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PCR Q&A: Johns Hopkins Team Uses Delta-PCR to Improve Molecular Testing of Certain Cancers


Dr Gocke picture.jpgNAME: Christopher Gocke

POSITION: Associate professor of pathology and oncology, interim laboratory director, molecular diagnostics laboratory, Johns Hopkins University School of Medicine

Clinical researchers from Johns Hopkins School of Medicine have developed a twist on PCR-based molecular diagnostic testing that improves their ability to detect chromosomal translocations and small insertion or deletion mutations that are characteristic of certain types of cancer.

Specifically, the method, dubbed ΔPCR, can ensure PCR specificity and identify individual breakpoints when diagnosing cancers such as follicular lymphoma and leukemia, for which PCR detection has traditionally been challenging due to widely distributed breakpoints, multiple mutations, low signal strength, and elevated background noise.

In a study published this month in the Journal of Molecular Diagnostics, the researchers demonstrated the ability of multiplex ΔPCR to identify, with a high degree of specificity and sensitivity, translocations with multiple breakpoints or partners by testing follicular lymphoma and control specimens from formalin-fixed, paraffin-embedded patient samples.

In addition, the scientists demonstrated how their method could be used to detect minor leukemic clones with internal tandem duplication mutations; and described how their technique could be used in other insertion/deletion and duplication mutations.

This week, PCR Insider caught up with Christopher Gocke, corresponding author on the study, to discuss the genesis of the ΔPCR method, how it works, and its potential commercial applications. The following is an edited version of the interview.

What was your group's motivation for developing ΔPCR?

We are a clinical laboratory performing diagnostic tests on patients. It is not uncommon to obtain a sample that is a mixture of normal and mutant, or abnormal and normal cells. In the case of a cancer, we'd be talking about a mutation. We also like to do things in as efficient a manner as possible. Generally that means a multiplexed assay, where we're looking at several analytes at once.

We have run into the situation where relatively low-intensity signals are difficult to interpret. Questions arise as to whether it is a background or contaminant band; or whether it is the real thing we're looking for, but just at a relatively low prevalence in the population. We developed this assay as a way of increasing our specificity and sensitivity.

Where did the ΔPCR name come from?

The test is three oligonucleotides per analyte in a PCR reaction, all at once. Two of the oligonucleotides are on one side of the target, and one oligo is on the opposite side. In the reaction, the two oligos on one side of the reaction are simultaneously amplified with the solitary oligo on the other side. That leads to two different sized PCR products from a single target. The difference in the size between them is rigidly defined by where we place those two oligos relative to each other. So if they're 10 bases apart, then the PCR products will differ in size by 10 bases. If they're 15 bases apart, the products will differ by 15. That number, the difference between the two, is what we've termed the Δ. For each assay, it's a particular defining constant.

The paper mentions that there are cases where translocation events must be detected at the DNA level by PCR because of the lack of a fusion transcript, and that ΔPCR can help. Why is this so challenging to do with standard PCR methods?

This situation is not uncommon in the diagnostic world. A translocation between two chromosomes has occurred, and no fusion mRNA is produced. The challenge is if the breakpoints of one or both of the partners in the translocation are not uniform. If they occur over a range of places within a chromosome, then it gets pretty challenging at a DNA level to figure out exactly where to place your diagnostic PCR primers. That generally leads to a situation where people do multiple PCRs to span a pretty large breakpoint. That's a situation where, if you're multiplexing it to be more efficient, you end up with a PCR product [that is] diagnostic of a translocation of an unknown size going in, because the size is going to be determined by exactly where the breakpoints lie. That varied size is a challenge, diagnostically, particularly when it's a fairly low amplitude or fairly low intensity distinguishing that from just a random background band.

The beauty of the ΔPCR approach is that we get an internal control in the form of that Δ, that second PCR product that's produced, that tells us: This is a specific product. It is of exactly the defined size that we'd expect; therefore it is a bona fide translocation, and not some artifact. That's how we increase the specificity of our reaction.

Does it matter what type of PCR reagents or detection methodologies you use for this?

It's not rigidly controlled with regard to that. We tend to use AmpliTaq Gold as our Taq polymerase, but in general it will work with any set of reagents.

The detection end of it is currently done by either gel electrophoresis or, in our case, by capillary electrophoresis. That serves to give you precise information about the Δ.

Is there another way to do the detection? Is this amenable to molecular diagnostic testing?

It's a common dilemma with microbiology or cancer, where you may be dealing with a relatively small amount of whatever it is you're interested in, in a background full of lots of other stuff. So the sensitivity and specificity of the assays really do matter.

There are other ways to overcome this problem, but they tend to be long and tedious. That's what the ΔPCR method really solves. It solves that problem of doing something rapidly, yet improving the sensitivity and specificity.

Are you routinely using this now as a molecular diagnostic test? Are there any modifications that you could make to improve the method?

We do use it. We have employed it in the diagnostic lab whenever this sort of issue comes up. And there are particular areas, as we described in the paper … for example, FLT3 mutation in myelogenous leukemia is a big player in the diagnostic field. Exactly the situation I've described comes up with that: Are low-intensity bands real, or is this an artifact? We do employ it regularly to discriminate.

It's also mentioned in the paper how, with some modifications, the method may be more broadly applicable, such as in analyzing the species-specific length polymorphisms in the internal transcribed spacer 2 region of the rRNA genes for rapid identification of fungal infection. What types of modifications would be needed?

Perhaps we were being a little too concrete there. Really it's just design of the oligos, design of the primers to target that specific question. We're not a microbiology lab, and it's not something we pursue. But we think it can be done.

Does this have commercial potential, and is your lab or the Johns Hopkins patent office pursuing that?

I think it does. It provides a need. It provides an inexpensive solution to a relatively common problem. The patent applications have been filed, but I don't know where the patent process stands right now.

Do you have any other studies in the pipeline using ΔPCR?

Yes, there are some things I can't divulge at the moment. But we are employing the method for the purpose as described, in more of a research mode, to speed our throughput. But it's been quite a useful technique for us.

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