Increased integrity of free circulating DNA in sera of patients with colorectal or periampullary cancer: direct quantitative PCR for ALU repeats.
BACKGROUND
Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic nondiseased cells, DNA released from dead cancer cells varies in size. We developed a novel method to measure the ratio of longer to shorter DNA fragments (DNA integrity) in serum as a potential biomarker for patients with colorectal cancer (CRC) or periampullary cancers (PACs).


METHODS
Sera from 32 patients with CRC (3 stage I, 14 stage II, 6 stage III, and 9 stage IV patients), 19 patients with PACs (2 stage I, 9 stage II, 1 stage III, and 7 stage IV patients), and 51 healthy volunteers were assessed by quantitative real-time PCR of ALU repeats (ALU-qPCR) with 2 sets of primers (115 and 247 bp) amplifying different lengths of DNA. We used serum directly as a template for ALU-qPCR without DNA purification. DNA integrity was determined as ratio of qPCR results of 247-bp ALU over 115-bp ALU.


RESULTS
ALU-qPCR had a detection limit of 0.01 pg of DNA. Eliminating DNA purification reduced technical artifacts and reagent/labor costs. Serum DNA integrity was significantly increased for stage I/II and III/IV CRC and stage I/II and III/IV PACs (P = 0.002, P = 0.006, P = 0.022, and P <0.0001, respectively). ROC curves for detecting CRC and PACs had areas under the curves of 0.78 and 0.80, respectively.


CONCLUSIONS
Direct ALU-qPCR is a robust, highly sensitive, and high-throughput method to measure serum DNA integrity. DNA integrity is a potential serum biomarker for detection and evaluation of CRC and PACs.
Umetani
Naoyuki
N
Department of Molecular Oncology and Division of Surgical Oncology, John Wayne Cancer Institute, Santa Monica, CA 90404, USA.
Kim
Joseph
J
Hiramatsu
Suzanne
S
Reber
Howard A
HA
Hines
Oscar J
OJ
Bilchik
Anton J
AJ
Hoon
Dave S B
DS
D000368:Aged; D014670:Ampulla of Vater; D014408:Biomarkers, Tumor / Q000097:blood*; D021441:Carcinoma, Pancreatic Ductal / Q000175:diagnosis* / Q000473:pathology; D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000473:pathology; D003138:Common Bile Duct Neoplasms / Q000175:diagnosis* / Q000473:pathology; D004247:DNA / Q000097:blood*; D053938:DNA Fragmentation; D004379:Duodenal Neoplasms / Q000175:diagnosis* / Q000473:pathology; D005260:Female; D006801:Humans; D008297:Male; D010190:Pancreatic Neoplasms / Q000175:diagnosis* / Q000473:pathology; D010865:Pilot Projects; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D014408:Biomarkers, Tumor; D004247:DNA