Colorectal cancer remains a significant cause of morbidity and mortality worldwide with a high incidence rate. The discovery of non-invasive biomarkers for CRC detection with adequate sensitivity and specificity is a major challenge to reduce cancer-related morbidity and mortality. New non-invasive tests are under research to meet the balance between the increase of sensitivity and decreasing the need for unnecessary colonoscopies .
Circulating free DNA has been suggested to be a promising tumor marker. However, as cfDNA levels may also get elevated in various non-malignant disorders, more specific approaches have been proposed. Among these approaches is the calculation of the DII. The DII describes the ratio of longer free DNA fragments to shorter free DNA fragments [6, 7]. In healthy individuals, cfDNA mainly originates from apoptotic cells which usually release DNA fragments of 185–200 bp. In contrast, cfDNA released from cancer cells is usually longer due to the pathologic cell death in tumors. Therefore, DNA integrity has the potential of being used for tumor detection and prognostic prediction . DII calculation using fragments from GAPDH has been described by Van Beers et al. , also Salvianti et al.  has investigated the determination of DII targeting sequences in amyloid precursor protein.
The use of ALU repeats for determination of DII was first proposed by Umetani et al. . ALU115 and ALU247 are 2 amplicons with a length of 115 and 247 bp. ALU115 and ALU247 are used to distinguish between DNA originating from apoptotic and necrotic cell death, respectively. Since the main source of short cfDNA (180–200 bp fragments) in healthy individuals has been attributed to apoptotic cells, a majority of longer DNA fragments (ALU247) could represent a biomarker for malignant tumor detection. The annealing sites of ALU115 are within the ALU247 ones, the ALU115 primers could amplify both shorter (truncated by apoptosis, i.e., ALU115) and longer DNA fragments (ALU247), so results of ALU115 quantitation represent the total amount of cfDNA. However, ALU247 primers amplify only longer DNA fragments; therefore, results of ALU247 quantitation represent amounts of DNA released from necrotic cell death. DII is calculated as the ratio of longer to shorter ALU fragments (ALU247/ALU115) .
In this study, we chose ALU115 and ALU247 repeats to calculate the DII in CRC patients. We aimed to assess the clinical utility of DII as a potential biomarker for CRC and to evaluate its correlation with CEA, the conventional marker used for prognosis, and follow-up of CRC patients.
We found higher DII in CRC patients than both pathological controls and healthy controls. DII was higher in early stage CRC patients compared to healthy controls and was also higher in the metastatic CRC patients compared to non-metastatic patients. Also, DII positively correlated with CEA, tumor size, and tumor stage.
Umetani et al.  investigated the DII in 32 CRC patients and 51 heathy controls in the USA and reported that DII was higher in CRC patients compared to healthy controls with an AUC of 0.78 for discriminating CRC patients from healthy individuals. Similarly, Leszinski et al.  assessed DII in 24 CRC patients, 11 patients with benign gastrointestinal diseases, and 24 healthy individuals in Germany, and reported higher DII in CRC patients compared to healthy controls with an AUC of 0.738 while no difference was observed between CRC patients and patients with benign gastrointestinal diseases. El-Gayar et al.  also assessed DII in 50 CRC patients and 20 healthy controls and reported that DII is higher in CRC patients compared to healthy individuals with AUC of 0.9.
As expected, the level of CEA was significantly higher in CRC patients than in both the healthy control group and the pathological control group. This came in consistent with El-Gayar and his coworkers . As for ALU 115 that represents absolute total DNA concentration, we found a significant increase in the level of absolute DNA concentration in CRC patients compared to healthy controls but non-significant difference was found between CRC patients and benign group. Similarly, El-Gayar et al.  and Hao et al.  found significantly higher levels of absolute DNA concentration in CRC group than healthy controls. However, in contrast to our findings, they found significantly higher levels of absolute DNA concentration in the CRC group compared to the benign group. This difference may be attributed to that previous studies were conducted on patients having poorly differentiated (grade III) tumors, with different histopathological states that might contribute to a significant increase in absolute cfDNA levels in CRC patients compared to the benign group. However, in our study, most patients had moderately differentiated (grade II) mucinous adenocarcinoma.
Additionally, we observed a significant increase in absolute DNA concentration in a benign group compared to the healthy control group. This finding came in agreement with Mead et al.  and contrasted by Bedin et al.  and El-Gayar et al. . Our findings are supported by the fact that any benign disease condition may be accompanied by some sort of inflammation that may cause elevation of total DNA in our pathological control group. However, the discrepancy with Bedin et al.  and El-Gayar et al.  in this regard may be attributed to difference in sample sizes, and Bedin and his colleagues  used plasma as source of cfDNA, the exclusion of presence of inflammatory conditions in both benign and healthy group was done by full history-taking only, and there were no investigations done to confirm absence of hidden inflammatory conditions in our study and other studies.
As for ALU 247 concentration, which represents necrotic long DNA fragments, there was a significantly higher concentration of ALU 247 in CRC patients compared to both healthy controls and pathological controls. No statistically significant difference was found between pathological controls and healthy controls. This came in agreement with Mead et al.  who found a significant difference between CRC patients compared to healthy controls.
To assess the clinical relevance of our studied markers as early markers for CRC, there was a statistically significant difference in the levels of DII and ALU247 between patients with early stages without evidence of metastasis (stages I and II) and controls. However, non-significant difference was found between the two groups as regards CEA and ALU115. This came in accordance with Umetani et al.  and Arakawa et al.  who found significantly higher levels of DII in patients with early stages of CRC than in healthy volunteers.
To predict the prognostic significance of CEA, ALU 115,247, and DII, we compared the previously mentioned markers with the status of metastasis; there were significantly higher levels of CEA and DII in patients with metastasis than those without metastasis. This suggests using both markers to differentiate between patients with early and late stages Also, a correlation study was done between the studied markers and both tumor size and stage. DII was found to have a significant positive correlation with tumor stage and tumor size while CEA was only found to have a significant positive correlation with tumor stage. Also, DII positively correlated with CEA. These findings came in agreement with El-Gayar et al.  and Arakawa et al. . No significant difference was found between the metastatic and non-metastatic groups as regards ALU 115 and ALU 247. Similarly, El-Gayar et al.  found a non-significant difference in ALU 115 between the two groups.