Antill Yoland C, Mitchell Gillian, Johnson Sandra A, Devereux Lisa, Milner Alvin, Phillips Kelly-Anne, Campbell Ian G
Victorian Breast Cancer Research Consortium Cancer Genetics Laboratory, Melbourne, Australia.
Cancer Epidemiol Biomarkers Prev. 2006 Jul;15(7):1396-8. doi: 10.1158/1055-9965.EPI-05-0986.
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.
据报道，乳腺导管灌洗（DL）液中的杂合性缺失（LOH）是恶性变化的潜在生物标志物。LOH的解读依赖于足够的DNA质量和数量。我们研究了BRCA1/2突变携带者DL样本中BRCA1/2基因座的LOH情况，同时评估了DNA数量的影响。
使用定量实时PCR估计DNA产量。使用荧光标记的微卫星标记确定DL DNA的等位基因状态，以受试者的淋巴细胞DNA作为对照。如果在重复实验中观察到相同结果，样本被评为始终杂合或显示LOH。此外，如果样本最初显示LOH，但在重复实验中显示杂合性或相反等位基因的LOH，则被评为“不一致的LOH”。
在11名BRCA1携带者中，46个导管可评估，在14名BRCA2携带者中，39个导管可评估。分别在BRCA1和BRCA2的17%和18%的导管中观察到LOH。在23个BRCA1（50%）和15个BRCA2（38%）样本中看到不一致的结果。对于BRCA1（P = 0.003）和BRCA2（P = 0.003），始终杂合的样本中的DNA产量显著高于在重复实验中不一致或显示LOH的样本。
DL样本之间的DNA数量差异很大，低产量可能会对LOH的解读产生不利影响。总之，LOH可能不是检测通过DL获得的样本中恶性变化早期阶段的合适方法。
