Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL), a New Method to Detect Translocations in Leukemia

Abstract

Here we present a novel method “Genomic inverse PCR for exploration of ligated breakpoints” (GIPFEL) that allows the sensitive detection of recurrent chromosomal translocations. This technique utilizes limited amounts of DNA as starting material and relies on PCR based quantification of unique DNA sequences that are created by circular ligation of restricted genomic DNA from translocation bearing cells. Because the complete potential breakpoint region is interrogated, a prior knowledge of the individual, specific interchromosomal fusion site is not required. We validated GIPFEL for the five most common gene fusions associated with childhood leukemia (MLL-AF4, MLL-AF9, MLL-ENL, ETV6-RUNX1, and TCF3-PBX1). A workflow of restriction digest, purification, ligation, removal of linear fragments and precipitation enriching for circular DNA was developed. GIPFEL allowed detection of translocation specific signature sequences down to a 10−4 dilution which is close to the theoretical limit. In a blinded proof-of-principle study utilizing DNA from cell lines and 144 children with B-precursor-ALL associated translocations this method was 100% specific with no false positive results. Sensitivity was 83%, 65%, and 24% for t(4;11), t(9;11) and t(11;19) respectively. Translocation t(12;21) was correctly detected in 64% and t(1;19) in 39% of the cases. In contrast to other methods, the characteristics of GIPFEL make it particularly attractive for prospective studies.

Insights into the prenatal origin of childhood acute lymphoblastic leukemia

“…To date, we have conducted the only study identifying ETV6-RUNX1 + cells via DNA quantification [7]. We used the novel GIPFEL technique [50], allowing for the indirect identification of chromosomal translocations at the DNA level. In this study, we identified 5% of healthy newborns to be ETV6-RUNX1 + .…”

Five percent of healthy newborns have an ETV6-RUNX1 fusion as revealed by DNA-based GIPFEL screening

“…It has been shown to be associated with monocytic AML phenotypes and proven to be of functional relevance for leukemogenesis. [13][14][15] It is interesting to note that RKIP loss has also been observed in a variety of solid cancers and has been described as a bona fide…”

“…Only DNA-based techniques can differentiate between breakpoints that are specific for each individual patient and localized in intronic regions. (B-E) The workflow of cord blood screening using a modified GIPFEL technique 15 is presented. (B) CD19 1 B cells were enriched from newborn cord blood.…”

“…To analyze the frequency of ETV6-RUNX1 1 preleukemic cells in newborns, we therefore developed a DNA-based method termed genomic inverse PCR for exploration of ligated breakpoints (GIPFEL) 15 because DNA, in contrast to RNA, is very stable. By DNA-based approaches, patient-specific amplification products of the translocation site that differ from one another in size and sequence are obtained and contaminations can be easily identified ( Figure 1A).…”

“…The incidence of ETV6-RUNX1 fusions may even exceed 5% because complex rearrangements, rearrangements outside of the known breakpoint regions, and not-yet-expanded cell clones (,10 24 ) evade detection by GIPFEL. 15 Thus, translocation-carrying clones (and potentially .1) are likely present in a high number of healthy individuals who will never develop leukemia. Similarly, other leukemia-or lymphoma-associated translocations have been detected in peripheral blood of healthy individuals (reviewed in Janz et al 17 ).…”