Real-time PCR quantification of plasma DNA in non-small cell lung cancer patients and healthy controls

Szpechcinski A, Dancewicz M, Kopinski P, Kowalewski J, Chorostowska-Wynimko, Chorostowska-Wynimko J

Description

Free-circulating DNA is present in minute amounts in plasma of healthy individuals, whereas increased levels are found in a number of malignant pathologies including non-small cell lung cancer (NSCLC). The objective of this research was the evaluation of the plasma DNA quantification capacity to distinguish between healthy subjects and non-small cell lung cancer (NSCLC) patients. Materials and methods Plasma samples were collected prospectively from 16 healthy volunteers and 30 untreated NSCLC patients (I-IIIA). Subsequently, free-circulating DNA extraction and quantitative real-time PCR analysis were performed. Results The values of plasma DNA concentration ranged from 0.9 up to 7.0 ng/ml in healthy individuals and from 1.5 up to 50 ng/ml in NSCLC patients before treatment. Cancer group showed several-fold higher mean free-circulating DNA concentration than that present in healthy subjects (mean 12.00 vs. 2.65 ng/ml; P < 0.001). A greater variability of plasma DNA concentrations was observed in NSCLC patients than in controls (SD 14.50 vs. 2.02, respectively). The area under the ROC curve was 0.87 (95% CI, 0.744 to 0.954, P < 0.001). Conclusion Non-small cell lung cancer is associated with elevated levels of cell-free DNA in plasma with respect to healthy controls. Real-time PCR method proved its utility in effective free-circulating DNA detection and quantification.

Sujets

• Lung cancer
• Plasma
• PCR en temps réel

Informations

• Publié par: biomed
• Publié le: 01 janvier 2009
• Nombre de lectures: 5
• Langue: English

Extrait

December 7, 2009

EUROPEAN JOURNAL OF MEDICAL RESEARCH

Eur J Med Res (2009) 14(Suppl. IV): 237-240

237

© I. Holzapfel Publishers 2009

R EAL - TIME PCR Q UANTIFICATION OF P LASMA DNA IN N ON - SMALL C ELL L UNG C ANCER P ATIENTS AND H EALTHY C ONTROLS

1 23 21 A. Szpechcinski , M. Dancewicz , P. Kopinski , J. Kowalewski , J. Chorostowska-Wynimko

1 Laboratory ofMolecular Diagnostics and Immunology, National Institute ofTuberculosis and Lung Diseases, Warsaw, Poland; 2 3 Department ofThoracic Surgery and Tumors, Center ofOncology andChair Gene Therapy, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz

Abstract Intr oduction:Free-circulating DNA is present in minute amounts in plasma ofhealthy individuals, whereas increased levels are found in a number ofma- lignant pathologies including non-small cell lung can- cer (NSCLC). The objective ofthis research was the evaluation ofthe plasma DNA quantification capacity to distinguish between healthy subjects and non-small cell lung cancer (NSCLC) patients. Material and methods:Plasma samples were collected prospectively from 16 healthy volunteers and 30 un- treated NSCLC patients (I-IIIA). Subsequently, free- circulating DNA extraction and quantitative real-time PCR analysis were performed. Results:The values ofplasma DNA concentration ranged from 0.9 up to 7.0 ng/ml in healthy individuals and from 1.5 up to 50 ng/ml in NSCLC patients be- fore treatment. Cancer group showed several-fold higher mean free-circulating DNA concentration than that present in healthy subjects (mean 12.00 vs. 2.65 ng/ml; P<0.001). A greater variability ofplasma DNA concentrations was observed in NSCLC patients than in controls (SD 14.50 vs. 2.02, respectively). The area under the ROC curve was 0.87 (95% CI, 0.744 to 0.954, P<0.001). Conclusion:Non-small cell lung cancer is associated with elevated levels ofcell-free DNA in plasma with respect to healthy controls. Real-time PCR method proved its utility in effective free-circulating DNA de- tection and quantification.

Key words:DNA quantification, free-circulating DNA, lung cancer, plasma, real-time PCR

I NTRODUCTION

The discovery ofextracellular DNA circulating in plasma, serum, and other body fluids influences can- cer genomics, as it allows non-invasive access to genet- ic material originating form diseased cells and tissues. Blood is easily and cheaply accessible material and its proper sampling doesn’t require additional personnel training, therefore it is ideal for extensive research projects on the clinical value offree-circulating DNA in lung cancer [1]. Free-circulating DNA is present in minute amounts in the plasma ofhealthy individuals. Increased levels

of circulatingDNA have been found in a number of pathologies, such as cancer, stroke, trauma, myocardial infarction, autoimmune disorders, chronic inflamma- tion, and pregnancy-associated complications [2]. The precise mechanism by which DNA is released into the bloodstream remains unknown. Recent reports demonstrated that a significant proportion ofcirculat- ing DNA is ofhematopoietic origin in healthy as well as in diseased individuals [3]. However, the detection of commongenetic (e.g., mutations, microsatellite al- terations) and epigenetic (e.g. promoter methylation) modifications in tumors and paired plasma/serum samples evidenced that in cancer patients part ofthe free-circulating DNA is oftumor origin [4]. It is believed that free-circulating DNA, by its ap- parent linkage with altered cell proliferation, apopto- sis, necrosis, angiogenesis and other cancer-related phenomena, might provide us with valuable biomark- ers for lung cancer diagnostics and therapy monitor- ing. Recent findings on correlation ofplasma DNA levels in lung cancer patients with disease stage, cancer histopathology, disease progression rate and response to therapy evidence the importance ofquantitative studies on free-circulating DNA [5]. Here, we present results ofreal-time qPCR plasma DNA measurement in non-small cell lung cancer patients and healthy con- trols as the promising introduction to further investi- gations.

M ATERIAL AND M ETHODS C OLLECTION OF P LASMA S AMPLES

Blood samples were collected from 16 healthy volun- teers and 30 patients with resectable (I-IIIA) non- small cell lung cancer (NSCLC) before any treatment. Blood was collected to 9-ml tubes containing EDTA as anticoagulant agent and processed within 1 h.Plas- ma was then separated from the cellular fraction by two centrifugations at 1000 x g for 10 min in 4ºC, and banked at -80ºC.

E XTRACTION AND R EAL - TIME PCR Q UANTIFICATION OF P LASMA DNA

DNA was extracted from 0.5 ml plasma aliquots with QIAmp DNA Blood Midi kit (Qiagen, Germany) ac-