Gastric cancer: immunohistochemical classification of molecular subtypes and their association with clinicopathological characteristics

The study population consists of 244 patients diagnosed with adenocarcinoma of the stomach, GOJ or distal oesophagus at the Turku University Hospital between years 1993 and 2012. The initial search in the clinical database of Auria Biobank retrieved tumour specimens from 437 patients. The exclusion criteria were carcinoma in situ (Tis, n = 23), insufficient sample material or indeterminate histology (n = 63), neuroendocrine histology (n = 3) and metastatic adenocarcinoma from a different organ (n = 6). This resulted in 190 patients with intestinal-type tumours and 152 patients with diffuse-type tumours. From these, all intestinal-type tumours and 54 representative diffuse-type tumours were included in the ngTMA. Among all of the patients, 11.9% (29/244) did receive preoperative chemotherapy (13 patients with intestinal-type and 16 with diffuse-type tumours). The type of surgery was total gastrectomy for 154 (63.1%) patients, subtotal gastrectomy or tumour resection for 72 (29.5%) patients and palliative surgery for 18 (7.4%) patients. The extent of surgery was determined as R0 (no residual tumour) for 180 (73.8%) patients, R1 (microscopic residual tumour) for 34 (13.9%) patients and R2 (macroscopic residual tumour) for 20 (8.2%) patients. The extent of surgery could not be determined for 10 (4.1%) patients. Tumour stage was assessed according to the current WHO Classification manual [10]. The median follow-up time of the patients was 125 months. All corresponding H&E slides have been reviewed for confirmation of diagnosis and adequacy of material. Relevant clinical information has been gathered from each case. The reporting of the study has been performed in compliance with the current recommendations [11]. The patient characteristics are presented in Table 1.

The ngTMA was created as follows [9]. The appropriate formalin-fixed paraffin-embedded (FFPE) tissue specimens were chosen based on clinical data and retrieved from the pathology archives. A representative haematoxylin-eosin (H&E) section containing areas of invasive carcinoma was selected from each tumour. New H&E slides were produced, scanned (Pannoramic P250, 3DHistech) and uploaded into the university digital microscopy web portal (casecenter.​utu.​fi). Each slide was viewed using Pannoramic Viewer software (3DHistech). Using the 1.0-mm annotation tool, annotations of different colours corresponding to various histological areas were placed onto each digital slide. Two annotations were placed in the centre of the tumour and two annotations in the periphery or invasive front of the tumour. The corresponding tissue cores were then transferred into the TMA blocks using an automated TMA instrument (TMA Grandmaster, 3DHistech) by overlaying each annotated digital slide with the corresponding tissue specimen. One tissue core containing benign tissue was selected from each tumour to act as a control. The constructed TMA blocks were sectioned, stained, scanned and uploaded into the web portal (casecenter.​utu.​fi), and each individual spot was scored by two pathologists (EB and NM). The resulting scores were combined with the clinical data for statistical analysis.

The link https://​seafile.​utu.​fi/​d/​7c4aa1964b/​ contains examples of our TMA results. The directory “TMA staining” contains low resolution images of all stainings performed on TMA block number 7, and “TMA map and score” contains the map and scores of the same TMA block. The directory “Example scoring” contains selected high resolution images of positive/negative and aberrant/wild-type stained tissue cores.

