5′-TGACTTCAAGGACTGTGAAC-3′ (Probe)*||Exon 8|
|5′-TCTTTTGAGCTGGTCTGAACGA-3′ (Rev)||Exon 8|
|6||5′-CAGATGCACAGCAGGAAGCA-3′ (Fwd)||Exon 7||Unpublished|
|5′-AAGTCACACTGGTATGGTT-3′ (Probe)||Exon 8|
|5′-AGAAAACCTTCGTTCACAGTCCTT-3′ (Rev)||Exon 8|
|7||5′-CAGATGCACAGCAGGAAGCA-3′ (Fwd)||Exon 7||Unpublished|
|5′-AAGTCACACTGGTATGGTT-3′ (Probe)*||Exon 8|
|5′-AGAAAACCTTCGTTCACAGTCCTT-3′ (Rev)||Exon 8|
|8||5′-CAGGCTGCAATAAGAGATATTTTAAGCT-3′ (Fwd)||Exon 7||Cilloni et al|
|5′-CTTACAGATGCACAGCAGGAAGCACACTG-3′ (Probe)||Exon 7|
|5′-GAAGTCACACTGGTATGGTTTCTCA-3′ (Rev)||Exon 8|
|9||5′-CGCTATTCGCAATCAGGGTTA-3′ (Fwd)||Exons 1-2||Van Dijk et al|
|5′-AGCACGGTCACCTTCGACGGGA-3′ (Probe)||Exon 2|
|5′-GGGCGTGTGACCGTAGCT-3′ (Rev)||Exon 2|
*Denotes minor groove binding probe; for the remaining assays FAM/TAMRA-labeled probes were used. (Ogawa H, Tamaki H, Ikegame K, et al: Blood 101:1698-1704, 2003; Osborne D, Frost L, Tobal K, et al: Bone Marrow Transplant 36:67-70, 2005; Østergaard M, Olesen LH, Hasle H, et al: Br J Haematol 125:590-600, 2004; Cilloni D, Gottardi E, De Micheli D, et al: Leukemia 16:2115-2121, 2002; Van Dijk JP, Knops GH, Van De Locht LT, et al: Br J Haematol 118:1027-1033, 2002)
|Dilution||Mean WT1 Cycle Threshold Value|
|Assay 1||Assay 2||Assay 3||Assay 4||Assay 5||Assay 6||Assay 7||Assay 8||Assay 9|
NOTE. Mean WT1 cycle threshold values observed following amplification of cDNA derived from serial dilutions of HL60 cells in peripheral blood lymphocytes. Assay 1 was found to amplify genomic DNA and therefore was excluded at this stage, along with Assays 4 and 6 which exhibited poor levels of sensitivity.
|Variable||Mean WT1 Cycle Threshold Value (for each plasmid dilution)|
|Assay 2||Assay 3||Assay 5||Assay 7||Assay 8||Assay 9|
|Plasmid serial dilution|
|1 × 106||28.50||19.23||21.70||20.25||21.61||19.14|
|1 × 105||31.92||22.60||25.61||23.93||25.30||22.42|
|1 × 103||39.18||29.63||33.42||31.16||32.63||29.50|
|1 × 102||42.78||32.78||37.36||34.64||36.47||32.75|
|1 × 101||45.82||36.08||41.08||38.26||39.96||36.35|
NOTE. For assays with maximal efficiency, the slope value is −3.3; therefore, Assays 3 and 9 were the most efficient, with the cycle threshold value equivalent to one copy (indicated by Intercept on y-axis) falling below the detection limit of 40 cycles. These assays were taken forward for further evaluation in phase III with Assay 8.