Comparative Evaluation of Serum Peroxiredoxin 2 (PRDX2), Serum Peroxiredoxin 4 (PRDX4), and Plasma Methylated Septin 9 (mSEPT9) Levels Against Conventional Biomarkers for Early Detection of Colorectal Cancer: A Study Protocol

Background: Colorectal cancer has been characterised as one of the major causes of morbidity and mortality due to cancer globally, and its load is increasing in India with a growing percentage of younger patients. Early intervention (by diagnosis) is associated with a substantial improvement in survival; however, current screening methods, such as colonoscopy, are expensive and invasive, and stool-based and conventional serum biomarkers demonstrate poor sensitivity to disease at an early stage. Oxidative stress and epigenetic modifications play a key role in colorectal carcinogenesis, and circulating indicators of these pathways can enhance non-invasive early diagnosis. The objective of this study is to compare the diagnostic accuracy of serum peroxiredoxin 2 (PRDX2), serum peroxiredoxin 4 (PRDX4), and plasma methylated septin 9 (mSEPT9) with conventional biomarkers for the early detection of colorectal cancer in an Indian population. Methods: This hospital-based case-control study will include 100 participants, including 50 histopathologically confirmed untreated early-stage colorectal cancer patients (stage I-II) and 50 age- and sex-matched healthy controls without malignancy or chronic inflammatory disease, based in a hospital. The sample size was determined based on the expected difference in diagnostic performance between novel and conventional biomarkers, as described in the Methods section. Venous blood will be gathered after overnight fasting and processed to get serum and plasma, which will be stored at −80°C until analysis. The study has received institutional ethics committee (IEC) approval. The levels of serum PRDX2, serum PRDX4, plasma mSEPT9, carcinoembryonic antigen 19-9, C-reactive protein, neutrophil gelatinase-associated lipocalin, interleukin-6, and estimated glomerular filtration rate will be determined either with standardised enzyme-linked immunosorbent assay procedures or with computation formulas. To evaluate individual and multi-markers, the receiver operating characteristic (ROC) curve will be used to evaluate the diagnostic performance, and multivariate logistic regression models will be used to evaluate the diagnostic performance of individual and multi-markers. The primary outcome will be the diagnostic accuracy of PRDX2, PRDX4, and mSEPT9 assessed using the area under the ROC curve, while secondary outcomes will include sensitivity, specificity, positive predictive value, negative predictive value, and the diagnostic performance of combined biomarker panels. Discussion: This protocol describes a systematic assessment of both oxidative stress and epigenetic biomarkers in addition to conventional biomarkers for the early detection of colorectal cancer in an Indian population. The study aims to improve the diagnostic sensitivity for early-stage colorectal cancer while maintaining feasibility for routine clinical laboratory practice by evaluating a minimally invasive blood-based panel comprising PRDX2, PRDX4, and mSEPT9. The findings of this study may provide preliminary evidence for the development of cost-effective, non-invasive screening strategies suitable for resource-limited settings and may support future validation studies and implementation research.