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FLT3 Pathway Mutation PCR Array

Human
 
qBiomarker Somatic Mutation PCR Array: Human FLT3 Pathway
Normal haematopoietic cells use complex systems to control proliferation, differentiation, and cell death. Proliferation control is in part accomplished through ligand-induced stimulation of receptor tyrosine kinases and their downstream signaling cascades. Mutations in the FMS-like tyrosine receptor kinase 3 (FLT3) gene have been found to be the most common genetic lesion in acute myeloid leukaemia (AML), occurring in approximately 25% of cases. Recent studies have established a link between FLT3 mutation status and response to FLT3 inhibitors that warrants further investigation.

The Human FLT3 Pathway qBiomarker Somatic Mutation PCR Array is a translational research tool that allows rapid and accurate profiling of the somatic mutation status of the FLT3 gene and key downstream signaling components. The utility of individual and multiple somatic mutation status information in identifying key signaling transduction disruptions has been demonstrated in numerous research studies. For example, the mutation status of the EGFR and KRAS genes can predict the physiological response to certain drugs targeting these molecules. The FLT3 Pathway qBiomarker Somatic Mutation PCR Array, with its comprehensive content coverage, is designed for studying mutations in the context of the FLT3 pathway and has the potential for discovering and verifying drug target biomarkers for a variety of human cancers involving the FLT3 signaling pathway and downstream effectors. This array covers 85 DNA sequence mutation assays designed to detect the most frequent, functionally verified, and biologically most significant mutations in the FLT3 pathway. These mutations were chosen from curated, comprehensive somatic mutation databases and peer-reviewed scientific literature. The simplicity of the product format and operating procedure allows routine somatic mutation profiling in any research laboratory with access to real-time PCR instruments.

The qBiomarker Somatic Mutation PCR Arrays are intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

 

Assay Functional Annotations How It Works References Resources
 
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FLT3 gene:
19 mutation assays are included in this panel to cover the most frequently identified FLT3 mutations. These include point mutations, insertion and deletion mutations in the juxtamembrane region of the coding sequence and in the activation loop portion of the sequence. An extracellular domain mutation p.S451F and an N-terminal kinase domain mutation A680V were also covered.

AKT gene:
The panel includes an assay for the best known AKT1 mutation, c.49G>A, p.E17K. This is a PH domain mutation that results in constitutive targeting of AKT1 to plasma emebrane.

BRAF gene:
Two classes of mutation assays are included. One class covers mutations that lead to increased BRAF kinase activity, such as the p.L597 and p.V600 mutations. The other class detects mutations that lead to impaired kinase activity, such as the p.G464V, p.G466V and p.G469A mutations.

KRAS gene:
16 KRAS mutation assays provide comprehensive analysis capacity for the most frequently occurring mutations in KRAS codon positions 12,13 and 61. Mutations at these positions result in reduced intrinsic GTPase activity and/or cause KRAS unresponsive to RasGAP. The p.L19F mutation assay is also included.

HRAS gene:
Similar to KRAS mutation assays, the >10 HRAS mutation assays on this panel aim to cover the most important HRAS mutations identified in cancers at codon 12, 13 and 61 positions.

NRAS gene:
12 NRAS mutation assays were included on the panel to cover codon positions 12, 13 and 61, as well as mutation p.A18T.

MEK1 gene:
4 assays for mutations with verified clinical significance in cancer were included on the panel. These mutations cluster in MEK1 N-terminal negative regulatory domain and an adjacent domain, and are all activating mutations (i.e. lead to upregulated intrinsic MEK1 kinase activity).

PIK3CA (phosphatidylinositol 3-kinase catalytic subunit) gene:
The mutation assays covered on this panel can detect 7 most frequently occurring PIK3CA mutations that belong to two classes: p.H1047 mutations, which are activating, kinase domain mutations; and mutations in P539-E545 region, which are helical domain mutations that mimic activation by growth factors.

