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Tech Notes : Jan 15, 2009 (Vol. 29, No. 2)

Finessing PI3 Kinase Assay Development

DxS Works to Overcome the Psuedogene Problem
  • Kevin Entwistle

The EGFR cell-signaling pathway plays a central role in processes such as cell growth, proliferation, survival, and differentiation. Understanding the biological role of this pathway and its effect in various cancer types has led to the development of several therapeutic approaches that target this pathway, including monoclonal antibodies that inhibit the EGF-receptor and block the activation of the signaling cascade, and small molecule inhibitors. 

When these drugs were put through clinical trials, however, it was discovered that a group of the population tested did not gain any benefit from the therapy. Further investigation into the mutation status of those people who showed no improvement with the drug revealed that mutations in genes involved in the EGFR cell signaling pathway, such as K-RAS, B-RAF, and PI3 kinase, affected the response to therapy. This has led to personalized medicine taking a more central role in selecting cancer therapies for an individual.

PI3 Kinase as a Target

Phosphatidylinositol 3-kinases (PI3K) are heterodimeric lipid kinases involved in cell signaling. There are three classes of PI3-kinases, the PIK3CA gene is found in class I. Class I PI3 kinase is made up of two specific parts, an 85kDa subunit, p85, which is an adaptor molecule that has no catalytic function but attaches the unit to the receptor, and a 110kDa subunit, p110, which triggers the phosphorylation events in the PI3K-AKT pathway. 

The active part of PI3K (p110) is encoded for by the gene PIK3CA. Activity of PI3 kinase was first reported to be associated with viral oncogenes in 1991 and, more recently, mutations in the PIK3CA gene have been identified in human cancers.

It has been suggested that the presence of PIK3CA mutations in a tumor can act as a prognostic factor and may predict the response to treatment. This makes the PIK3CA gene attractive as a potential target for anticancer therapies. 

There are three mutation hotspots in the PIK3CA gene: E542K and E545K in exon 9, and H1047R in exon 20, these mutations represent >80% of all PIK3CA mutations described. The frequency of PIK3CA mutations in colon, breast, lung, and melanoma cancers have been estimated at 10.2%, 38.7%, 1.9%, and 2.9%, respectively. 

DxS has developed a real-time PCR assay using its technologies to detect mutations in the three hotspots of the PIK3CA gene and also one rare mutation, E545D, also found in exon 9. DxS has previously developed mutation detection kits using the same technologies for other genes in the EGFR pathway such as EGFR, K-RAS, and B-RAF. 

The DxS PI3K Mutation Test Kit follows the same format as other kits in the DxS product portfolio using Amplification Refractory Mutation System (ARMS®; AstraZeneca) and Scorpions® (DxS) technologies.

Developing the Assay

DxS produces real-time PCR assays that  yield results in less than three hours from DNA extraction, and can detect down to 1% mutation in a background of wild-type DNA. The kits contain a standard (containing synthetic templates of all the mutations detected in the kit, which is used to show that each assay is working efficiently), a control assay (to assess quantity and quality of the DNA sample, this is an ARMS and Scorpions reaction that amplifies a region in the gene of interest that is not affected by mutations), and mutation assays (individual ARMS and Scorpions primers designed to only detect the mutation screened for by that kit).

Each assay (control and mutation) is multiplexed with an exogenous control to ensure the reaction is working efficiently and to allow detection of any inhibitors.  In a real-time PCR assay a positive reaction is detected by accumulation of a fluorescent signal. The DxS kits use ARMS for discrimination and Scorpions for detection. 

ARMS technology is based on the principle that primer extension is efficient when the 3´ terminal base of a primer matches its target, whereas extension is inefficient or nonexistent when the terminal base is mismatched. Primers are designed against the mutation of interest, and an amplicon is produced only if the mutation is present.

Scorpions probes are bifunctional molecules that contain a PCR amplification primer attached to a probe sequence. During PCR (Figure 1), the primer is extended to form an amplicon, and the probe, which contains an amplicon-specific sequence, binds to its target to produce a fluorescent signal. The amount of fluorescence will be proportional to the amount of amplicon generated. 

