Between January 2015 and August 2022, 750 patients who underwent PET/CT with a diagnosis or suspicion of gastric adenocarcinoma in our hospital were retrospectively screened. A total of 117 patients, including 83 males and 34 females, were included in the study. The inclusion criteria were confirmed pathological diagnosis of gastric adenocarcinoma, HER2 test analysis, and no prior treatment before imaging. Staging was performed based on the TNM classification for carcinoma of the stomach according to the 8th edition of the American Joint Committee on Cancer guidelines [12]. In patients with, total resection material surgical staging was possible. Patients with distant metastasis were classified as stage 4.
Patients with a second primary malignancy and those who did not undergo HER2 testing were excluded from the study.
18F- FDG PET/CT examination included the evaluation of SUVmax, SUVmean, metabolic tumor volume (MTV), total lesion glycolysis (TLG), and the presence of distant metastases. Pathological data were examined, including HER2 status, perineural invasion, local invasion, vascular invasion, tumor size and location, lymph node metastasis, histological subtype, and histological component. Age and gender were obtained by the institutional records system, and patient survival status and date of death were obtained from the national death notification system (DNS).
Histopathological Method:
All specimens underwent HER-2 immunohistochemical (IHC) staining. Cases with suspicious HER-2 scores (score 2) and positive scores (score 3) were further investigated for HER-2 gene amplification using the dual silver in situ hybridization (SISH) method.
Immunohistochemistry:
Two-micron thick sections were obtained from formalin-fixed and paraffin-embedded tissues. The sections were placed on positively charged electrostatic slides (Isotherm Technical Laboratory Glass Materials) and incubated at 58ºC for approximately 4 hours until the paraffin dissolved. All sections were placed in an automated IHC staining device (Ventana, Benchmark, Ultra). Anti-HER-2/neu (4B5) rabbit monoclonal antibody (Ventana, Catalog No. 790–2991) and the compatible Ultraview Universal DAB Detection Kit were used for IHC staining.
In-situ Hybridization (ISH):
Two-micron thick sections obtained from paraffin-embedded tissues were placed on positively charged electrostatic slides (Isotherm Technical Laboratory Glass Materials) and incubated at 58ºC for approximately 4 hours until the paraffin dissolved. All sections were then transferred to an automated staining device (Ventana, Benchmark, Ultra) and the Ventana inform HER2 Dual ISH Automated System was applied. The Ultraview SISH DNP Detection Kit was used for HER-2 gene detection, resulting in black signals through silver deposition, while the Ultraview Red ISH DIG Detection Kit was used for chromosome 17, resulting in red signals using Naftol and Fast Red.
IHC Evaluation:
Hofmann et al. published the scoring system for the HER-2 (4B5 clone) IHC marker evaluation [13, 14]. The staining pattern, location, intensity, and extent were considered. Membranous staining with a complete/basolateral/lateral pattern was considered positive. The negative evaluation was given to cytoplasmic, nuclear, luminal, or basal staining. The results were reported as negative (score 0 and score 1), suspicious (score 2), and positive (score 3).
ISH Evaluation:
For cases with suspicious (score 2) and positive (score 3) HER-2 IHC results, HER-2 gene amplification was investigated using the dual silver in situ hybridization (SISH) method [15, 16]. In the identified neoplastic cell group, gene and chromosome signals were counted in 40 consecutive cells at x40 magnification. Cases with an average HER-2 gene copy number ≥ 6 or an HER-2 gene/chromosome 17 ratio of 2 and above were considered positive. Those with an HER-2 gene/chromosome 17 ratio below 2 were considered negative. Cells showing both gene and chromosome signals were included in the evaluation. Amplified tumor cells with overlapping gene signals were assessed by comparing them to a normal signal size. Signals touching each other or closer than one signal size were considered "paired signals" and counted as a single signal. In samples with an expected signal in the internal control cells, multiple overlapping signals in tumor cells that could not be counted separately were considered clusters. A small cluster was considered as 6 signals, and a large cluster as 12 signals. Cells without signals, cells with signals in a single color, and signals observed outside the cells were not included in the evaluation.
18 F-FDG PET/CT Imaging Technique:
After fasting for a minimum of six hours, all patients underwent PET/CT, and their blood sugar levels were assessed. An intravenous dose of 8–12 mCi (29–44 MBq; or approximately 8.1 MBq of FDG per kilogram of body weight) of 18F-FDG was administered when the serum glucose level was less than 200 ng/dl. Whole-body PET/CT imaging was performed using a full-ring high-resolution (HI-REZ) LSO PET and a six-slice CT scanner (Siemens Biograph 6, Chicago, IL, USA). The procedure included a non-enhanced CT scan followed by positron emission tomography scanning. The PET image datasets were created using the ordered subsets expectation maximization approach combined with CT-based attenuation correction was used to iteratively recreate PET image datasets.
18F-FDG PET/CT Image Analysis:
A General Electric Advantage Workstation (AW workstation Volume Viewer 3 software; GE Healthcare, Waukesha, WI, USA) was used for all image analysis. In three planes (transaxial, coronal, and sagittal), the maximum intensity projection and attenuation-corrected PET/CT fusion images were assessed. The tumor’s maximum SUV, mean SUV(SUVmean), MTV, and TLG were noted. Using the formula [injected dose(MBq) body weight (g)], maximum SUV were determined based on body weight. To determine MTV, the tumor contours were semi-automatically identified by employing a threshold of 42% of the SUVmax within the lesion. The SUVmean was multiplied by the MTV to compute total lesion glycolysis. A volumetric zone of interest was defined to encompass the main tumor completely. On checking the sagittal and coronal images, the volumetric region of interest border was semi-automatically adjusted if the volume extended beyond the borders of the primary lesion.
Ethics Committee Approval:
The study was authorized by the local ethics committee of Prof. Dr. Cemil Tascioglu City Hospital in Istanbul, Turkey (2023/19), and informed consent was received from all patients who participated in this study. All procedures in human-participant studies were carried out in compliance with the institutional research committee's ethical standards and the Helsinki Declaration of 1964.
Statistical Analysis:
SPSS 25.0 for Windows software was used for statistical analysis. Descriptive statistics were provided for categorical variables in terms of frequency and percentage, and for numerical variables in terms of mean, standard deviation, minimum, maximum, and median. The proportions between groups were compared using the Chi-square test. Since the numerical variables did not meet the assumption of normal distribution, independent group comparisons were conducted using the Mann-Whitney U test, and multiple group comparisons were conducted using the Kruskal-Wallis test. A statistical alpha significance level of p < 0.05 was accepted.