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FLT3-ITD (with Allelic Ratio)

Background

The FLT3 gene encodes a tyrosine kinase receptor that regulates proliferation and differentiation of hematopoietic stem cells. An internal tandem duplication (ITD) of varying size and is thought to cause a conformational change in the juxtamembrane domain of the FLT3 receptor leading to ligand independent receptor dimerization and thus unregulated receptor kinase activity.

Clinical Significance

An internal tandem duplication (ITD) within exons 14 and 15 of FLT3 has been found to be an independent prognostic factor for poor outcome in both pediatric1 and adult2 acute myeloid leukemia (AML) patients. The prognostic significance of FLT3/ITD may be modified by the allelic ratio. Ratios of 0.4 or greater in pediatric patients3 and 0.5 or greater in adults4 may be a significant prognostic factor for poor outcome.

Method

DNA is extracted from peripheral blood, bone marrow or tissue samples. Polymerase chain reaction (PCR) amplifies the target sequence in exons 14 and 15 of the FLT3 gene. Capillary electrophoresis of the PCR product reveals a molecular weight band higher than the wild-type FLT3 PCR product when an ITD is present. The FLT3-ITD allelic ratio is calculated as the ratio of the peak height of the mutant product to the peak height of the wild-type product.

Patient samples are run in duplicate with appropriate positive and negative controls. In addition, a multiplex QC PCR assay is run in tandem with FLT3-ITD samples to ensure high quality and intact DNA. The QC primers are analyzed to detect and control for any DNA degradation. This is especially important with tissue samples. 

References

Blood (2001); 97(1): 89-94
Blood (1999); 93(9): 3074-3080
Blood (2006); 108(12): 3654-3661


FLT3 TKD (D835/I836 Point Mutation) 

Background

The FLT3 tyrosine kinase domain (TKD) mutations are small mutations in the activation loop of FLT3, representing point mutations in codon D835 and codon I836. This assay does not detect other point mutations throughout the TKD nor the FLT3-ITD.

Clinical Significance

Consensus has not yet been reached on the prognostic value of FLT3 TKD mutations at diagnosis, but the presence/absence of a D835/I836 mutation may help direct treatment routes. Research suggests TKD mutations are associated with the emergence of drug resistance during tyrosine kinase inhibitor (TKI) therapy.

Methods

DNA is extracted from Peripheral blood, bone marrow, or tissue samples. Polymerase chain reaction (PCR) is used to amplify the genes of interest. The forward primer is labeled with a fluorophore to enable detection during capillary electrophoresis. The PCR product is digested with the EcoRV restriction enzyme and analyzed by capillary electrophoresis. All positive samples are sent for Sanger sequencing to determine the exact mutation present.

References

Cancer (2014); 120(14): 2142-2149
Blood (2008); 111(3): 1552-1559
JMD (2003); 5(2): 96-102


NPM1 Insertion Mutation  

Background

The NPM1 gene encodes a phosphoprotein that is commonly expressed and highly conserved. An overall insertion of four base pairs causes a frameshift that leads to the aberrant localization of the NPM1 protein in the cytoplasm. Several different mutations have been observed for NPM1, however they are all insertions of four base pairs overall and result in the same frameshift. 

Clinical Significance

Research has shown that patients positive for the NPM1 insertion mutations (NPM1m) who test negative for FLT3-ITD have a trend toward better event-free survival (EFS) and overall survival (OS). Patients who test positive for FLT3-ITD show no significant difference in EFS or OS, despite the presence of absence of NPM1m

Method

DNA is extracted from peripheral blood, bone marrow or tissue samples and the NPM1 transcript is amplified by polymerase chain reaction (PCR). Patient samples are run in duplicate with the appropriate positive and negative controls. A PCR reaction, using primers flanking exon 12 is used to detect four nucleotide insertions near the C-terminus region of the gene. A labeled probe is attached to the forward primer for fluorescence detection by capillary gel electrophoresis. Analysis of the PCR product reveals a molecular weight band four base pairs higher than the normal NPM1 wild-type band when an insertion mutation is present. The wild-type band is present in both normal and mutation patient samples, and is used as the endogenous control.

