Study design
This study included 45 patients with non-distant metastatic pathologically proven gastric carcinoma. The study was carried out in the Pathology, Clinical Pathology, and Clinical Oncology Departments during the period between January 2015 and December 2016. Patients were followed up until December 2018.
Patient characteristics and inclusion criteria
All included patients were free of distant metastases at the beginning of the study. Patients have age between 18 and 70 years, Karnofsky performance status ≥ 70, adequate bone marrow reserve (hemoglobin ≥ 10 g/dL, white blood cell count ≥ 3.5 × 109/L, and platelets ≥ 100 × 109/L), and good renal function (creatinine clearance ≥ 60 mL/min).
Patients were excluded from this study if they had metastases, altered mental status, dementia, or any psychiatric condition that affects understanding and impedes informed consent. Also, we excluded patients who had secondary malignancy or non-malignant systemic disease that precluded them from receiving chemotherapy (e.g., uncontrolled active infection, persistent immune-compromised states, congestive heart failure, any clinically significant cardiac arrhythmia). Patients who were pregnant and with clinically significant pleural effusions or ascites were also excluded from this study.
The protocol was approved by the Institutional Ethics Committee, and before the initiation of any treatment, all patients signed an informed consent.
Treatment protocol and follow-up
All patients had undergone surgery with lymph node dissection and received more than four cycles of adjuvant chemotherapy. The regimen of chemotherapy was fluorouracil and/or cisplatin/oxaliplatin which consisted of 2000 mg/m2 (days 1 and 2) fluorouracil IV continuous infusion over 48 h and 50 mg/m2 (IV day 1) cisplatin, and this cycle was repeated every 14 days or oxaliplatin 85 mg/m2 (IV day 1), leucovorin 400 mg/m2 (IV day 1), fluorouracil 400 mg/m2 (IV push day 1), and fluorouracil 1200 mg/m2 (IV day 1, 2) continuous infusion over 24 h cycled every 14 days. Supportive care as growth factors, blood transfusions, and administration of antiemetics and analgesics were included, while prophylactic use of growth factors was not recommended.
The follow-up program consisted of physical examination and regular abdominal CT scan every 3–6 months for the first 2 years after operation. TNM stages were classified according to the American Joint Committee on Cancer (AJCC) [8].
Tissue samples
From each participant in this study, gastric tissue specimens obtained by the surgical excision were sent to the Pathology Department for histopathological evaluation and immunohistochemical (IHC) staining. Samples from tumor center and adjacent non-cancerous tissue (at least 5 cm from the tumor) were then stored frozen at − 80 °C till genetically investigated [9].
Histopathologic evaluation
Gastric carcinoma specimens were fixed in 10% neutral buffered formalin then paraffin blocks were prepared. Examination of hematoxylin and eosin (H&E)-stained sections was carried out to confirm the diagnosis of GC. Cases were histologically classified and graded according to the World Health Organization (WHO) [10].
Immunohistochemical staining
Sections from GC tissue, on positively charged slides, were dried for 30 min at 37 °C. Deparaffinization and antigen retrieval were performed in a Dako PT Link unit. Both high and low pH EnVisionTM FLEX Target Retrieval Solutions were used reaching 97 °C for 20 min. Dako Autostainer Link 48 automated slide stainer was used for immunostaining. We used Oct4 mouse monoclonal antibody (clone MRQ-10, 1:30 dilution, Cell Marque, Rocklin, CA, USA), Ki-67 mouse monoclonal antibody (clone MIB-1, 1:100 dilution, Dako, Glostrup, Denmark), and VEGF mouse monoclonal antibody (M7273, 1:50 dilution, Dako, Glostrup, Denmark). Shortly, the slides were incubated with primary antibodies for 20–30 min following treatment with peroxidase-blocking reagent for 5 min then incubation with horseradish peroxidase (HRP) polymer reagent for 20 min and diaminobenzidine (DAB) chromogen/substrate working solution for 10 min. Hematoxylin was applied for counterstaining.