PTEN gene:
Included on the panel are 6 most commonly detected PTEN loss-of-function mutations that are due to either truncation (p.R233* and p.R130*) or point mutation-caused phosphatase inactivation (p.R130 and p.R173 mutations).

 

Assay Functional Annotations How It Works References Resources
 

Overview of the qBiomarker Somatic Mutation PCR Array / Assay Protocol

Overview of the qBiomarker Somatic Mutation PCR Array / Assay Protocol.
The procedure involves DNA extraction (QIAGEN QIAamp DNA Mini Kit or FFPE Tissue Kit is recommended), an optional amplification (QIAGEN REPLI-g kit or REPLI-g UltraFast kit is recommended) step for DNA isolated from fresh samples, qPCR detection on qBiomarker Somatic Mutation PCR Arrays or Assays, and data analysis (using the qBiomarker Somatic Mutation Data Analysis Template). An optional DNA sample QC step immediately before the detection array or assay setup allows the user to qualify the DNA samples.

Principle of Mutant Discrimination with ARMS®

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Assay Functional Annotations How It Works References Resources
 
  1. Prognostic significance of FLT3 internal tandem duplication and tyrosine kinase domain mutations in acute promyelocytic leukemia: a systematic review. Beitinjaneh A, Jang S, Roukoz H, Majhail NS. Leuk Res. 2010 Jul; 34(7):831-6. Epub 2010 Jan 21.
  2. The PKB/AKT pathway in cancer.Carnero A. Curr Pharm Des. 2010 Jan; 16(1):34-44.
  3. BRAF, a target in melanoma: implications for solid tumor drug development. Flaherty KT, McArthur G. Cancer. 2010 Jul 13. [Epub ahead of print].
  4. Clinical relevance of KRAS in human cancers. Jancík S, Drábek J, Radzioch D, Hajdúch M.
    J Biomed Biotechnol. 2010; 2010:150960.
  5. Oncogenic Ras in tumour progression and metastasis. Giehl K. Biol Chem. 2005 Mar; 386(3):193-205.
  6. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Emery CM, Vijayendran KG, Zipser MC, Sawyer AM, Niu L, Kim JJ, Hatton C, Chopra R, Oberholzer PA, Karpova MB, MacConaill LE, Zhang J, Gray NS, Sellers WR, Dummer R, Garraway LA. Proc Natl Acad Sci U S A. 2009 Dec 1;106(48):20411-6.
  7. PIK3CA mutations in human solid tumors: role in sensitivity to various therapeutic approaches. Ligresti G, Militello L, Steelman LS, Cavallaro A, Basile F, Nicoletti F, Stivala F, McCubrey JA, Libra M. Cell Cycle. 2009 May 1; 8(9):1352-8.
  8. Oncogenic mutations as predictive factors in colorectal cancer. Ličvre A, Blons H, Laurent-Puig P. Oncogene. 2010 May 27; 29(21):3033-43.
  9. PI(3)King Apart PTEN's Role in Cancer. Zhang S, Yu D. Clin Cancer Res. 2010 Jul 8. [Epub ahead of print].
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Assay Functional Annotations How It Works References Resources
 

User Manual qBiomarker Somatic Mutation PCR Array System (PDF)
Data Analysis qBiomarker Somatic Mutation PCR Array Data Analysis Software
Application Data Detection Limits, Cancer Pathways
FAQ Frequently Asked Questions about Somatic Mutation Assays and Arrays
Webinar qBiomarker Somatic Mutation Analysis: Real-World Application Data
Slide Presentation> Presentation about qBiomarker Somatic Mutation Assays and Arrays (PDF)
Scientific Poster A Novel Tool for Pathway-Focused Cancer Mutation Profiling (PDF)
Presented at American Association for Cancer Research 2011
White Paper Rapid and accurate cancer somatic mutation profiling with the qBiomarker Somatic Mutation PCR Array (PDF)
Product Profile For screening biology-focused panels of gene mutations (PDF)

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