Loss of Heterozygosity at the BRCA1 and BRCA2 Loci Detected in Ductal Lavage Fluid from BRCA Gene Mutation Carriers and Controls

Women carrying pathogenic BRCA gene mutations are at increased risk for developing breast cancer under the age of 50 years (1). It is well recognized that mammograms are less sensitive in younger women who have more radiodense breast tissue, and although alternative imaging modalities, such as magnetic resonance imaging, have shown promise, there is still a clear need for better risk assessment and earlier breast cancer detection in this high risk group (2, 3).

Somatic loss of heterozygosity (LOH), the loss of a normal functioning allele at a heterozygous locus, is a frequent genetic event in the multistep process of breast cancer tumor development and progression (4). Carriers of germ-line mutations in tumor suppressor genes, such as BRCA1 or BRCA2, are at risk of acquired loss of the wild-type allele, one of the common mechanisms of inactivation in hereditary breast cancer (5).

Ductal lavage allows repeated minimally invasive sampling of breast duct fluid, and sufficient exfoliated epithelial cells for cytologic diagnosis may be collected (6). More than 60 women from BRCA gene mutation carrying families are taking part in the ductal research program at The Royal Marsden NHS Foundation Trust evaluating the usefulness of nipple aspiration and ductal lavage as risk assessment tools. We are doing a variety of molecular analyses on the ductal fluid collected in the search for surrogate biomarkers of risk. The purpose of this study is to evaluate the frequency of LOH at the BRCA1 and BRCA2 loci detected in free DNA from the ductal lavage fluid of BRCA gene mutation carriers and controls, to assess the reproducibility of doing these microsatellite analyses, and to compare the LOH rate with that at a control locus of uncommon loss in breast cancer.

Prospective ethics committee approval was gained for this study assessing epithelial cell atypia and potential biomarkers in the ductal lavage fluid of BRCA gene mutation carriers and predictive genetic test negative controls. Informed consent was gained from all subjects. Ductal lavage was attempted on all healthy unaffected breasts, and where possible, multiple ducts in the same breast were cannulated. Thirty-three ductal lavage specimens were available for LOH analysis from 17 women, of whom 14 were BRCA mutation carriers (5 BRCA1 and 9 BRCA2) and 3 were predictive test negative controls. Five ductal lavage samples were repeat samples, collected 1 year apart, from 3 BRCA carriers. Five of the BRCA2 carriers had previously contralateral breast cancer but were currently disease free.

Ductal lavage was done as described previously with the modification that cannulation of both nipple aspirate fluid–yielding and nipple aspirate non fluid–yielding ducts was attempted (6). Ductal lavage samples were centrifuged at 1,500 rpm for 10 minutes at 4°C, and the Shandon cytospin technique was used to produce two slides for cytologic assessment. Slides with <10 epithelial cells were classed as inadequate cellular material for diagnosis. Slides with sufficient epithelial cells were further categorized as benign ductal epithelial cells, mildly atypical epithelial cells, markedly atypical cells, or malignant epithelial cells. Free DNA was extracted from 200 μL ductal lavage supernatant, using the DNeasy Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μL buffer. Two aliquots of free DNA were extracted separately for reproducibility studies assessing intraaliquot and interaliquot reproducibility. DNA concentration was measured using a NanoDrop ND-1000 spectrophotometer.