Patient samples are run in duplicate with appropriate positive and negative controls. In addition, a multiplex QC PCR assay is run in tandem with NPM1 samples to ensure high quality and intact DNA. The QC primers are analyzed to detect and control for any DNA degradation. This is especially important with tissue samples. 

References

Blood (2007); 110(3); 979-895
Haematologica (2007); 92(4): 519-532
Blood (2007); 109(3): 874-885
NEJM (2005); 352(3): 254-266
NEJM (2008); 358(18): 1909-1918 


CEBPα Mutation Detection

Background

The CCAAT/enhancer binding protein (CEBPα) transcription factor is required for normal myeloid differentiation. Mutations of the CEBPα gene are believed to cause a loss of function and altered expression of the p30 CEBPα protein, which defines a distinct subclass of AML. The mutant CEBPα gene is associated with a favorable prognosis in AML.

Clinical Significance

Among the various types of CEBPα mutations, only bZIP is screened in this assay. CEBPα mutations predict a favorable prognosis in AML with normal cytogenetics in that they are associated with lower relapse rate and improved survival.

Method

Peripheral blood, bone marrow, or tissue sample is extracted to DNA and the CEBPα bZIP region is amplified by polymerase chain reaction (PCR). A fluorescent-labeled probe is attached to the forward primer for fluorescence detection by capillary gel electrophoresis. The wild-type band is present in both normal and mutation patient samples, and is used as the endogenous control. Presence of bands larger than the wild type peak is indicative of an insertion and/or duplication mutation. Presence of peaks smaller than the wild type product is indicative of deletion mutations. Presence of a product larger or smaller than the wild type is considered a positive result for CEBPα  bZIP mutation.

Patient samples are run in duplicate with appropriate positive and negative controls. In addition, a multiplex QC PCR assay is run in tandem with CEBPa samples to ensure high quality and intact DNA. The QC primers are analyzed to detect and control for any DNA degradation. This is especially important with tissue samples. 

References

NEJM (2008); 358(18):1909-1918
Blood Cells, Molecules and Disease (2008); 40(3): 401-405
Blood (2009); 113(26): 6558-6566


IDH2 (Exon 4; R140, R172)

Clinical Significance

IDH2 mutations are seen in 9-19% of AML patients, with the majority of the mutations at two hotspots (R140 and R172) that carry unique prognostic significance. Retrospective studies have shown that IDH2 mutations are associated with poor overall survival or leukemia-free survival in myelodysplastic syndrome (MDS) patients. IDH2 inhibitors targeting these IDH2 mutated enzymes are in various stages of development and clinical trials. The NCI and U.S. Intergroups are conducting clinical trials where the IDH2 inhibitor will be used in patients with these mutations.

Methods

Peripheral blood or bone marrow aspirate is processed to white blood cells by density gradient centrifugation. DNA is extracted and amplified by polymerase chain reaction (PCR), using primers flanking exon 4. The amplified PCR product is followed by bidirectional sequencing, using dye-terminator technology. Sequencing data is analyzed, using Mutation Surveyor® software and mutations  at  positions R140 and R172 are reported clinically.

References

Ther Adv Hematol. (2018); 9(7): 163-173
Blood (2017); 130(6): 722-731
J Clin Oncol. (2010); 28(22): 3636-3643
J Clin Oncol. (2010);  28(14): 2348-2355
JMD (2011): 13(6): 678-686


Quantitative BCR/ABL, p210 (% IS)

Background

The Philadelphia chromosome (Ph) is the product of the reciprocal translocation between the ABL gene on chromosome 9, and the BCR gene on chromosome 22, resulting in deregulated ABL tyrosine kinase activity. The breakpoints in the BCR gene are predominately in the major breakpoint cluster region (M-BCR) of chromosome 22, and transcripts from the M-BCR encode a BCR/ABL protein of 210kDA, called p210 BCR/ABL. Patients can have transcripts from one or multiple breakpoints. This assay detects both e13a2 (b2a2) and e14a2 (b3a2) breakpoints, but does not distinguish between the two.

