Application of COLD-PCR for improved detection of KRAS mutations in clinical samples.
KRAS mutations have been detected in approximately 30% of all human tumors, and have been shown to predict response to some targeted therapies. The most common KRAS mutation-detection strategy consists of conventional PCR and direct sequencing. This approach has a 10-20% detection sensitivity depending on whether pyrosequencing or Sanger sequencing is used. To improve detection sensitivity, we compared our conventional method with the recently described co-amplification-at-lower denaturation-temperature PCR (COLD-PCR) method, which selectively amplifies minority alleles. In COLD-PCR, the critical denaturation temperature is lowered to 80 degrees C (vs 94 degrees C in conventional PCR). The sensitivity of COLD-PCR was determined by assessing serial dilutions. Fifty clinical samples were used, including 20 fresh bone-marrow aspirate specimens and the formalin-fixed paraffin-embedded (FFPE) tissue of 30 solid tumors. Implementation of COLD-PCR was straightforward and required no additional cost for reagents or instruments. The method was specific and reproducible. COLD-PCR successfully detected mutations in all samples that were positive by conventional PCR, and enhanced the mutant-to-wild-type ratio by >4.74-fold, increasing the mutation detection sensitivity to 1.5%. The enhancement of mutation detection by COLD-PCR inversely correlated with the tumor-cell percentage in a sample. In conclusion, we validated the utility and superior sensitivity of COLD-PCR for detecting KRAS mutations in a variety of hematopoietic and solid tumors using either fresh or fixed, paraffin-embedded tissue.
Zuo
Zhuang
Z
Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, Texas 77054, USA.
Chen
Su S
SS
Chandra
Pranil K
PK
Galbincea
John M
JM
Soape
Matthew
M
Doan
Steven
S
Barkoh
Bedia A
BA
Koeppen
Hartmut
H
Medeiros
L Jeffrey
LJ
Luthra
Rajyalakshmi
R
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D003080:Cold Temperature; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins