Increased Free Circulating DNA Integrity Index as a Serum Biomarker in Patients with Colorectal Carcinoma

Abstract

Background: Cell-free DNA circulating in blood is a candidate biomarker for malignant tumors. Unlike uniformly truncated DNA released from apoptotic non diseased cells, DNA released from necrotic cancer cells varies in size. Objectives: To measure the DNA integrity index in serum and the absolute DNA concentration to assess their clinical utility as potential serum biomarkers for colorectal carcinoma (CRC) compared to CEA and CA19-9. Materials and Methods: Fifty patients with CRC, 10 with benign colonic polyps and 20 healthy sex and age matched volunteers, were investigated by real time PCR of ALU repeats (ALU q-PCR) using two sets of primers (115 and 247 bp) amplifying different lengths of DNA fragments. The DNA integrity index was calculated as the ratio of q-PCR results of ALU 247/ ALU 115bp. Results: Serum DNA integrity was statistically significantly higher in CRC patients compared to the benign and control groups (p<0.001). ROC curves for differentiating CRC patients from normal controls and benign groups had areas under curves of 0.90 and 0.85 respectively. Conclusions: The DNA integrity index is superior to the absolute DNA concentration as a potential serum biomarker for screening and diagnosis of CRC. It may also serve as an indicator for monitoring the progression of CRC patients. Combining CEA and CA19-9 with either of the genetic markers studied is better than either of them alone.

Methodological, biological and clinical aspects of circulating free DNA in metastatic colorectal cancer

“…In general, the levels are higher in mCRC patients than in healthy controls, as shown in Table 1 [15,20,47,58,[63][64][65][66][67][68][69][70][71][72]; and the levels have some correlation to stage as reported in a limited number of studies [15,41,68,73] in primary CRC. These findings give important biological information, and researchers suggest that cfDNA has potential as a diagnostic or screening tool; a potential that is currently being investigated by several groups.…”

“…The size of DNA fragments seems to hold important biological information. This has been explored by several groups advocating that a cfDNA integrity index should be used in clinical investigations [15,20,53]. Although the potential that lies in detection and measurement of the tumor-specific DNA has been established, the clinical value of total cfDNA quantification is still being debated.…”

“…It is therefore relevant to analyze the relation between cfDNA and CEA. As illustrated from the summary in Table 2, there is considerable variation in the measures, methods and parameter used to compare or combine the information from CEA and cfDNA analysis [15,20,32,33,39,41,[65][66][67][68][69][70][71][72][81][82][83]. The table includes studies identified by a systematic literature search of circulating DNA in CRC (all stages) as illustrated by Supplementary Figure 1.…”

“…Another stand of research explores the potential clinical value of measuring the cfDNA and the emergence of recent technologies has enabled investigation of relevant cohorts of cancer patients in clinical settings. Several studies indicate a potential prognostic, predictive and diagnostic value of measuring the cfDNA, which calls for reliable validation [14][15][16][17][18][19][20][21][22][23].…”

Cell‐free nucleic acids are present in blood products and regulate genes of innate immune response

“…25 As CFNAs are derived from the nuclear genome of blood, hepatic, or placental cells, Alu sequences were used as a CFNA marker. Because cell-free Alu 81, Alu 115, and Alu 247 were previously reported in plasma, 26 they were tested as relevant CFNA markers. The three different Alu sequences were found in blood product-derived CFNA, but Alu 115 qPCR was the most effective (not shown) and then chosen as the CFNA marker.…”