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German Group Develops New PCR Method to Detect Chromosomal Translocations at DNA Level

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NEW YORK (GenomeWeb) — Researchers from three German institutions have developed a PCR-based method to sensitively detect recurrent leukemia-associated chromosomal translocations at the DNA level without prior knowledge of specific breakpoints.

The method, called genomic inverse PCR for exploration of ligated breakpoints (GIPFEL), has advantages over PCR-based methods that use RNA/cDNA as a template and over well-established DNA-based methods such as long-range PCR, according to the researchers.

As such, the technique may be ideal for applications such as screening cord blood samples to detect so-called pre-leukemic events in healthy newborns that might portend the subsequent onset of childhood leukemia — an endeavor the team is currently exploring with a Danish academic hospital.

Arndt Borkhardt, a hematology and clinical immunology researcher at the University Children's Hospital at Heinrich Heine University, along with colleagues at Friedrich Alexander University and Hannover Medical School, published their method in August in PLOS One.

Borkhardt and colleagues were seeking a more dependable method to sensitively detect recurrent chromosomal translocations, which have become essential for diagnosing, stratifying, prognosing, and planning treatment for leukemia patients.

In particular, "the main purpose of this method is to screen cord blood samples from healthy newborns …for the presence of such chromosomal translocation breakpoints … in pre-leukemic cells," Borkhardt told PCR Insider in an email. "There are data … that state that 1 percent of all healthy newborns have … one of the fusion breakpoints present in the cord blood at birth, and only a very small [number] of them will later develop leukemia."

Borkhardt added that it is important to better elucidate the frequency of these pre-leukemic events in healthy newborns with the goal of eventually screening for infants at risk of developing leukemia later in life.

For this application, cytogenetics and fluorescent in situ hybridization are fairly reliable but cumbersome and time-consuming, the researchers noted in their paper. Meantime, PCR-based methods that use RNA as a template have emerged as a promising alternative, but the labile nature of RNA often precludes successful amplification from stored or aged samples, the researchers noted.

Finally, DNA-based PCR methods, such as long-range PCR, have shown promise for these types of applications, but have limited sensitivity and can become overly complex due to the inordinate number of primer pairs typically used to amplify genomic breakpoints that are usually distributed over large stretches of sequence.

"RT-PCR methods … are not very well suited because of the fact that RT-PCR for chromosomal translocation always gives the same signals, the same PCR amplification product for all the patients," Borkhardt said. "That's quite prone to contamination — when you have one positive patient, that might be the source of contamination."

Meantime, on the DNA level, the difference between well-established methods like long-range PCR and GIPFEL is "mainly sensitivity … because we [showed] much better sensitivity as compared to so-called long-range PCR. In order to screen healthy newborns or samples, generally speaking, with very low amounts of cells with a translocation diluted among many healthy cells … of course we need a sensitive method."

As described in the PLOS One paper, the GIPFEL method takes advantage of the fact that genomic breakpoints are usually confined to defined chromosomal regions, and involves restriction digestion of genomic DNA followed by circularization of resulting fragments, thus dividing even large breakpoints into a manageable number of DNA circles.

"Only cells with translocations will form a signature circle that is uniquely characteristic for the nature of the underlying genomic aberration," the researchers explained. "These circles can be quantified by real-time PCR because the sequence of the corresponding ligation joint can be derived from the known genomic sequence and the respective location of the restriction sites within the breakpoint region."

To evaluate GIPFEL under ideal conditions, the researchers used it to try and detect known chromosomal breakpoints from three cell lines mixed in various rations with control cells. Under these conditions, GIPFEL was able to detect signature circles for all translocations down to a dilution of one in 10-4 — equivalent to a calculated presence of 19 target molecules per PCR reaction, according to the paper.

Borkhardt told PCR Insider that the team actually believes the theoretical sensitivity limit is 10-5, or approximately one translocation-bearing cell among 100,000 normal cells.

The team then further validated its method on actual patient samples — DNA obtained from clinical repositories. Specifically, they tested GIPFEL's ability to detect the five most frequent translocations in childhood leukemia: t(4;11), t(9;11), t(11;19), t(12;21), and t(1;19).

GIPFEL demonstrated 100 percent specificity with no false-positive results, and was still able to obtain results at dilutions between 10-3 and 10-4. Accuracy in detecting the various translocations was lower — ranging from 24 percent to 83 percent — but this was to be expected, the researchers noted.

"The success rate of this a priori method is not always the same," Borkhardt said. "[Some] translocations are more easily detectable compared to the others. This is simply because … [some] breakpoints are more easily amplified by PCR, and there are more complicated breakpoints. [What's] important is that each patient has an individual breakpoint — an individual marker on the DNA level, like a fingerprint of the leukemic cell."

Nevertheless, the team is currently trying to push the method's sensitivity by enriching for pre-leukemic cells prior to applying GIPFEL. "That's not ready yet," Borkhardt said. "But it's still much better sensitivity compared to long-range DNA PCR."

In general, the researchers noted that the new method may be a valuable tool in prospective settings for applications such as identifying translocation-positive secondary malignancies in patients that have been exposed to topoisomerase inhibitors during treatment of non-blood-related neoplastic diseases; or screening for the appearance of translocation positive clones in individuals that have been exposed to ionizing radiation.

However, Borkhardt believes the technique's sweet spot could be newborn pre-leukemia screening, and his lab is currently working with that of Kjeld Schmiegelow, a pediatric oncology specialist at the University Hospital Rigshospitalet in Copenhagen, to pursue this application.

As part of this, the groups are looking to implement the aforementioned enrichment step, and may explore the use of droplet digital PCR to improve throughput of the method by simultaneously screening for multiple translocations.

Schmiegelow, Borkhardt said, "has a large cord blood bank, and wants to [screen it] using our method. He has stored thousands of cord blood from healthy newborns, and is keen to know how many of those cord bloods have these pre-leukemic [cells] or cells with translocations. I'm sure we have to think about some more automated way to do it."

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