Identifying TP53 Copy Number Variations in Hematologic Malignancies with a Digital PCR Method

Tumor protein p53 (TP53) is a well-known tumor suppressor gene, of which allelic status has widely been raising concern in recent years. Copy number (CN) loss in this gene results in either haploinsufficiency or loss of function. Though detection methods like next generation sequencing (NGS) or array-based comparative genomic hybridization (aCGH) can be applied, the accurate and cost-effective identification of copy number variation (CNV) remains a challenge for in-hospital laboratories.

In this study, we developed a digital PCR method to quantify the TP53 copy number in hematologic malignancies. Two Taqman probes were designed to be placed at the 5th and 7th exons of TP53 gene, while another one was placed at the RPP30 gene. By performing the experiments with the DNA of 102 healthy checkup individuals and two leukemia cell lines, we established the characteristics of the assay performance, including the limits of blank (LOB), the limits of detection (LOD), the linearities, and the coefficients of variation at the LOD levels. Forty-two samples from patients newly diagnosed with leukemia, lymphoma, myeloma, or myelodysplastic syndrome were further tested for validation. The results were then compared with other reports related to their allelic statuses of TP53.

The lower LOB of the exon 5 and exon 7 were revealed to be 1.756 and 1.836 copies per genome, respectively, while the upper limits were 2.008 and 2.041. The LOD for CN loss of two exons were 1.692 and 1.777 copies per genome, respectively. Taking NGS results as reference, 1.716 and 1.786 copies per genome for exon 5 and exon 7, respectively, were decided as the cutoff values for CN loss using the receiver operator curve (ROC) method. The areas under curve (AUC) for both exons reached 1.

All in all, we consider dPCR an excellent tool for identifying TP53 CNV status in hematologic malignancies.