Case series

103 healthy donors enrolled from the Blood Transfusion Unit of Morgagni-Pierantoni Hospital in Forlì, and 128 non-small cell lung cancer patients recruited from the Department of Diseases of the Thorax of the same hospital were analysed.

Median age was 69 (range 38-86) years for patients and 63 years (range 32-85) for controls. Of the 128 patients, 92 were male and 36 were female, while of the 103 healthy donors, 68 were male and 35 were female. 65 tumours were adenocarcinoma (ADC), 52 squamous cell carcinoma (SCC), 6 large cell carcinoma and 5 poorly differentiated carcinoma. On the basis of TNM classification, 50 tumours were stage I, 22 were stage II, 25 were stage III and 31 were stage IV. Among patients, 31 had never smoked and 97 were either current smokers (n = 61) (at least 20 packs of cigarette/year) or had stopped smoking a few years previously (n = 36). Among healthy donors, 34 had never smoked and 69 were either current smokers (n = 45) or had stopped smoking some years previously (n = 24).

Blood samples were taken after obtaining informed consent from all individuals. The study protocol was reviewed and approved by the local Ethics Committee.

Methodologies

Determination of free circulating DNA

DNA was extracted from 1 ml of serum by QIamp DNA Mini Kit (Qiagen, Hilden, Germany) and then quantified using a Real-Time quantitative PCR assay based on SYBR Green I dye chemistry and MyiQ Single Color Real-Time PCR Detection System (BioRad, Hercules, California, USA). DNA amplification was performed using the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) (sequence primers were 5’- ACC CAG AAG ACT GTG GAT GG - 3’ and 5’- TTC AGC TCA GGG ATG ACC TT- 3’), and the absolute concentration of target DNA was calculated on a standard curve using concentrations ranging from 25 to 0.01 ng of DNA from the peripheral blood of a healthy donor.

RNA extraction and COX-2 gene amplification

2.5 ml of peripheral blood from controls and patients was collected in PAX-Gene blood RNA tubes (Qiagen), and RNA was extracted by PAX-Gene blood RNA kit (Qiagen). 500 ng of RNA was reverse-transcribed and Real-Time PCR was performed using the MyiQ Single Color Real-Time PCR Detection System (BioRad) and SYBR Green I dye chemistry. The stably expressed endogenous b2-microglobulin gene was amplified as a control for quality and quantity of input RNA.

Sequences of primers were forward 5’- CGC TAC TCT CTC TTT CTG GC - 3’ and reverse 5’- AGA CAC ATA GCA ATT CAG GAA AT -3’ for b2-microglobulin, and forward 5’- GTT CTC CTG CCT ACT GGA AGC C -3’ and reverse 5’- GCT CTG GAT CTG GAA CAC TGA ATG-3’ for COX-2.

The relative expression of COX-2 mRNA was obtained using Gene Expression Macro Software (Version 1.1) (BioRad) employing an optimised comparative Ct (∆∆Ct) value method.

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