Detection of a K-ras mutation in urine of patients with colorectal cancer.
Author(s): Su YH, Wang M, Aiamkitsumrit B, Brenner DE, Block TM
Publication: Cancer Biomark, 2005, Vol. 1, Page 177-82
PubMed ID: 17192038 PubMed Review Paper? No
Purpose of Paper
This paper characterized the size of DNA isolated from usine and semi-quantitatively compared the amplification of long and short DNA fragments from urine that was stored on ice for up to 24 h. The authors also compared KRAS mutation detection success among matched tissue, urine, and plasma specimens from patients with colorectal carcinoma or adenomatous polyps.
Conclusion of Paper
High molecular weight DNA, as well as DNA of 150-250 bp, was present in urine collected from all five patients. The abundance of the “long” PCR product was greater when urine was stored on ice for 8 h than in matched specimens that were stored on ice for ≤4 h., which the authors attribute to genomic DNA contamination from urinary tract cell lysis. Levels of the “short” PCR product were unchanged when urine was stored for up to 8 h on ice; however, the authors state that this likely reflects a slight loss of the short amplicon within the cell-free component given that an increase in amplification would be expected with the genomic contamination that was observed. Neither the short nor long PCR product was amplifiable from urine stored on ice for 24 h. The KRAS mutation was detectable in both the tumor and urine specimen, but only wildtype KRAS was detected in the normal adjacent specimen. During validation, a KRAS mutation was detected in all five colorectal cancer tissue specimens and all five matched urine specimens, but only 2 of the matched plasma specimens. KRAS mutations were detected in 13 of polyp specimens, with mutations detected in 10 and 8 of the matched urine and plasma specimens, respectively. Interestingly, one of the two polyp specimens without a KRAS mutation had a mutation detected in the matched plasma and urine specimens. The authors conclude that urine may be a more sensitive biospecimen type for colorectal cancer detection than plasma.
Studies
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Study Purpose
This study characterized the size of DNA isolated from urine and semi-quantitatively compared the amplification of long and short DNA fragments from urine that was stored on ice for up to 24 h. The authors also compared KRAS mutation detection success among matched tissue, urine, and plasma specimens from patients with colorectal carcinoma or adenomatous polyps. To characterize the size of DNA isolated from urine, DNA was isolated from urine specimens that were collected from five patients (diagnosis not specified) using the Wizard DNA Isolation Kit and visualized by electrophoresis. To investigate the stability of DNA in urine, a specimen was collected from a volunteer (diagnosis not specified), mixed with EDTA (10 mM final concentration, pH 8.0), aliquoted, and stored on ice for 0, 2, 4, 8, or 24 h before freezing. DNA was isolated from the supernatant using the Wizard DNA Isolation Kit, and a short 81 bp fragment of KRAS and a “long” 400 bp fragment of alpha-fetoprotein were amplified by PCR and visualized by electrophoresis. To investigate the suitability of urine to detect KRAS mutations in colorectal cancer specimens, DNA was extracted from tumor, normal adjacent tissue, and urine specimens of a patient with colorectal cancer that harbored a mutation in KRAS codon 12. The codon 12 mutation was detected by amplification of KRAS codon 12 followed by melting analysis. The assay was further validated using DNA that was extracted from tumor containing paraffin-embedded tissue sections, urine, and plasma specimens from five colorectal carcinoma patients and fifteen patients with adenomatous polyps.
Summary of Findings:
High molecular weight DNA, as well as DNA of 150-250 bp, was present in the urine from all five patients. The abundance of the “long” PCR product was greater when urine was stored on ice for 8 h than in matched specimens that were stored on ice for ≤4 h., which the authors attribute to genomic DNA contamination from urinary tract cell lysis. Levels of the “short” PCR product were unchanged when urine was stored for up to 8 h on ice; however, the authors state that this likely reflects a slight loss of the short amplicon within the cell-free component given that an increase in amplification would be expected with the genomic contamination that was observed. Neither the short nor long PCR product was amplifiable from urine stored on ice for 24 h. The KRAS mutation was detectable in both the tumor and urine specimen, but only wildtype KRAS was detected in the n ormal adjacent specimen. IDuring validation, a KRAS mutation was detected in all five colorectal cancer tissue specimens and all five matched urine specimens, but only 2 of the matched plasma specimens. KRAS mutations were detected in 13 of the polyp specimens, with mutations detected in 10 and 8 of the matched urine and plasma specimens, respectively. Interestingly, one of the two polyp specimens without a KRAS mutation had a mutation detected in the matched plasma and urine specimens. The authors conclude that urine may be a more sensitive biospecimen type for colorectal cancer detection than plasma.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Neoplastic - Carcinoma
- Not specified
- Neoplastic - Benign
Platform:
Analyte Technology Platform DNA Electrophoresis DNA PCR Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Biospecimen location Plasma
Tissue
Urine
Tumor
Normal adjacent tissue
Preaquisition Diagnosis/ patient condition Colorectal carcinoma
Adenomatous polyps
Storage Storage duration 0 h
2 h
4 h
8 h
24 h
PCR Specific Length of gene fragment 80 bp
400 bp