Epstein-Barr virus positivity (EBV(+)) and high-microsatellite instability (MSI-H) have been identified as molecular subgroups in gastric carcinoma. The aim of our study was to determine the prognostic and predictive relevance of these subgroups in the context of platinum/5-fluorouracil (5-FU) based preoperative chemotherapy (CTx). Additionally, we investigated the clinical relevance of the low-MSI (MSI-L) phenotype. We analysed 760 adenocarcinomas of the stomach or the gastro-oesophageal junction encompassing 143 biopsies before CTx and 617 resected tumours (291 without and 326 after CTx). EBV was determined by PCR and in situ hybridisation for selected cases. MSI was analysed by PCR using five microsatellite markers and classified as MSI-H and MSI-L. Frequencies of EBV(+), MSI-H and MSI-L in the biopsies before CTx were 4.2, 10.5 and 4.9% respectively. EBV(+) or MSI-H did not correlate with response, but MSI-L was associated with better response (p = 0.011). In the resected tumours, frequencies of EBV(+), MSI-H and MSI-L were 3.9, 9.6 and 4.5% respectively. Overall survival (OS) was significantly different in the non-CTx group (p = 0.014). Patients with EBV(+) tumours showed the best OS, followed by MSI-H. MSI-L was significantly associated with worse OS (hazard ratio [HR], 2.21; 95% confidence interval [CI], 1.21-4.04, p = 0.01). In the resected tumours after CTx, MSI-H was also associated with increased OS (HR, 0.54; 95% CI, 0.26-1.09, p = 0.085). In multivariable analysis, molecular classification was an independent prognostic factor in the completely resected (R0) non-CTx group (p = 0.035).In conclusion, MSI-H and EBV(+) are not predictive of response to neoadjuvant platinum/5-FU based CTx, but they are indicative of a good prognosis. In particular, MSI-H indicates a favourable prognosis irrespective of treatment with CTx. MSI-L predicts good response to CTx and its negative prognostic effect for patients treated with surgery alone suggests that MSI-L might help to identify patients with potentially high-benefit from preoperative CTx.

Background Response to immunotherapy in gastric cancer is associated with microsatellite instability (or mismatch repair deficiency) and Epstein-Barr virus (EBV) positivity. We therefore aimed to develop and validate deep learningbased classifiers to detect microsatellite instability and EBV status from routine histology slides. MethodsIn this retrospective, multicentre study, we collected tissue samples from ten cohorts of patients with gastric cancer from seven countries (South Korea, Switzerland, Japan, Italy, Germany, the UK and the USA). We trained a deep learning-based classifier to detect microsatellite instability and EBV positivity from digitised, haematoxylin and eosin stained resection slides without annotating tumour containing regions. The performance of the classifier was assessed by within-cohort cross-validation in all ten cohorts and by external validation, for which we split the cohorts into a five-cohort training dataset and a five-cohort test dataset. We measured the area under the receiver operating curve (AUROC) for detection of microsatellite instability and EBV status. Microsatellite instability and EBV status were determined to be detectable if the lower bound of the 95% CI for the AUROC was above 0•5. FindingsAcross the ten cohorts, our analysis included 2823 patients with known microsatellite instability status and 2685 patients with known EBV status. In the within-cohort cross-validation, the deep learning-based classifier could detect microsatellite instability status in nine of ten cohorts, with AUROCs ranging from 0•597 (95% CI 0•522-0•737) to 0•836 (0•795-0•880) and EBV status in five of eight cohorts, with AUROCs ranging from 0•819 (0•752-0•841) to 0•897 (0•513-0•966). Training a classifier on the pooled training dataset and testing it on the five remaining cohorts resulted in high classification performance with AUROCs ranging from 0•723 (95% CI 0•676-0•794) to 0•863 (0•747-0•969) for detection of microsatellite instability and from 0•672 (0•403-0•989) to 0•859 (0•823-0•919) for detection of EBV status. Interpretation Classifiers became increasingly robust when trained on pooled cohorts. After prospective validation, this deep learning-based tissue classification system could be used as an inexpensive predictive biomarker for immunotherapy in gastric cancer. Funding German Cancer Aid and German Federal Ministry of Health.

