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Abstract Colorectal cancer (CRC) is a term used for cancer that starts in the colon or the rectum. CRC incidence and mortality rates vary markedly around the world. However, rates are substantially higher in males than in females, representing the third most commonly diagnosed cancer in males and the second in females. The importance of screening tools to identify early stage CRC is acknowledged worldwide. Colonoscopy is currently considered to be the gold standard for CRC screening. However, it is estimated that many individuals over 50 years of age have refused to undergo this test due to the invasiveness of the procedure. Screening with established tumor markers for gastrointestinal cancers, such as CEA or CA19-9, has limited efficiency because of erratic detection and increased levels in benign disorders. Circulating cell free DNA (ccf-DNA) is a double-stranded molecule of low molecular weight which, although mainly fragmented in 70-200 base pairs (bp), also has pieces up to 21 kilobases in length. ccf- DNA can be found in healthy persons, persons with non-malignant diseases, as well as persons with various malignancies. In healthy individuals, apoptosis and necrosis of lymphocytes and other nucleated cells are mainly involved in the release of circulating nucleic acids into the blood. Apoptosis leads to DNA degradation in which chromosomal DNA is first cleaved into large fragments (50-300 kb) and then into multiples of nucleosomal units (180-200 bp). While in cancer patients, ccf-DNA most likely result from tumor necrosis, but other suggested mechanisms include lysis of circulating Summary and Conclusion 124 cancer cells or of micro-metastases, or due to active release. Necrosis generates a spectrum of DNA fragments with different strand lengths, mostly large DNA fragments, because of random and incomplete digestion of genomic DNA by DNases. DNA integrity that potentially mirrors the relation between the necrotic and overall cell death rate, can be defined as the ratio of the concentrations of longer DNA fragments (ALU 247) to shorter DNA fragments (ALU 115). The integrity index is increased at the early stages of CRC and is a promising molecular biomarker for detecting CRC. The current study was carried out by cooperation between Medical Biochemistry, General Surgery & Clinical Oncology and Nuclear Medicine Departments, Faculty of Medicine, Menoufia University. The aim of this study was to study whether the concentrations and integrity index of circulating cell free DNA (ccf-DNA) in serum can be used as a diagnostic biomarker for colorectal cancer (CRC) patients. This study involved 80 individuals, 40 CRC patients, 20 patients with benign diseases in colon and rectum and 20 healthy persons served as controls. Full history and clinical examination were made to every subject. Laboratory investigations were also carried out to all individuals including analysis of CA19-9 level by ELISA technique and ccf-DNA through detection of long (247 bp) and short (115 bp) DNA fragments in serum by real-time quantitative PCR by amplifying the ALU repeats (ALU-qPCR). The results of the present study can be summerized as follows: A significantly higher level of serum CA 19-9 concentrations were observed in CRC patients and patients with benign diseases in comparison to control group. Summary and Conclusion 125 A significantly higher level of serum ALU 115 and ALU 247 concentrations were observed in CRC patients and patients with benign diseases in comparison to control group. Significantly higher levels of DNA integrity (ALU247/115) were observed in CRC patients and patients with benign diseases in comparison to control group. There was significant statistical difference among studied CRC patients in tumor stage and size as regards ALU 247 serum levels. There was significant statistical difference among studied CRC patients in tumor stage and size as regards DNA integrity (ALU247/115). There was significant negative correlation between ALU 115 serum levels and each of the size of tumor and the invasiveness of the primary tumor (T stage). There was significant positive correlation between ALU 115 serum levels and the grade of tumor. There was significant negative correlation between DNA integrity (ALU247/115) and the invasiveness of the primary tumor (T stage). There was significant positive correlation between DNA integrity (ALU247/115) and each of size of tumor and the number of local and regional lymph nodes containing metastatic cancer (N stage). There was significant positive correlation between CA19-9 serum levels and each of the invasiveness of the primary tumor (T stage) and distant metastasis (M stage). Summary and Conclusion 126 The diagnostic accuracy for distinguishing primary CRC patients from normal controls by ALU 115 serum levels was (85%), with sensitivity (83%) , specificity (90%), positive predictive value (96%) and negative predictive value (64%) at cut off point of 426 ng/ml. The diagnostic accuracy for distinguishing primary CRC patients from normal controls by ALU 247 serum levels was (93%), with sensitivity (93%), specificity (90%), positive predictive value (97%) and negative predictive value (82%) at cut off point of 135 ng/ml. The diagnostic accuracy for distinguishing primary CRC patients from normal controls by DNA integrity (ALU247/115) was (61%), with sensitivity (75%), specificity (20%), positive predictive value (74%) and negative predictive value (21%) at cut off point of 0.42. |