IHC reactions were performed on 4-μm paraffin sections of each tumour on the TMA slides with BenchMark XT (Ventana/Roche). For TP53, a ready-to-use antibody clone Bp53-11 (Ventana/Roche) was used, and the protocol included mild (30 min) CC1 pretreatment together with 28-min antibody incubation. Signal detection was performed with ultraView universal DAB Detection Kit (Ventana/Roche). For MLH1, an antibody clone G168-15 (BD Pharmingen) was used at 1:5 dilution together with standard (60 min) CC1 pretreatment and 36-min antibody incubation. The signal was detected with ultraView Universal DAB Detection Kit and amplification kit. For MSH2, an antibody clone G219-1129 (BD Pharmingen) was used at 1:200 dilution together with standard CC1 pretreatment and 28-min antibody incubation. The signal was detected with ultraView Universal DAB Detection Kit. For MSH6, an antibody clone EP49 (Epitomoc) was used at 1:200 dilution together with standard CC1 pretreatment and 32-min antibody incubation. The signal was detected with ultraView Universal DAB Detection Kit. For PMS2, a ready-to-use antibody clone EPR3947 (Ventana/Roche) was used together with extended (90 min) CC1 pretreatment, 44-min antibody incubation and 12-min HQ LINKER and HRP MULTIMER enhancements. The signal was detected with OptiView Universal DAB Detection Kit and amplification kit. For EBV, a ready-to-use EBER (EBV-encoded small RNA) probe (Ventana/Roche) was used together with ISH-Protease 3 pretreatment for 28 min and 1-h probe incubation. The signal was detected with ISH iVIEW Blue Detection Kit. For E-cadherin, an antibody clone NCH-38 (Agilent Technologies) was used at 1:100 dilution together with standard CC1 pretreatment and 32-min antibody incubation. The signal was detected with ultraView Universal DAB Detection Kit and amplification kit. A complete loss of or strong diffuse TP53 nuclear positivity was classified as aberrant TP53 expression. EBER ISH was scored either positive or negative according to the nuclear reaction. A tumour was classified as MSI if at least one of the markers (MLH1, MSH2, MSH6 and PMS2) showed a complete loss of nuclear reactivity together with positive background reaction in benign epithelium, smooth muscle cells and lymphocytes. Negative nuclear reactivity with negative background was considered controversial and not used for classification Loss of membranous reactivity or only faint cytoplasmic reaction was classified as aberrant E-cadherin expression (Fig. 1a). The methods for EGFR and HER2 IHC and silver in situ hybridisation (SISH) have been described previously [12‐15]. In short, with EGFR the scoring was based on the most intense membranous or membranous+cytoplasmic staining (0, negative; 1+, weak; 2+, moderate; 3+, strong). Specimens were classified as IHC high if showing 2+ or 3+ membranous or membranous+cytoplasmic staining intensity in ≥ 10% of tumour cells. With HER2 IHC, specimens showing 2+ or 3+ membranous staining in ≥ 10% of tumour cells were classified as IHC high. The IHC high samples were further analysed with SISH. The EGFR and HER2 IHC and SISH were performed on whole tissue sections.

Statistical analyses were performed with IBM SPSS Statistics for Windows, version 21.0 (IBM Corporation, Armonk, NY). Frequency table data were analysed using Pearson’s χ ² test or Fisher’s exact test for categorical variables and 2 × 2 tables were used to calculate odds ratios (OR). The Kaplan-Meier method and log-rank test as well as Cox’s proportional hazards regression model were used for univariate survival analysis. Multivariate survival analysis was performed by Cox’s proportional hazards regression model. Recurrence-free survival (RFS) was calculated from the time of diagnosis to the time of first recurrence, death of any cause or to the last follow-up date. Only recurrences ≥ 6 months after the time of diagnosis were considered relevant. Detection of a local or distant recurrence < 6 months from the diagnosis was considered likely to present an initially advanced disease. Patients without disease recurrence ≥ 6 months after diagnosis were considered curatively treated. Overall survival (OS) was calculated from the time of diagnosis to the time of death of any cause or the last follow-up date. Five patients (2.0%) who had received trastuzumab treatment for recurrent cancer were excluded from the OS analysis and additionally 13 patients with stage IV disease (5.3%) from the RFS analysis. All statistical tests were two-sided, and p values under 0.05 were considered statistically significant.

EBV, MSI and TP53 were analysed in 238 tumours and E-cadherin in 232 tumours. In the other tumours, the markers could not be evaluated due to insufficient tissue material. EBV RNA was found to be present in 17/186 (9.1%) of the intestinal tumours, while none of the diffuse tumours was EBV positive (Fisher’s exact test, p = 0.028; RR 0.91, 95% CI 0.87–0.95). MSI was detected in 19/186 (10.2%) of the intestinal tumours, while none of the diffuse tumours was found to have MSI phenotype (Fisher’s exact test, p = 0.017; RR 0.90, 95% CI 0.86–0.94). Aberrant TP53 expression was observed to be significantly more common among intestinal-type (103/186, 55.4%) than diffuse-type tumours (10/52, 19.2%) (Fisher’s exact test, p < 0.0001; OR 5.21, 95% CI 2.47–11.0). Ninety-four tumours (39.5%) were found to be EBV negative, MSS and TP53 wild-type. Of these, 52/186 (28.0%) tumours had intestinal-type and 42/52 (80.8%) tumours had diffuse-type histology (Fisher’s exact test, p < 0.0001; OR 0.09, 95% CI 0.04–0.20). None of the MSI tumours had both EBV positivity and aberrant TP53 expression. Among the intestinal-type tumours, 3/183 (1.6%) tumours had aberrant E-cadherin expression, whereas among the diffuse-type tumours, aberrant E-cadherin expression could be seen in 25/49 (51.0%) tumours. Among the EBV negative, MSS and TP53 wild-type tumours, 21/39 (53.8%) of the diffuse-type tumours but none of the intestinal-type tumours (n = 51) had aberrant E-cadherin expression.

