Detection of LOH and Mitochondrial DNA Alterations in Ductal Lavage and Nipple Aspirate Fluids from High-risk Patients

Abstract

We describe a method for the isolation of free DNA from ductal lavage (DL) and nipple aspirate fluid (NAF), and its evaluation for the presence of LOH at the BRCA1 and FHIT genes and for mitochondrial DNA (mtDNA) mutations at the D310 marker, to improve early detection of breast cancer. We evaluated 26 DL and six NAF samples from 14 women of known BRCA1 status, who have no clinical evidence of breast tumors: nine mutation carriers and five non-carriers. LOH studies at the BRCA1 locus were possible in 19/26 DL samples, and at the FHIT locus in 16/26 samples. In 4/9 mutation carriers we found LOH at the BRCA1 allele, and in two of these we also found LOH at the FHIT allele. In one of the mutation carriers with BRCA1 LOH, invasive breast cancer was subsequently detected, and the tumor showed the same LOH as the DL. In one of the true negatives, BRCA1 and FHIT LOH were detected. The mitochondrial studies were possible in all 26 DL samples and a somatic mutation was found in 3/9 carriers, two of whom also had LOH at the BRCA1 locus, and in none of the non-carriers. mtDNA mutation evaluation was possible in 4/6 NAF samples. The NAF and DL results were concordant. One NAF sample from a BRCA1 patient showed a mtDNA mutation. Our data demonstrates the feasibility of performing molecular studies using the free DNA present in the ductal fluid, while the intact cells can be used for cytologic studies.

Loss of Heterozygosity at theBRCA1andBRCA2Loci Detected in Ductal Lavage Fluid fromBRCAGene Mutation Carriers and Controls

“…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.…”

Mitochondrial DNA mutations in human cancer

“…In another study, identical changes were detected in four of four urine sediments from patients with bladder cancer and in three of three fine needle aspirates of patients with breast cancer (Parrella et al, 2003). Nipple aspirate fluid has also mtDNA mutations in human cancer A Chatterjee et al yielded mitochondrial C-tract alterations in breast cancer patients (Isaacs et al, 2004) In another study, urine from five patients with bladder cancer and duodenal aspirates from four patients with pancreatic cancer were tested using the Mitochip array (Maitra et al, 2004). At least one cancer associated mitochondrial mutation was found in six of the nine body fluid samples.…”

Ductal Lavage of Fluid-Yielding and Non–Fluid-Yielding Ducts in BRCA1 and BRCA2 Mutation Carriers and Other Women at High Inherited Breast Cancer Risk

“…For these reasons, evaluation of breast duct cells is an emerging technique for breast cancer risk assessment, and for the discovery of potential biomarkers which may serve as intermediate end points in trials of cancer prevention agents (28)(29)(30)(31)(32)(33)(34). Various minimally invasive methods exist for collecting breast duct cells, including nipple suction aspiration and random periareolar fine-needle aspiration; the finding of atypia in cells collected by each of these techniques has been associated with increased subsequent breast cancer risk (35,36).…”