The combination of ARMS and Scorpions technologies in a real-time PCR assay are ideal for mutation detection in tumor samples. This is because tumors are heterogenic, in that when the biopsy is taken there will be tumor cells and normal healthy cells, but the mutation may only be present in a few cells of the tumor biopsy. 

This means that there will be limited material available that will contain the mutation. The combination of ARMS and Scorpions allows 1% mutant to be detected in a background of wild-type, meaning it is more likely to detect the mutation compared to sequencing, which has a sensitivity of approximately 20%. 

ARMS and Scorpions technologies are used to obtain Ct values in real-time PCR.  The Ct (cycle threshold) is defined as the number of cycles required for the fluorescent signal to cross the threshold (i.e., exceed background level). Ct levels are inversely proportional to the amount of target nucleic acid in the sample (i.e., the lower the Ct level the greater the amount of target nucleic acid in the sample). 

The assays work on delta-Ct values, which is the difference between two Ct values. A control assay is run with every sample that produces a Ct value for the amount of the gene present in the sample. A separate Ct value is produced from the specific mutation assay, and the delta-Ct is the difference between the mutation Ct and the control Ct. Cut-off limits have been validated, and the delta-Ct from the sample is compared to the cut-off value, if the value is equal to or less than the cut-off then the sample is deemed positive for that mutation, if the delta-Ct is greater than the cut-off value the sample is negative or beyond the limits of the kit. Figure 2 shows typical results with the PI3K assay.

The ARMS and Scorpions primers were designed for the specific regions in the PIK3CA gene that will be detected by the assay. The format of DxS’ other kits were followed.

The Psuedogene Problem

It is known that the PI3 kinase gene has a pseudogene. Pseudogenes are believed to be relatives of functional genes due to their homology to real genes but lack of functionality compared to their relative, which has occurred over many evolutionary cycles. Pseudogenes occur as a result of two main processes: duplication and retrotransposition.

As natural selection affects individual genes, beneficial mutations in a gene that improve an organism’s fitness tend to be preserved, whereas a sequence change that impairs a gene’s function leads to it being discarded. As pseudogenes are classed as junk DNA and most commonly found in the intergenic regions, they are released from this selection pressure and are free to accumulate all kinds of mutations, including changes that would be deleterious to normal genes.

The activating mutations in the PI3 kinase oncogene promote cellular replication, so the cells bearing these mutations have a growth advantage whereas similar mutations in the pseudogene have no affect on cell fitness and viability.  

In designing the ARMS and Scorpions primers for the PI3K assay, the pseudogene needed to be considered. In the region of mutations in exon 9 of the PI3 kinase gene, there is a high level of homolgy with the pseudogene making it difficult to design primers to selectively amplify the true gene over the pseudogene. The particular selectivity of ARMS allows specific yet sensitive amplification of only mutant variants of the true gene (Figure 3). In exon 20, the homology is much less significant and primer design to differentiate between true gene and pseudogene is less problematic.

DxS has designed an assay in kit form to detect the most common mutations seen in the PIK3CA gene that correlate with tumor progression in numerous types of cancer. Previously, an alternative method for detection of these mutations was sequencing, however, this technology has its limitations in that it is labor intensive and has a low sensitivity of 15–25%. The combination of ARMS and Scorpions technology in a real-time PCR assay has overcome the limitations of sequencing and the assay can detect down to 1% mutant in a background of wild-type DNA.

The PI3K-AKT pathway is an attractive target for anticancer therapies, and this assay allows detection of the most common mutations, which will aid in the development of targeted therapies for individuals affected by these cancers. Due to the presence of a pseudogene that shows a lot of homology with exon 9 of the PI3 kinase gene, ARMS and Scorpions primers have had to be specifically designed to ensure that they only detect the mutations seen in the actual gene and not the pseudogene. The DxS PI3K assay achieves this goal.