ORIGINAL ARTICLE Journal of the College of Physicians and Surgeons Pakistan 2021, Vol. 31(10): 1174-1178 1174 Detection of Kras Gene in Colorectal Cancer Patients through Liquid Biopsy: A Cost-effective Method Hafiz Syed Muhammad Osama Jafri, Shamim Mushtaq and Saeeda Baig Department of Biochemistry, Basic Health Sciences, Dr. Ziauddin University, Karachi, Pakistan ABSTRACT Objective: To detect the Kras gene through liquid biopsy, a less invasive technique in diagnosed colorectal cancer patients. Study Design: Cross-sectional study. Place and Duration of Study: Department of Oncology, Dr. Ziauddin Hospital and Bait-us-Sukoon Cancer Hospital, Karachi, from 2019 to 2020. Methodology: Circulating tumor DNA (ctDNA) in colorectal cancer patients was extracted through magnetic bead technique using MagMAX cell free DNA kit (Thermofisher, Uk). The frequency of K ras gene was quantified using a real-time polymerase chain reaction (RT-PCR) assay (qPCR). ANOVA and Chi-square tests were utilised for statistical analysis. Results: Mean threshold cycle (CT) of Kras gene showed significantly higher expression 15.6 ± 1.82 (p=0.001) in stage IV CRC cases compared to early stages (19.53 ± 18.223.7 ± 2.9 and 19.8 ± 2.69 of stage 1, 2 and 3, respectively. Similarly, ΔCT mean of Kras gene at stage IV showed significantly higher expression of 2.48 ± 1.40 (0.048), compared to 2.39 ± 0.6, 3.12 ± 0.68 and 3.15 ± 0.41 of stage 1, 2 and 3, respectively. Males (n=40, 55%) showed significant association (p=0.001) with CRC compared to females (n=33, 45%). Categorisation of tumor types within different age groups revealed that colon cancer was more frequent (n=11, 15.1%) in the 41-50 age group, while rectal cancer was more frequent (n= 11, 15.1%) in the 41-50 age group, while rectal cancer was more in the 51-60 age group (n=11, 15.1%). Conclusion: Kras gene was detected with significantly increased levels in plasma of CRC patients at advanced stages. This confirms that liquid biopsy can be used to detect K ras gene in ctDNA of CRC patients through a magnetic bead based tech- nique. Key Words: Liquid biopsy, Circulating tumor DNA, KRAS, Colorectal cancer, Real-time polymerase chain reaction. How to cite this article: Jafri HSMO, Mushtaq S, Baig S. Detection of Kras Gene in Colorectal Cancer Patients through Liquid Biopsy: A Cost-effective Method. J Coll Physicians Surg Pak 2021; 31(10):1174-1178. INTRODUCTION Colorectal cancer (CRC) globally is one of the main stream cancer causing mortalities in both genders being the 7 th most common cancer in women, while 4 th in men according to Globacan statistics with low prevalence in Asia and Africa. 1 Colorectal cancer is a highly diversified, heterogeneous and complex disease containing cells of the same histological type with different genotypes or cell subtypes. 2 Effective monitoring and management are, therefore, extremely essential for the regular evaluation of the disease. Kras, aproto-oncogene,havingdualfunction,playsamajorrole both in inhibition of cancer as well as in carcinogenesis. Correspondence to: Prof. Dr. Saeeda Baig, Department of Biochemistry, Basic Health Sciences, Dr. Ziauddin Univer- sity, Clifton, Karachi, Pakistan E-mail: saeeda.baig@zu.edu.pk ..................................................... Received: June 23, 2021; Revised: September 09, 2021; Accepted: September 20, 2021 DOI: https://doi.org/10.29271/jcpsp.2021.10.1174 Mutation of Kras on codons 12, 13 transforms it into an onco- gene, identified in the colorectal cancer (CRC; 25-45%), lung cancer (35%), and breast cancer (5-10%). 3 Kras, a prominent member of the Ras family, plays a major role in healthy tissue signalling through cycling of GTPase between active GTP, K ras GTP and Kras GDP, which is inactive formations. Mutations in Kras can impair GTPase’s function, leading to permanent activa- tion of AKT / mTOR / PI3K and MEK / ERK / RAF, the downstream signalling pathways. Thus, the ongoing activation of K ras ulti- mately develops into malignancies. 4 Liquid biopsy, an outstanding molecular diagnostic method, which can detect tumor DNA fragments, known as circulating tumorDNA(ctDNA),floatingintheblood(plasma)withminimal invasiveness and is highly responsive tool for CRC patients . ctDNA is found in a number of body fluids, including blood plasma,synovialfluid,andcerebrospinalfluid,originatingfrom cancer cells that have undergone cell death; it is composed of single or double stranded DNA. 5 The ctDNA has increasingly progressed from research to clinical use as the first option for liquid biopsy, because its testing is less invasive and replica- ble/reproducible, 6 detecting gene mutations in a convenient manner.Inaddition,anothertheoreticalbenefitofliquidbiopsy