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Johns Hopkins Team Develops TD-PCR Method to Detect Tandem Duplication Mutations in AML Patients

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Clinical researchers from Johns Hopkins University School of Medicine have developed a technique called tandem duplication, or TD-PCR, for detecting internal tandem duplications of the FLT3 gene, which has been associated with a poor prognosis in acute myeloid leukemia patients

According to the researchers, the assay has a high degree of sensitivity and specificity, with a limit of detection approaching just a single mutant molecule, and as such may prove useful as a tool to monitor minimal residual disease in AML patients.

To that end, the group is now conducting clinical trials — one with an undisclosed pharma and one with an undisclosed research cooperative — to test whether the assay can help detect minimal residual disease at an early stage in order to provide a better prognosis than is currently possible, Christopher Gocke, an associate professor of pathology and oncology at the JHU School of Medicine, told PCR Insider.

In 2011, Gocke, who is also director of hematology molecular diagnostics at the JHU School of Medicine, spoke with PCR Insider about a method developed in his lab called Delta-PCR, following the publication of a paper in the Journal of Molecular Diagnostics describing how that method could be used to detect chromosomal translocations and small insertion or deletion mutations with a high degree of specificity and sensitivity (PCR Insider 1/27/2011).

That method was able to improve the limit of detection of leukemia cells in a background of wild-type cells, and Gocke and colleagues showed that using it to detect minor leukemia mutants with an internal tandem duplication mutant allelic burden of less than 1 percent could be clinically significant.

The limit of detection of the recently developed TD-PCR method is even better than that of Delta-PCR, according to Gocke.

"I think with this advance we are basically at the single-mutant molecule detection level," Gocke said. "We're not talking 1 percent or 0.10 percent sensitivity limit of detection, as we were before, but probably 10- or 100-fold better than that."

TD-PCR is a modified version of inverse PCR, in which restriction enzyme-digested DNA circularized by ligation allows adjacent PCR primers that face away from each other to amplify a product across the restriction site, the researchers explained in a paper published in the September 2013 issue of Diagnostic Molecular Pathology.

In TD-PCR, the researchers wrote, template-specific PCR products are only amplified from the mutant allele by a forward primer and reverse external and internal primers. "No template-specific amplification occurs from the wild-type allele or if one or both primers lie outside the duplicated segment," they wrote. In contrast, in standard PCR "template-specific products are amplified from both the smaller wild-type allele and the larger mutant allele, resulting in competition for amplification and reduced mutant signal."

In the recently published study, Gocke and colleagues demonstrated a limit of detection for their assay of a few molecules of internal tandem duplication mutants, and used the assay to confirm ITD mutation of an amplicon that was undetectable by capillary electrophoresis. In addition, the researchers used TD-PCR to detect ITD mutations in two of 77 patients previously reported as negative for ITD mutation with a standard PCR assay.

Gocke said that the increased level of sensitivity is important in detecting minimal residual disease in AML patients who are harboring an FLT3 mutation and being treated with an inhibitor drug, of which several are currently being developed. And he noted that the current method for detecting that minimal residual disease — an assay from personalized medicine company InVivoScribe — is somewhat limited.

"We're interested in pushing those limits … because the current paradigm for leukemia testing is … you have an inhibitor drug, and you want to measure how much leukemia is left," he said. And the current methods for detecting that have their limits — about 5 percent or so mutant molecules in a background of 95 percent wild-type molecules."

That limit of detection is acceptable "when you're making the initial diagnosis where you've got lots and lots of leukemia blasts present," Gocke said. "But for this minimal residual disease state — and particularly one that's induced by a tyrosine kinase inhibitor directed at FLT3 — 5 percent is nowhere near what's needed."

One shortcoming of the method as described in the recently published paper is that it was only able to pick up a relatively small fraction of all possible ITDs. However, some more recent modifications to the assay now allows the researchers to detect "a much larger fraction … of all possible or all known ITDs — probably 75 or 80 percent of them," Gocke said.

Johns Hopkins has filed for a provisional patent surrounding the TD-PCR method. Gocke also noted that his group is in the early stages of two clinical trials — "one with a drug company, and one with one of the large cooperative groups to see whether our detection of minimal residual disease in these patients makes any difference in their outcome," he said. "And although it's too preliminary for me to give you the details, our initial study looks very exciting. We have a very interesting ability to discriminate patients who will relapse from those who won't."

The new method may also be promising for prognosing other cancers hallmarked by internal tandem duplications.

"They are not uncommon … in other tyrosine kinases," Gocke said. "[Name] a tyrosine kinase, and I'm sure that an ITD has been identified in it, whether that be C-kit, or EGFR, or a bunch of others … usually at pretty infrequent levels. Only a percent or so of the mutations that are found in those genes are ITDs, and those genes don't necessarily have to be in leukemias — they can be in other types of tumors, too."

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