Among the intestinal-type tumours, aberrant TP53 expression was more common in EBV negative than in EBV positive tumours (Fisher’s exact test, p < 0.0001; OR 0.041, 95% CI 0.01–0.32). Tumours with aberrant TP53 expression were also more frequently MSS than MSI (Fisher’s exact test, p = 0.003; OR 5.46, 95% CI 1.74–17.2). No association was found between aberrant E-cadherin expression and either EBV, TP53 or MSI status. Among the diffuse-type tumours, testing for statistical significance was not applicable for EBV or MSI (Table 2, Fig. 1b).

EGFR and HER2 protein expression levels were evaluated in 183 intestinal-type adenocarcinomas. Moderate/strong EGFR protein expression was found in 59 (32.2%) of the tumours and 25 (13.7%) of the tumours had moderate/strong HER2 protein expression. Among these, EGFR gene amplification was detected in 27/59 (14.8% of the whole study material) tumours and HER2 gene amplification in 24/25 (13.1% of the whole study material) tumours. No significant associations were observed between EGFR/HER2 protein expression level or gene amplification and TP53/EBV/MSI status. The majority of the EGFR (n = 17) or HER2 (n = 15) gene amplifications were found in the group of EBV negative and MSS tumours with aberrant TP53. Most of the co-amplifications (n = 5) were also found in this subgroup (Fig. 1b). The co-localisation of aberrant TP53 expression and either EGFR or HER2 gene amplification was detected more often in the proximal (distal oesophagus/GOJ/cardia) than distal (corpus/antrum/pylorus) intestinal-type tumours (Fisher’s exact test, p = 0.019; OR 2.83, 95% CI 1.21–6.61).

Among the intestinal-type tumours, aberrant TP53 expression was more frequent in proximal than distal tumours (Fisher’s exact test, p = 0.002; OR 2.71, 95% CI 1.47–5.00). In contrast, MSI phenotype was more frequent in distally located tumours (Fisher’s exact test, p = 0.003; OR 7.10, 95% CI 1.59–31.7). EBV positive tumours were more common among male than female patients (Fisher’s exact test, p = 0.035; OR 4.57, 95% CI 1.01–20.6), whereas MSI tumours were more common among female than male patients (Fisher’s exact test, p = 0.042; OR 2.89, 95% CI 1.07–7.36). EBV positive tumours were also more often poorly differentiated than well or moderately differentiated (Fisher’s exact test, p < 0.0001; OR 0.08, 95% CI 0.02–0.37) and most often located in the gastric corpus (Fisher’s exact test, p = 0.011). No significant associations were observed between the EBV negative/MSS/TP53 wild-type tumours and the examined clinicopathological variables. Among diffuse-type tumours, no significant associations were found with the examined variables (Table 3).

In univariate survival analysis for intestinal tumours, the presence of MSI was associated with longer overall survival (OS, median) (124.6 versus 28.7 months, log-rank test, p = 0.040; Cox test, p = 0.043, HR 0.54, 95% CI 0.30–0.98) but not with recurrence-free survival (RFS). In intestinal tumours, increasing depth of tumour invasion was associated with shorter RFS and OS (RFS log-rank test, p = 0.045; Cox test, p = 0.046, HR 1.53, 95% CI 1.01–2.32; OS log-rank test, p = 0.030; Cox test, p = 0.031, HR 1.54, 95% CI 1.04–2.28). Similarly, increasing tumour stage was associated with shorter RFS and OS (RFS log-rank test, p = 0.019; Cox test, p = 0.020, HR 1.56, 95% CI 1.07–2.26; OS log-rank test, p < 0.0001; Cox test p < 0.0001, HR 1.84, 95% CI 1.32–2.57). In addition, patient age above median at the time of diagnosis was associated with shorter RFS and OS (RFS log-rank test, p = 0.006; Cox test, p = 0.006, HR 1.67, 95% CI 1.16–2.42; OS log-rank test, p = 0.026; Cox test p = 0.027, HR 1.46, 95% CI 1.04–2.03). No significant associations were observed between TP53 or EBV status and survival.

The multivariate model for OS included the following variables: patient age at diagnosis (below versus above median, median 72.3 years), postoperative T (T1–T2 versus T3–T4), tumour stage (I–II versus III–IV) and MMR status (MSS versus MSI, for intestinal tumours only). Among intestinal tumours, MSI status was found to be predictive for longer OS (Cox test, p = 0.015, HR 0.46, 95% CI 0.25–0.86) while patient age above median (Cox test, p = 0.009, HR 1.57, 95% CI 1.12–2.21) and increasing tumour stage (Cox test, p = 0.036, HR 1.50, 95% CI 1.03–2.18) were predictive for shorter OS. In diffuse-type tumours, patient age above median remained as a single predictive factor for shorter OS (Cox test, p = 0.030, HR 2.29, 95% CI 1.08–4.83) (Table 4).