WT1 expression and NPM1 mutations as biomarkers for minimal residual disease (MRD) monitoring in AML patients

The AML Global Portal highlights the value of AML biomarkers WT1 expression and NPM1 mutation for MRD monitoring in AML patients

The AML_Global_Portal is an evidence-based online resource committed to informing and educating health care providers about the latest cutting-edge advancements in acute myeloid leukemia (AML). The portal aims to help advance understanding and share expertise in the field of AML, and to spread this knowledge globally. The value of two biomarkers, Wilms’ tumor gene 1 (WT1) expression and mutation in the gene encoding nucleophosmin (NPM1), to predict relapse in treated acute myeloid Leukemia (AML) patients was recently reviewed in two separate posts on the AML Global portal (1, 2). These posts respectively review independent studies which conclude that WT1_is_a_suitable_indicator of MRD in AML (1), and that the presence of MRD determined by quantitation of mutated NPM1_transcipts  provides powerful independent prognostic information (2).

WT1 expression as suitable MRD indicator to stratify high-risk AML patients and determine appropriate treatment intensity

WT1 is a transcription factor that plays an important role in cellular development and cell survival. WT1 may act as either a tumor suppressor or an oncogene (3), depending on the cell type in which it is expressed. WT1 is overexpressed in 90% of AML cases and is mutated in approximately 10% of AML cases (4). Recent evidence supports WT1 expression as a marker for minimal residual disease (MRD) in AML patients (1).

There is growing evidence that detecting and monitoring MRD using molecular-based approaches provides powerful independent prognostic information (5) and interest is increasing in using MRD detection to provide early clinical end points and to inform patient management. However, there are challenges in implementation and assessment of MRD in clinical practice. Recent data supports WT1 expression as a suitable indicator of MRD in AML and may be a useful biomarker to predict and manage relapse following allogeneic stem cell transplantation (allo-SCT) treatment (1), currently considered the only efficacious therapy for high-risk patients with AML.

Quantitation of NPM1-mutated transcripts using RT-qPCR to determine presence of MRD and predict relapse

NPM1 is a phosphoprotein which moves between the nucleus and the cytoplasm and is involved in regulation of the ARF/p53 pathway. Mutations in NPM1 cause alterations at the C-terminus of the NPM mutant proteins (6, 7). NPM1 mutations status emerged as a stable marker of MRD already ten years ago, and highly sensitive, specific and reliable real-time quantitative PCR assays for either DNA or RNA have been developed, which are able to monitor all the NPM1 mutation variants in adult AML (9). Current studies confirm quantitation of NPM1-mutated transcripts are able to determine the presence of MRD, and provide powerful independent prognostic information (10).

The ipsogen product line from QIAGEN offers a broad portfolio of high quality biomarker testing solutions for oncohematology, including kits targeting WT1 expression and NPM1 mutations. For WT1 expression, the ipsogen WT1 ProfileQuant Kit (24) CE* provides reliable and sensitive quantification of WT1 gene transcripts relative to ABL1 control gene expression, with a limit of detection (LOD) of 13.08 WT1 normalized copy number (NCN) (11). The components have been validated together in the context of a collaborative study led by a group of experts from the European Leukemia Net (ELN) consortium (8). For clinical research on NPM1 mutations, ipsogen NPM1 MutaScreen Kit (24) RUO** uses real-time PCR clamp technology to define NPM1 mutational status (mutated or wild type) and identify NPM1 mutation type (A, B or D) simultaneously (Figure 1). Depending on the predetermined NPM1 genotype of the sample to be analyzed, the ipsogen NPM1 mut A MutaQuant Kit (24) RUO** and the ipsogen NPM1 mut B&D MutaQuant_Kit (24) RUO** respectively allow quantification of NPM1 mutA transcripts, or separate quantification of NPM1 mutB and NPM1 mutD transcripts, by real-time quantitative PCR (qPCR) relative to ABL1 control gene expression (9) (Figure 2).

Figure 1

Figure 1. Reliable detection of total and mutated NPM1 (12). Detection of (A) total NPM1 and (B) total NPM1, total mutated NPM1, NPM1 mut A, NPM1 mut B and NPM1 mut D. (click on image to enlarge).

Figure 2

Figure 2. Sensitive detection of NPM1 mut A and ABL standards (13). Detection of (A) NPM1 mut A standards (M1 to M5) with 101, 102, 103, 105, and 106 copies/5 μl and (B) ABL standards (C1, C2, C3) with 103, 104, and 105 copies/5 μl. (click on image to enlarge).

Find out more about the ipsogen portfolio of solutions available for research use only in the US and products available in in Europe.

*Available for IVD use in Europe only; available in the US for research use only.

**Available for research use only.

The ratio of copy number (CN) values gives the normalized copy number (NCN) expressed per 104 ABL1 copies.

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References:

  1. 1. Wilms’ Tumor gene (WT1) expression and Minimal Residual Disease in Acute Myeloid Leukemia. AML Global Portal post, August 23, 2016 Link
  2. 2. Sequential RT-qPCR Monitoring in Prediction of Relapse among Patients with NPM1 Mutations. AML Global Portal post, August 25, 2016 Link
  3. 3. Yang, L. et al. (2007) A tumor suppressor and oncogene: the WT1 story- Leukemia 21: 868–876. Link
  4. 4. Rein L.A. and Chao, N.J. (2014) WT1 vaccination in acute myeloid leukemia: new methods of implementing adoptive immunotherapy. Expert Opin Investig Drugs 23:417-26. Link
  5. 5. Grimwade, D. and Freeman, S.D. (2014) Defining minimal residual disease in acute myeloid leukemia: which platforms are ready for “prime time”? Blood 124:3345-3355. Link
  6. 6. Falini, B., et al. (2005). Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 352: 254–266. Link
  7. 7. Nakagawa, M, et al. (2005) Nucleophosmin in acute myelogenous leukemia. N Engl J Med 352: 1819–1820. Link
  8. 8. Cilloni, D. et al. (2009). 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. J Clin Oncol. 27:5195-201. Link
  9. 9. Gorello, P. et al. (2006). Quantitative assessment of minimal residual disease in acute myeloid leukemia carrying nucleophosmin (NPM1) gene mutations. Leukemia 20: 1103–1108. Link
  10. 10. Ivey A. et al. (2016) Assessment of Minimal Residual Disease in Standard-Risk AML. N Engl J Med 374:422-33. Link
  11. 11. ipsogen WT1 ProfileQuant Kit (ELN*) Handbook. Link
  12. 12. ipsogen NPM1 MutaScreen Kit product details Link
  13. 13. ipsogen NPM1 mut A, B&D MutaQuant Kits product details Link
Kathryn Collinet

Kathryn Collinet, PhD, is a Technical and Marketing Writer for Personalized Healthcare and Oncology at QIAGEN. She trained as a molecular biologist at the University of Barcelona and the Institute for Research in Biomedicine, where she studied DNA and protein modifications and their influence on chromatin conformation and gene expression. Since 2011 Kathryn has been working in marketing communications for the scientific information and molecular diagnostics industries. Kathryn has a passion for delivering knowledge and insights about molecular and clinical technologies, and their power to impact research and healthcare.

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