Methodology  

The study will be initiated after obtaining approval from the Institutional Scientific Committee and the Human Ethics Committee at Regional Cancer Centre,  Thiruvananthapuram. All patients underwent thorough physical examination and  staging work up as per the institution protocol. Investigations done will be complete  blood counts with differentials, renal function tests, liver function tests, tissue  diagnosis , CT of  thorax, abdomen and pelvis. Decision regarding Surgery will be made at the multidisciplinary tumor board based on the clinical stage and patient condition. Patients satisfying the inclusion criteria will be selected for the study.  A written informed consent will be taken from the included patients prior to the  procedure. 

The comparison among pre-surgery and 4week post surgery cfDNA and DNA integrity index will be done. 

Method of assessing cfDNA and DNA integrity index  

Peripheral blood (10 ml) will be collected in sterile EDTA-coated vials at baseline (before surgery) and  4 weeks after surgery.    

The plasma will be separated by two sequential centrifugation at 1000 g, 4°C and stored at −80°C. cfDNA will be extracted from plasma samples using the QIAamp Circulating Nucleic Acid Kit (Qiagen, Valencia, CA, USA) following manufacturer instructions. Quantity and quality of plasma DNA will be assessed by quantitative real-time PCR (QRTPCR) by Quantstudio 6 Flex real-time PCR system (ABI, USA) using human beta-actin gene as a reference gene. By using a standard curve, it is possible to calculate the absolute concentration of target DNA in a sample. A standard curve will be constructed using five-fold serial dilutions of known concentrations of DNA (range of 0.01-100 ng). Each PCR reaction mixture consists of 10 μl master mix TAKARAâ„¢ SYBRâ„¢ Green PCR Master Mix (TAKARA, USA), 1.0 μl each primer (0.4 mM), 2 μl water, and 6μl of extracted DNA. The thermal cycling conditions comprise cycles at 95°C for 10 min and 40 cycles at 95°C for 10 s and at 60°C for 60 s followed by melt curve analysis. Sample DNA concentration will be extrapolated from the standard. A colon cancer cell line will be used as a control, and a negative no template control will be included in each run. DNA integrity index will be calculated as the ratio between the 394 and 99 bp amplicons of the β-actin gene. The primer sequences are as follows: Common forward primer: 5′-CCACACTGTGCCCATCTACG-3′, Reverse primer (β-actin 99 bp):  5′AGGATCTTCATGAGGAGTCAGTCAG-3′, Reverse primer (β-actin 394 bp): 5′- TTAGCTTCCACAGCACAGCC- 3′.

Plasma DNA integrity

Increased DNA integrity in plasma, derived from threshold cycle numbers assessed by quantitative real-time PCR (qRTPCR) for 2 amplicons (394 bp and 99 bp) of a specific gene, is reported to indicate the presence of cancers. The premise is that DNA released from necrotic malignant cells varies in size, whereas DNA released from apoptotic cells is uniformly truncated into 185- to 200-bp fragments. Because the main source of free circulating DNA in healthy individuals is apoptotic cells, a preponderance of longer DNA fragments could be a marker for malignant tumor detection. DNA integrity will be calculated as - 394 bp amplicons of the β-actin gene／99bp amplicons of the β-actin gene.

Statistical analysis

The categorical variables will be presented using frequencies and percentages,  continuous variables will be presented using mean, standard deviation, median and interquartile range. The  association between two categorical variables will be assessed using Fisher’s exact test.  The change in cfDNA levels will be tested using paired t test or Wilcoxon Signed rank test as appropriate. The significance difference in means between two groups will be tested using Students t test for normally distributed variables, otherwise Mann Whitney U test will be used. Univariate and multivariate logistic  regression models will be used to predict the risk associated with cfDNA levels. The strength of association between cfDNA and CEA will be assessed using the Karl Pearson correlation coefficient. A p-value of <0.05 will be considered to be significant. 

References:

1. Elshimali YI, Khaddour H, Sarkissyan M, Wu Y, Vadgama JV. The Clinical Utilisation of Circulating Cell Free DNA (CCFDNA) in Blood of Cancer Patients. Int J Mol Sci. 2013 Sept 13;14(9):18925–58.

2. Salem, R., Ahmed, R., Shaheen, K. et al. DNA integrity index as a potential molecular biomarker in colorectal cancer. Egypt J Med Hum Genet 21, 38 (2020)

3. Rosen, A.W., Gögenur, M., Paulsen, I.W. et al. Perioperative changes in cell-free DNA for patients undergoing surgery for colon cancer. BMC Gastroenterol 22, 168 (2022). 

4. Henriksen TV, Reinert T, Christensen E, Sethi H, Birkenkamp-Demtröder K, Gögenur M, Gögenur I, Zimmermann BG; IMPROVE Study Group, Dyrskjøt L, Andersen CL. The effect of surgical trauma on circulating free DNA levels in cancer patients-implications for studies of circulating tumor DNA. Mol Oncol. 2020 Aug;14(8):1670-1679. doi: 10.1002/1878-0261.12729. Epub 2020 Jun 16. PMID: 32471011; PMCID: PMC7400779.