Loss of heterozygosity analysis in ductal lavage samples from BRCA1 and BRCA2 carriers: a cautionary tale

Loss of heterozygosity (LOH) in breast ductal lavage (DL) fluid has been reported to be a potential biomarker of malignant change. Interpretation of LOH is reliant on sufficient quality and quantity of DNA. We investigated LOH of the BRCA1/2 loci in DL samples from BRCA1/2 mutation carriers, while also assessing the effect of DNA quantity.

DNA yield was estimated using quantitative real-time PCR. Allelic status of DL DNA was determined using fluorescently tagged microsatellite markers with the subject's lymphocytic DNA serving as a control. Samples were scored as consistently heterozygous or as demonstrating LOH if the same result was observed in replicate experiments. Additionally, samples were scored as "discordant LOH" if they initially showed LOH, but in replicate experiments either showed heterozygosity or LOH of the opposite allele.

In 11 BRCA1 carriers, 46 ducts were assessable, and 39 ducts from 14 BRCA2 carriers were assessable. LOH was observed in 17% and 18% of ducts from BRCA1 and BRCA2, respectively. Discordant results were seen in 23 BRCA1 (50%) and 15 BRCA2 (38%) samples. DNA yield was significantly greater in samples that were consistently heterozygous than those that were either discordant or showed LOH in replicate experiments for both BRCA1 (P = 0.003) and BRCA2 (P = 0.003).

DNA quantity is highly variable between DL samples, with low yields likely to detrimentally affect the interpretation of LOH. In conclusion, LOH may not be an adequate method to detect the early stages of malignant change in samples obtained via DL.