Determination of the Origin of the t(1;19) TCF3-PBX1 Fusion By Genomic Inverse PCR for Exploration of Ligated Breakpoints (GIPFEL)

Pediatric acute lymphoblastic leukemia (ALL) is characterized by recurrent chromosomal translocations. The translocation t(1;19) that fuses the gene encoding the basic helix-loop-helix transcription factor TCF3 with the gene encoding the homeodomain protein PBX1 is the second most common one occurring in approximately 5-10% of precursor B ALL cases. Backtracking of clonotypic TCF3-PBX1 translocations that were identified in leukemia patients by PCR amplification of Guthrie cards from these individuals provided weak evidence for a prenatal origin of a minority of TCF3-PBX1 translocations (2 of 15 cases). The presence of N-nucleotides at the recombination junction, IGH rearrangements and the specific JH and DH segment usage indirectly supported a postnatal origin of the majority of translocations, but could not definitely date the fusion event during development (Wiemels et al. PNAS 2002).

We recently developed a novel, DNA-based screening technique (genomic inverse PCR for exploration of ligated breakpoints, GIPFEL) for the detection of translocations without prior knowledge of the exact breakpoint (Fueller et al. PLOS ONE 2015). By GIPFEL screening of 1,000 umbilical cord blood samples, we confirmed a high prevalence (≥5%) of the most frequent ALL associated translocation, t(12;21), in healthy newborns (Schaefer et al. BLOOD 2018). This translocation was 500-fold more frequent than the corresponding leukemia incidence (1/10,000) indicating a low penetrance of the leukemic fusion and a greater importance of secondary oncogenic events. In order to trace the origin of the TCF3-PBX1 fusion and to assess the risk of children bearing the translocation to develop leukemia, we collected 340 cord blood samples of healthy newborns and subjected them to GIPFEL screening.

The GIPFEL technique uses stable DNA as a sample and detects a translocation by inverse PCR after restriction enzyme digest of the DNA and circularization of fragments by ligation. For t(1;19) screening, DNA was isolated from CD19⁺ enriched mononuclear cells, digested with the enzyme MfeI and ligated. Remaining linear DNA fragments were removed by exonuclease digest. After ethanol precipitation of the DNA circles a partially multiplexed, semi-nested PCR was carried out to quantify all possible ligation/junction products specific for the translocation. Samples that screened positive underwent one further demultiplexed PCR, agarose gel electrophoresis and Sanger sequencing to validate the result. An internal PBX1 genomic ligation product served as a positive control. TCF3-PBX1 positive cells at a frequency ≥10^-4 to 10^-5 would be detected by the GIPFEL method.

Of the 340 screened cord bloods, 292 are currently undergoing evaluation and 48 are validated. So far, none of the 48 samples was positive for the TCF3-PBX1 translocation. In case all 340 cord bloods are negative, the result could suggest that TCF3-PBX1 translocations occur very rarely prenatally and that they have a high oncogenic penetrance if they arise in utero, although cooperating secondary mutations are clearly necessary. This would be in line with the strong capability of the TCF3-PBX1 oncoprotein to transform many cell types in vitro and with the generation of diverse (although late occurring) tumors observed in TCF3-PBX1 transgenic mice (reviewed in Aspland et al. Oncogene 2001). These results would support the previous finding of clonotypic TCF3-PBX1 transcripts in 2 of 15 Guthrie cards derived from individuals who later developed leukemia (Wiemels et al. PNAS 2002).

Complete results of GIPFEL screening of 340 newborns will be available and presented at the conference. Although the number of healthy newborns investigated is still low, these results will help to determine the origin of the t(1;19) TCF3-PBX1 fusion.