We analyzed 87 sera collected from patients before the surgical treatment of gastric cancer (n = 49, 56%) and esophagogastric junction cancer (n=38, 44%) consecutive diagnosed in II Chair and Department of General and Gastrointestinal Surgery and Surgical Oncology of the Alimentary Tract I Lublin. The control group (compatible in terms of age and sex with the study group) consisted of 40 healthy donors. Moreover we analyzed corresponding available formalin-fixed paraffin-embedded (FFPE) tumor samples (n=70). Forty five (64%) tissue samples were taken before operation and without preoperative treatment and 25 (36%) samples were taken from patients who undergone neoadjuvant treatment (without trastuzumab) before surgery.
The study patients group consisted of 72 men (83%) and 15 women (17%). The median age of cancer patients was 62±8.7years (range: 39-75). Demographic and clinical data are presented in Table 1.
Circulating free DNA (cf-DNA) was isolated from serum samples of patients and healthy individuals with Quick-cfDNA Serum and Plasma DNA Miniprep Kit (Zymo Research, USA) according to manufacturers’ instructions. DNA from FFPE tissues was extracted using QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) according to manufacturers’ instructions. The quality and quantity of DNA was assessed using BioPhotometer UV/Vis Spectrophotometer (Eppendorf, Germany). Samples were stored in -20oC until qPCR (quantitative PCR) was performed.
HER2 CNV assessment by qPCR method
The HER2 gene copy number was determined by qPCR method. The internal control (housekeeping gene) was the RNaseP gene (Applied Biosystems, USA). PCR was performed using Illumina Eco Real-Time PCR equipment (Illumina Inc, San Diego, USA). PCR mixture contained: 5 µl of Genotyping MasterMix (Life Technologies, USA), 2 µl of DNA (5 ng/µl) and 0.5 µl of TaqMan CNV Assay (Hs02428732_cn, Applied Biosystems, USA) or internal control assay, and 3.5 µl of nuclease free water. Following conditions were applied: denaturation and enzyme activation: 95oC for 10 minutes, and 40 cycles: 95oC for 15 seconds, 62oC for 2 minutes. CNV was scored by 2-ΔΔCt method.
HER2 gene copy number analysis by FISH method
The PathVysion HER-2 DNA Probe Kit (PathVysion Kit, Abbot Molecular, USA) was used to detect HER2 gene amplification by fluorescence in situ hybridization technique. Paraffin-Pretreatment and Post-Hybridization Wash Buffer Kit (Abbot Molecular, USA) was also used for the pre-staining procedure. The ProbeChek HER-2/neu Normal Signal Ratio Slides (Abbot Molecular, USA) as well as ProbeChek HER-2/neu Control Slides (Abbot Molecular, USA) were used for each experiment. The paraffin sections of 3-5 μm thick were cut and mounted on positively charged glass slides. The unstained specimen and control slides were baked overnight at 64°C. Afterwards, the slides were immersed two times in xylene for 10 minutes and dehydrated twice in 100% ethanol for 5 minutes, and then dehydrated in 80% and 70% ethanol for 2 minutes each at ambient temperature and allowed to dry (app. 2 minutes). Then, the slides were incubated in 0.2 N HCl for 20 minutes and washed in purified water for 5 minutes. After removing excess water from slides, they were incubated for 30 minutes in Vysis Pretreatment Solution, which had been previously warmed to 80°C. Afterwards, the slides were incubated in purified water for 3 minutes and in Vysis Wash Buffer for 10 minutes. After removing excess wash buffer from slides, they were incubated for 30 minutes in Vysis Pepsine Solution previously warmed to 37°C. Then, the slides were washed in Vysis Wash Buffer for 10 minutes in 4°C, and in Vysis Wash Buffer for 10 minutes in room temperature. The slides were placed in a dark room. 10 μl of probe mixture was applied to a slide and immediately covered by a coverslip and sealed with rubber cement. The slides were placed for 5 minutes on a hotplate at 72°C and then at 37°C for overnight hybridization. At the end of the hybridization period, the rubber cement was removed from the slides and they were placed in Wash Buffer at ambient temperature to allow the coverslips to float off the slides. Afterwards, the slides were immersed for 5 minutes in Wash Buffer previously warmed at 74°C and air-dried in a dark room. 10 μl of DAPI counterstain was applied to the target area, covered by a coverslip, and the specimens were examined in fluorescence microscope (Nikon Eclipse 55i, Japan).
The PathVysion HER-2 DNA Probe Kit consists of two labeled DNA probes. The LSI (locus specific identifier) HER2 probe that spans the entire HER-2 gene is labeled in Spectrum Orange. The CEP17 probe is labelled in Spectrum Green and hybridizes to the alpha satellite DNA located at the centromere of chromosome 17 (17p11.1-q11.1). Inclusion of the CEP17 probe allows for determination of the relative copy number of the HER2 gene. We determined and recorded the number of LSI HER-2/neu and CEP17 counts in 20 nuclei in one region of interest (ROI) and this counting was repeated three times in different ROI. To calculate the final result, we used the following ratio (R): total signals from HER2 gene locus to total signals from CEP17 locus. The results were reported as follow: lack of HER2 gene amplification if the ratio was <2.2 and presence of HER2 gene amplification if the ratio was ≥2.2 .
Expression of HER2 protein examination by IHC method
Pre-diluted PATHWAY anti-HER-2/neu (clone 4B5) Rabbit Monoclonal Primary Antibody (Ventana, USA) was used for the semi-quantitative detection of HER2 antigen in sections of FFPE tissue on the fully automated VENTANA BenchMark IHC slide staining instrument. IHC staining procedures were performed according to the reagent kit manufacturer's recommendations. UltraView Universal DAB Detection Kit were used as a detection system. Hematoxylin counterstaining was incorporated in the staining protocol. As a negative control rabbit monoclonal negative control immunoglobulin was used (Ventana, USA). A semi-quantitative assessmet of HER2 protein expression was performed under light microscopy by two pathologists.
Statistical analysis was made using Statistica 13.1 software (TIBCO Software, USA). Pearson's chi-squared test of independence was used whether observations consisting of measures on variables are independent of each other. U-Mann-Whitney test was used to compare medians of analyzed parameters in different groups. Spearman correlation test was used to determine the relationship between particular factors in studied groups. ROC curve (Receiver Operating Characteristic) analysis was made to assess the utility of HER2 CNV assessment in liquid biopsy as a diagnostic test. The results were considered statistically significant at p<0.05.