Structural variations in mammary carcinomas and their detection in cell-free plasma DNA in a clinical dog model.

Abstract

1537 Background: Mammary tumors are the most frequent cancers in female dogs exhibiting a variety of histopathological differences to which a dearth of information about their genomic status exists. Massive parallel sequencing (MPS) of tumor, white blood cell and associated cell-free DNA from plasma revealed the utility of the dog in modeling the dynamics of cell-free cancer DNA. Methods: Five tumors of three different histological subtypes were evaluated. MPS of tumors was performed in comparison to the respective white blood cell DNA of each animal. Copy number and structural variations were evaluated and those only present in tumors were validated using digital droplet PCR (ddPCR). Results: Using mate-pair sequencing chromosomal aneuploidies were found in two tumors, frequent smaller deletions were found in one, inter-chromosomal fusions in one other, whereas noteworthy one tumor was devoid of any of these anomalies. The detected structural variations affect several known cancer associated genes such as BRCA1, cMYC, and KIT. One deletion of the proximal end of CFA27, harboring the tumor suppressor gene PFDN5 was detected in four of the five tumors. Using ddPCR, this deletion was validated and detected in 50% of tumors (N=20).One breakpoint specific ddPCRs was established for each of four tumors. In all cases tumor specific DNA was detected in the plasma prior to surgery and accounted for 1 to 24 % of the cfDNA. In one animal the ddPCR detected tumor specific DNA in cfDNA still present >1 year after surgery; subsequently a lung metastasis was found as cause. Furthermore, paired-end sequencing of extracted cfDNA and subsequent depth of coverage analysis proved that copy-number variations of the tumor are reflected by the cfDNA. Conclusions: The similarities of patterns of copy-number imbalances (e.g. chromosomal and segmental aneuploidy subtypes) of canine and human mammary cancers and their correlation in cfDNA supports the use of the dog as a model for using MPS for screening and discovery of individual cancer biomarkers. The ddPCR technology offers rapid, cost effective and sensitive confirmation and monitoring. The possibility exists that ddPCR can be expanded to screening for common cancer related variants.