Fluorescence-based PCR amplification of three BRCA1 polymorphic microsatellite marker, two intragenic (D17S855 and D17S1322) and one telomeric marker (D17S1325), and four markers flanking the BRCA2 locus, one centromeric (D13S260) and three telomeric (D13S171, D13S1493, and D13S267), was done. Intraaliquot and interaliquot reproducibility was evaluated using the BRCA1 intragenic marker D17S855 in a subset of 21 ductal lavage samples, where at least two informative marker results were available. The chromosomal region 5q21, using marker D5S346 close to the region of the APC gene, was chosen as a representative control locus, as it has been found not to be a site of preferential allelic loss in breast cancer (4). Markers were assessed for informativeness using genomic DNA extracted from the subject's peripheral blood lymphocytes. PCR for the BRCA microsatellite markers was conducted in 15 μL reactions containing 1.5 to 3.0 mmol/L magnesium chloride (optimized for each primer set), deoxynucleotide triphosphate mix (250 μmol/L each of dATP, dGTP, dTTP, and dCTP), 1 unit Taq polymerase (AmpliTaq Gold, Applied Biosystems, Foster City, CA), 1.5 μL of each primer (final optical density 0.1), and 5 μL extracted DNA from the ductal lavage samples (equating to ∼10 ng DNA). PCR for the APC locus was done as described by Zauber et al. (7) Genotyping and analysis were done using an ABI 3100 Genetic Analyser and Genotyper version 3.7 (Applied Biosystems).

LOH was defined as a 50% decrease in the allelic height ratio in DNA from an individual duct compared with the height ratio of the alleles in blood lymphocyte DNA. Allelic loss was determined using the normalized allelic ratio equation [LOH ratio = (D1)(N2) / (D2)(N1)], where D1 and D2 are the heights of the smaller and larger allelic peaks, respectively, from the duct and N1 and N2 are the heights of the allelic peaks from blood lymphocyte DNA (8). Ducts for which the calculated LOH ratio was ≤0.5 or ≥2.0 were scored as LOH. The LOH rate was defined as the number of samples in which LOH was shown divided by the total number of informative sample results available for each microsatellite marker. We deemed the LOH result to be reproducible if the D17S855 marker results were concordant (both heterozygous or both showing loss of the same allele). We defined inconsistency of the LOH result as a discrepancy between replicate experiments as to whether LOH was present or absent or as a discrepancy in which of the two alleles was lost.

In this study, we used free DNA extracted from the ductal lavage supernatant to allow the relatively limited cellular fraction of the ductal lavage sample to be retained for cytologic assessment and other molecular studies. Free tumor DNA has been isolated from various body fluids, including serum, plasma, urine, and peritoneal fluid (9, 10). The observation that levels of cell-free DNA are higher in the body fluids of cancer patients compared with healthy controls has led to interest in its use in the screening and early diagnosis of cancer (11).

We investigated whether LOH at the BRCA loci identified in free DNA from ductal lavage fluid could be used as a reproducible marker of breast cancer risk in healthy BRCA carriers. The mean ages of the BRCA carriers and controls at their first ductal lavage visit were 46.1 years (range, 35.1-62.8 years) and 49.7 years (range, 41.4-52.2 years), respectively. Thirty-three ductal lavage samples were obtained from 17 women (5 BRCA1 carriers, 9 BRCA2 carriers, and 3 controls). Twenty-three samples (69.7%) showed benign cytology. One ductal lavage sample from a BRCA1 carrier showed mild atypia (subject 2). A further BRCA carrier had previously had atypia identified in both breasts by nipple aspiration cytology and has subsequently had mild atypia identified in a ductal lavage sample taken from the right breast (subject 9). The remaining nine ductal lavage samples contained insufficient ductal epithelial cells for cytologic diagnosis.

We were able to isolate sufficient free DNA from all samples, including apparently acellular samples, to do PCR amplification for at least one marker. LOH was found in 9 of 22 (40.9%) and 9 of 16 (56.3%) ductal lavage samples from BRCA carriers using the D17S855 and D17S1322 BRCA1 markers, respectively, but in none of the 3 informative control samples. For the marker, D17S1325, 8 of 22 (36.4%) informative ductal samples from BRCA carriers showed LOH and 1 of the 3 informative control samples. Eight of 13 (61.5%) informative ductal lavage samples from BRCA carriers showed LOH for the centromeric BRCA2 microsatellite marker D13S260. Only 1 control sample was informative for this marker, and LOH was found. LOH was found in 6 of 12 (50%), 8 of 17 (47.1%), and 9 of 20 (45%) of informative ductal lavage samples assessed using the D13S171, D13S1493, and D13S267 BRCA2 markers, respectively. LOH was found in 3 of 4 informative samples from control women using the D13S171 marker and 1 of 2 using the D13S267 marker. No informative results were available for the control samples using the D13S1493 marker. We found LOH at the APC locus in 5 of 27 (18.5%) ductal lavage samples from BRCA carriers but none of the control samples (see Table 1).

