Abstract 715: Detection of a p53 codon 249 hotspot mutation in the urine of the patients with hepatocellular carcinoma

Abstract

Abstract Hepatocellular carcinoma (HCC), the 5th most frequent cancer worldwide, has a 5-year survival rate of 14% because it is difficult to diagnose early. The goal of this project is to construct a panel of circulation-derived DNA markers to use in a urine test for the early detection of HCC to improve its prognosis. The HCC-specific p53 codon 249T mutation was the first candidate DNA marker used to explore the criteria needed to detect HCC-derived DNA markers in urine of patients with HCC in a sensitive, noninvasive manner. We showed previously that urine contains circulation-derived DNA fragments that are mostly fewer than 300 bp, designated as low-molecular-weight (LMW) urine DNA. The LMW urine DNA contains DNA from tumor tissues when tumors are present. The tumor-derived DNA fragments offer the potential to develop absolutely noninvasive urine tests for the detection of any cancer with known DNA biomarkers. A locked nucleic acid clamp-mediated PCR assay for the p53 249T mutation, targeting only 41 nucleotides of the template, followed by melting curve analysis, was developed and tested using patient urine samples. Total urine DNA samples from 17 patients with HCC were fractionated into high-molecular-weight (HMW) (>1 kb, mostly cell-associated) DNA from the urinary tract and LMW (< 1 kb, mostly circulation derived) DNA and subjected to the p53 249T mutation assay. Encouragingly, samples from 9 of 17 patients with HCC contained detectable p53 249T mutation in the LMW urine DNA fraction and only 1 of 17 matching HMW urine DNA fraction samples was found to contain p53 249T mutated DNA. These findings suggest that the assay target template size of 41bp is suitable to detect HCC-derived DNA markers in urine and that the p53 249T mutation detected in LMW urine DNA is derived from the circulation. Next, we tested a larger number of samples of LMW urine DNA from patients with HCC. Overall, we detected the p53 249T mutation in 50.8% of the LMW urine DNA samples from patients with HCC (60/118). Of the 49 subjects whose urine was positive for the p53 mutation before surgery, the p53 mutation was no longer detectable in the urine of 29 of those patients after surgical removal of the tumor. This result suggested that the p53 249T mutations detected in the urine before surgery were derived from the surgically removed HCC. For controls, we tested LMW urine DNA from patients with hepatitis and cirrhosis and from normal subjects or patients with colorectal cancer and found that 0 of 32 normal (0%), 0 of 31 colorectal cancer (0%), 7 of 69 hepatitis (10.1%), and 6 of 47cirrhosis (12.8%) samples contained detectable amounts of p53 249T mutated DNA. A urine test that includes multiple DNA markers for HCC screening and recurrence monitoring is currently being explored. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 715. doi:1538-7445.AM2012-715