Clinical Significance

BCR/ABL levels are correlated with response in CML. In the IRIS trial, the level of BCR/ABL at 12 months post initiation of treatment with a Tyrosine Kinase Inhibitor (TKI) was associated with outcome. In this trial, a 3-log reduction (BCR/ABL ≤ 0.1% (IS)) from baseline was defined as a major molecular response (MMR). Increasing BCR/ABL levels may indicate a poor response or loss of response (resistance) to TKI therapy. In this case, mutation screening of the ABL kinase domain may assist in determining treatment routes.

Methods

The Cepheid GeneXpert combines RNA extraction, nucleic acid amplification and detection of the BCR/ABL target using real-time reverse transcription polymerase chain reaction (RT-PCR). The GeneXpert extracts the RNA from the sample, then performs a nested, multiplex assay with primers designed to detect the p210 BCR/ABL translocations (e13a2 and e14a2) and the ABL gene. ABL is the endogenous control and evaluates RNA quality, normalizes total RNA, and also may detect sample inhibition. Quantitation of BCR/ABL is determined by a percent ratio of BCR/ABL to ABL and results are reported according to International Standard (IS).

References

Blood (1995); 85(9): 2632-2638
JNCCN (2018);16(9):1108-1135
Blood (2010) 116(19):3758-3765


ABL Kinase Mutational Analysis for TKI Resistance

Background

Tyrosine kinase inhibitors (TKI) are widely used in the treatment of Chronic Myeloid Leukemia (CML) and Philadelphia Positive Acute Lymphocytic Leukemia (Ph+ALL). TKIs block the ATP binding site in the ABL kinase domain, maintaining the protein in an inactive conformation. Point mutations within the ABL kinase domain prevent the binding of TKIs, resulting in clinical resistance. Identification of ABL kinase mutations can result in early detection of drug resistance.

Clinical Significance

The presence of an ABL kinase point mutation is closely linked to the development of tyrosine kinase inhibitor (TKI) resistance1. In some cases, low levels of an ABL kinase mutation will be detected in a patient responding to TKI therapy and will not necessarily evolve into resistance. Also, ABL kinase mutations are not the only mechanisms of resistance to TKI therapy, therefore the presence or absence of a mutation must be placed in the clinical context. For help in the management of TKI resistance, please consult the most recent NCCN Guidelines for Chronic Myelogenous Leukemia or Acute Lymphoblastic Leukemia at:  http://www.nccn.org/professionals/physician_gls/f_guidelines.asp.

Methods

Peripheral blood or bone marrow aspirates are extracted to RNA and the BCR/ABL transcript is amplified by a reverse transcription polymerase chain reaction (RT-PCR). The ABL kinase domain is further amplified in a nested PCR reaction and is sequenced using Dye terminator chemistry. The ABL sequence is then compared to a wild-type reference sequence to identify mutations.

Assay Considerations

This assay detects mutations in the ABL Kinase Domain, ranging from amino acids 235 to 4863. Allelic ratios are estimated for all positive results, however this assay is not considered quantitative.  The limit of detection for most mutations is about 20%, but may be lower in some cases. It should be noted that stochastic variations may influence the sensitivity and precision of the assay, especially when levels of BCR/ABL transcripts are low.

References

Blood (2002); 99(9): 3472-3475
Blood (2003); 102(1): 276-283
Methods in Molecular Medicine (2006); 125: 93-106
Med Oncol (2012); 29(3): 2136-2142
Blood (2011); 118(19): 5250-5254