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Clinical Chemistry 52: 1062-1069, 2006; 10.1373/clinchem.2006.068577
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(Clinical Chemistry. 2006;52:1062-1069.)
© 2006 American Association for Clinical Chemistry, Inc.

Cancer Diagnostics

Increased Integrity of Free Circulating DNA in Sera of Patients with Colorectal or Periampullary Cancer: Direct Quantitative PCR for ALU Repeats

Naoyuki Umetani1, Joseph Kim1,2, Suzanne Hiramatsu1, Howard A. Reber3, Oscar J. Hines3, Anton J. Bilchik2 and Dave S. B. Hoon1,a

1 Department of Molecular Oncology and 2 Division of Surgical Oncology, John Wayne Cancer Institute, Santa Monica, CA.
3 Section of Gastrointestinal Surgery, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA.

aAddress correspondence to this author at: Department of Molecular Oncology, John Wayne Cancer Institute, 2200 Santa Monica Blvd, Santa Monica, CA 90404. Fax 310-449-5282; e-mail hoon{at}jwci.org.

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.




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