Identification of loss of heterozygosity on circulating free DNA in peripheral blood of prostate cancer patients: potential and technical improvements.
BACKGROUND
Accurate identification of loss of heterozygosity (LOH) on circulating free DNA is often restricted by technical limitations such as poor quality and quantity of tumor-specific DNA and contamination by normal DNA. However, plasma DNA may harbor tumor-specific genetic alterations and could therefore be an interesting target for noninvasive examinations of tumor DNA.


METHODS
By PCR-based fluorescence microsatellite analysis using 12 polymorphic markers, we investigated LOH on cell-free DNA in blood plasma from 59 patients with localized prostate cancer (PCa) and 12 with metastatic disease (MPCa). In addition, plasma DNA from 21 PCa patients was fractionated into high- and low-molecular-weight DNA by 2 different column systems. To avoid appearance of artificial allelic loss and stabilize the amplification, TMAC (tetramethylammonium chloride) was added to each PCR.


RESULTS
Overall incidences of LOH at all markers analyzed were 10% in PCa and 12% in MPCa samples. Highest frequencies were found at markers D11S898 (28%) in PCa and D6S1631 (27%) in MPCa. Statistical evaluation showed significant associations between LOH and increasing Gleason scores for the marker combinations D6S1631*D8S286*D9S171 (P = 0.03) and D8S286*D9S171 (P = 0.05). Fractionation of plasma DNA resulted in a higher overall LOH frequency in the low-molecular-weight DNA fraction (23%) compared with the high-molecular-weight DNA (7%).


CONCLUSIONS
LOH analysis of circulating DNA can provide tumor-specific genetic information on PCa patients. Fractionation of plasma DNA and addition of TMAC improved LOH detection and general assay performance.
Müller
Imke
I
Institute of Tumor Biology, University Medical Center Hamburg-Eppendorf, Germany.
Beeger
Cord
C
Alix-Panabières
Catherine
C
Rebillard
Xavier
X
Pantel
Klaus
K
Schwarzenbach
Heidi
H
D004247:DNA / Q000097:blood*; D005453:Fluorescence; D006801:Humans; D019656:Loss of Heterozygosity; D008297:Male; D018895:Microsatellite Repeats; D016133:Polymerase Chain Reaction; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology
D004247:DNA