One previous study has reported a LOH rate of 58% at the BRCA1 locus using free DNA from the ductal lavage supernatant of known BRCA1 carriers (12). Although Isaacs et al. reported that the LOH results were consistent using the same DNA preparations, reproducibility between different aliquots was not commented upon. We found that LOH results were consistent between separately extracted aliquots of DNA for 14 of the 21 (66.7%) ductal lavage samples analysed. For 6 of the 7 samples where there was inconsistency, there was interaliquot variation between LOH and heterozygous status. The remaining sample from subject 9, a BRCA2 carrier, showed complete loss of the smaller allele in the first aliquot and loss of the larger allele in the second (see Fig. 1). Replicate PCRs were done to assess intraaliquot reproducibility in 10 ductal lavage samples, and results were consistent in 90% of the repeat experiments.

We examined whether LOH status varied over a 1-year period by examining repeat ductal lavage samples taken from three BRCA carriers. The LOH results were entirely consistent between the two time points for the APC locus. For the BRCA1 marker, D17S855, one ductal lavage sample showed loss of the smaller allele at the first ductal lavage visit and heterozygosity in the sample from the second visit, one duct showed persistent loss of the smaller allele over time, and two further ducts remained heterozygous over time. We found no increase in the LOH rate in the ductal lavage fluid obtained from the contralateral breasts of those BRCA2 carriers who had previously had breast cancer in their other breast. Two BRCA carriers had current or previous atypia but did not show a consistent pattern of LOH at the BRCA loci in ductal lavage samples from the ducts in which atypia was found.

There was a trend toward a lower DNA concentration in ductal lavage samples showing LOH [mean, 2.8 ng/μL; (range, 1.2-3.8 ng/μL)] compared with samples in which heterozygosity was retained (mean, 4.1 ng/μL; range, 1.3-11.8 ng/μL), suggesting that some of the LOH found may relate to the relatively low DNA yield from this material and the degree of fragmentation of the DNA close to marker sites. The interaliquot discrepancy in LOH result seen may in part be attributable to sampling variation of a mixed cell population, confounding the detection of free DNA from a relatively small atypical or malignant tumor cell population within a background of normal ductal cells. Furthermore, our finding of LOH at the BRCA loci in predictive test negative controls, at assumed population risk for developing breast cancer, raises the possibility that the result may not reflect true molecular events but rather a methodologic artifact.

We conclude that doing microsatellite marker analyses on free DNA from ductal lavage supernatant is possible, but we found unexpectedly high levels of LOH at the BRCA loci considering these samples were collected from apparently healthy women, although they are at increased risk for developing breast cancer. The rate of LOH at the APC locus was lower but was not negligible (18.5%). If loss of the wild-type allele at the relevant BRCA locus in germ-line BRCA mutation carriers represents an early event in breast tumorigenesis, analysis of free DNA from ductal lavage fluid may offer a useful alternative surrogate marker of risk particularly where samples are insufficient for cytologic diagnosis. However, the degree of reproducibility of these assays found in this study and that of Antill et al. (13) raises questions about the potential of this technique as a robust risk assessment tool in the evaluation of women with a genetic predisposition to breast cancer. Further studies are required to optimize the reproducibility of the methods and to evaluate the specificity and predictive value of LOH in ductal lavage fluid for subsequent breast cancer development.

We thank Dr. Gillian Mitchell who was instrumental in setting up the ductal program at The Royal Marsden NHS Foundation Trust, Audrey Ardern-Jones and Elly Lynch who identified suitable women to take part in the study, Dr Timothy Blackburn for his helpful comments on the article, and all the women who kindly took part in this study.