Evaluation of immunohistochemical staining
Oct4 expression was detected as a nuclear staining in gastric carcinoma cells. Oct4 was scored by multiplying the percentage of positive tumor cells and the staining intensity [11]. As regards Oct4 percentage, no positive tumor cells were graded 0; < 10% positive tumor cells, 1; 10–50% positive tumor cells, 2; and > 50% positive tumor cells, 3. Staining intensity was scored as follows: 0, no staining; 1, weak staining; 2, modest staining; and 3, strong staining. The final scores obtained were 0, 1, 2, 3, 4, 6, and 9. Tumors with scores ≤ 4 were considered low expression while scores ≥ 6 were regarded as high expression.
Positive Ki-67 expression was defined as brownish staining in the nuclei of 10% or more of tumor cells [12]. Cytoplasmic VEGF staining was regarded as positive when the percentage of stained tumor cell was 10 or more [13].
Quantitative reverse transcription PCR
RNA extraction
The RNA was extracted from each stored frozen gastric tissue using RNA extraction kit (RNeasy mini kit, Qiagen, Hilden, Germany, Catalog no 74104) according to the manufacturer’s protocol. RNA yields were assayed quantitatively by measuring the absorbance at 260 nm on Jenway UV/Visible Spectrophotometer 6305, Staffordshire, UK.
Reverse transcription
The RNA yields were subjected to reverse transcription into cDNA using QuantiTect® Reverse Transcription (Qiagen, Hilden, Germany, Catalog no 205311), where the entire genomic DNA elimination reaction (14 μL) containing 500 ng of the template RNA mixed with 1 μL of Quantiscript Reverse Transcriptase, 4 μL of Quantiscript RT Buffer 5×, and 1 μL of RT primer mix, and incubated for 15 min at 42 °C then inactivated for 3 min at 95 °C according to the manufacturer’s instructions.
Relative quantitation Oct4 mRNA expression
RT-PCR amplifications with relative quantitation of Oct4 mRNA expression were performed using TaqMan gene expression assay kit (Thermo Scientific, Waltham, MA, USA). In a 20-μL total volume, mixture of 10.0 μL of 2× TaqMan® Universal PCR Master Mix II and 1.0 μL of 20× gene expression assay mix containing Oct4 primers (forward primer: 5-AGCAAAACCCGGAGGAGT-3; reverse primer: 5-CCACATCGGCCTGTGTATATC-3), with FAM-labeled probe (5-FAM-TGCAGGCCCGAAAGAGAAAGCG-3) and 5 μL of cDNA template (equivalent to 25 ng RNA), together with endogenous control (GAPDH) assay was used for each sample (forward primer: 5-ACCACAGTCCATGCCATCCAC-3; reverse primer: 5-TCCACCACCCTGTTGCTGTA-3). The plate was applied on Real-Time PCR System (Applied Biosystems, step I version) with the following thermal profile: hold at 95 °C for 10 min followed by 40 cycles (denaturation 95 °C for 15 s and annealing/extension at 60 °C for 1 min). The cycle threshold (CT) was obtained for the gene using Applied Biosystems, step I version, software analysis modules, and the expression of the gene was relatively quantified using the equation 2−ΔΔCt [14].
Statistical analysis
Statistical analysis was performed using Statistical Package for Social Science (SPSS version 23). Data were expressed as frequencies for categorical variables whereas continuous variables were expressed as mean ± SD or median and range. For comparing categorical variables, chi-square (χ2), Fisher’s exact, and Monte Carlo tests were applied. Continuous variables were compared using Student t test for normally distributed data, whereas Mann-Whitney and Kruskal-Wallis tests were performed for non-normally distributed ones. For survival analysis, overall survival (OS) rates were calculated as the interval between the date of diagnosis and the date of death or the last follow-up. Disease-free survival rates were calculated from the date of diagnosis to the date of disease recurrence and/or distant metastasis. Survival curves were built up using Kaplan-Meier method, and the exact log-rank test was used to evaluate the significance of the differences between the groups. p values of < 0.05 were considered statistically significant.