Compared to other malignancies, there is a lack of easy-to-evaluate biomarkers for gastric cancer, which is associated with an adverse clinical outcome in many cases. Here, we present Stroma AReactive Invasion Front Areas (SARIFA) as a new histological prognostic marker. We defined SARIFA as the direct contact between a cluster of tumor glands/cells comprising at least five tumor cells and inconspicuous surrounding adipose tissue at the invasion front. A total of 480 adenocarcinomas of the stomach and the gastroesophageal junction from two different collections were classified according to SARIFA. To understand the potential underlying mechanisms, a transcriptome analysis was conducted using digital spatial profiling (DSP). It was found that 20% of the tumors were SARIFA-positive. Kappa values between the three pathologists were good in both collections: 0.74 and 0.78. Patients who presented SARIFA-positive tumors had a significantly lower overall survival in Collections A (median: 20.0 versus 44.0 months; p = 0.014, n = 160) and B (median: 15.0 versus 41.0 months; p < 0.0001, n = 320). SARIFA positivity emerged as a negative independent prognostic factor for overall survival (HR 1.638, 95% CI 1.153-2.326, p = 0.006). Using DSP, the most upregulated genes in SARIFA-positive cases were those associated with triglyceride catabolism and endogenous sterols. COL15A1, FABP2, and FABP4 were differentially expressed in positive cases. At the protein level, the expression of proteins related to lipid metabolism was confirmed. SARIFA combines low inter-observer variability, minimal effort, and high prognostic relevance, and is therefore an extremely promising biomarker related to tumor-promoting adipocytes in gastric cancer.

Many studies have used histomorphological features to more precisely predict the prognosis of patients with colon cancer, focusing on tumor budding, poorly differentiated clusters, and the tumor–stroma ratio. Here, we introduce SARIFA: Stroma AReactive Invasion Front Area(s). We defined SARIFA as the direct contact between a tumor gland/tumor cell cluster (≥5 cells) and inconspicuous surrounding adipose tissue in the invasion front. In this retrospective, single-center study, we classified 449 adipose-infiltrative adenocarcinomas (not otherwise specified) from two groups based on SARIFA and found 25% of all tumors to be SARIFA-positive. Kappa values between the two pathologists were good/very good: 0.77 and 0.87. Patients with SARIFA-positive tumors had a significantly shorter colon-cancer-specific survival (p = 0.008, group A), absence of metastasis, and overall survival (p < 0.001, p = 0.003, group B). SARIFA was significantly associated with adverse features such as pT4 stage, lymph node metastasis, tumor budding, and higher tumor grade. Moreover, SARIFA was confirmed as an independent prognostic indicator for colon-cancer-specific survival (p = 0.011, group A). SARIFA assessment was very quick (<1 min). Because of low interobserver variability and good prognostic significance, SARIFA seems to be a promising histomorphological prognostic indicator in adipose-infiltrative adenocarcinomas of the colon. Further studies should validate our results and also determine whether SARIFA is a universal prognostic indicator in solid cancers.

We aimed to investigate patients with gastric/gastro-esophageal adenocarcinomas for sex- and age-specific differences regarding overall survival (OS) and response to neoadjuvant chemotherapy (CTx) under consideration of tumor specific molecular subtypes. Overall, 717 patients were analyzed, including 426 patients treated with and 291 treated without neoadjuvant CTx. Microsatellite instability (MSI) and Epstein-Barr virus positivity (EBV+) were determined previously. Females demonstrated a significantly increased OS (p = 0.035), particularly in the subgroup treated with CTx (p = 0.054). No significant differences regarding age were found. In the molecular subgroups, no sex-related differences were observed in the non-CTx group. However in the CTx group, females with MSI-high (H) tumors showed the best OS (p = 0.043), followed by the male MSI-H (p = 0.198) and female MSS (p = 0.114) compared to the male MSS group as reference. The interaction between sex and MSI in this patient group was noticeable (p = 0.053) and was included as a relevant factor in multivariable analyses. In conclusion, our results show an effect of sex on OS in gastric/gastro-esophageal cancer specifically for patients treated with neoadjuvant CTx. The superior survival of women with MSI-H tumors after neoadjuvant CTx implies that combined consideration of these factors could contribute to an individualized treatment of the patients.