Optimization of circulating cell-free DNA recovery for KRAS mutation and HPV detection in plasma.
BACKGROUND
The precise analysis of tumour markers in blood such as circulating cell-free DNA (cfDNA) could have a significant impact in facilitating monitoring of patients after initial therapy. Although high levels of total cfDNA in plasma of cancer patients are consistently demonstrated, a low sensitivity of DNA alterations is reported.


OBJECTIVE
The major question regards the recovery of tumour-specific cfDNA such as KRAS mutated DNA and cancer-associated type 16 of human papillomavirus (HPV16).


METHODS
TaqMan technology was used for detection of KRAS mutation, HPV16 and to quantify cfDNA in blood plasma.


RESULTS
Comparison of four different column-based commercial kits shows that the cfDNA purification carried out by the Genomic Mini AX Body Fluids kit and the QIAamp Circulating Nucleic Acid kit gave us the possibility to improve the sensitivity of detection of KRAS mutation and HPV16. The optimized method was used to follow the reduction in cancer-specific cfDNA after therapy. We found that large volume extractions with low volume of DNA eluate enabled trace amounts of tumour-specific cfDNA from cancer patients to be effectively identified.


CONCLUSIONS
Data presented in this study facilitate detection of tumour-specific cfDNA and improve standards needed for the implementation of cfDNA technology into routine clinical practice.
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
Agnieszka M
AM
Mazurek
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
A
A
Fiszer-Kierzkowska
I Radiotherapy Clinic, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
T
T
Rutkowski
I Radiotherapy Clinic, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
K
K
Składowski
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
M
M
Pierzyna
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
D
D
Scieglińska
Department of Radiotherapy, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
G
G
Woźniak
Department of Radiotherapy, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
G
G
Głowacki
Department of Radiotherapy, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
R
R
Kawczyński
Center for Translational Research and Molecular Biology of Cancer, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Gliwice Branch, Gliwice, Poland.
E
E
Małusecka
D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D002289:Carcinoma, Non-Small-Cell Lung / Q000097:blood / Q000235:genetics / Q000821:virology; D002853:Chromatography, Liquid; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000097:blood / Q000235:genetics / Q000302:isolation & purification*; D006258:Head and Neck Neoplasms / Q000097:blood / Q000235:genetics / Q000821:virology; D052162:Human papillomavirus 16 / Q000235:genetics*; D006801:Humans; D008175:Lung Neoplasms / Q000097:blood / Q000235:genetics / Q000821:virology; D020125:Mutation, Missense; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins / Q000235:genetics*
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D004279:DNA, Viral; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins
Circulating cell-free DNA; KRAS mutation; cancer; human papillomavirus; isolation method; plasma