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Background Recently, we presented Stroma AReactive Invasion Front Areas (SARIFA) as a new histomorphologic negative prognostic biomarker in gastric cancer. It is defined as direct contact between tumor cells and fat cells. The aim of this study was to further elucidate the underlying genomic, transcriptional, and immunological mechanisms of the SARIFA phenomenon. Methods To address these questions, SARIFA was classified on H&E-stained tissue sections of three cohorts: an external cohort ( n  = 489, prognostic validation), the TCGA-STAD cohort ( n  = 194, genomic and transcriptomic analysis), and a local cohort ( n  = 60, digital spatial profiling (whole transcriptome) and double RNA in situ hybridization/immunostaining of cytokines). Results SARIFA status proved to be an independent negative prognostic factor for overall survival in an external cohort of gastric carcinomas. In TCGA-STAD cohort, SARIFA is not driven by distinct genomic alterations, whereas the gene expression analyses showed an upregulation of FABP4 in SARIFA-positive tumors. In addition, the transcriptional regulations of white adipocyte differentiation, triglyceride metabolism, and catabolism were upregulated in pathway analyses. In the DSP analysis of SARIFA-positive tumors, FABP4 and the transcriptional regulation of white adipocyte differentiation were upregulated in macrophages. Additionally, a significantly lower expression of the cytokines IL6 and TNFα was observed at the invasion front. Conclusions SARIFA proves to be a strong negative prognostic biomarker in advanced gastric cancer, implicating an interaction of tumor cells with tumor-promoting adipocytes with crucial changes in tumor cell metabolism. SARIFA is not driven by tumor genetics but is very likely driven by an altered immune response as a causative mechanism.

Background The activity of the epidermal growth factor receptor (EGFR)-directed monoclonal antibody cetuximab combined with oxaliplatin/leucovorin/5-fluorouracil (FUFOX) was assessed in first-line metastatic gastric and oesophago-gastric junction (OGJ) cancer in a prospective phase II study showing a promising objective tumour response rate of 65% and a low mutation frequency of KRAS (3%). The aim of the correlative tumour tissue studies was to investigate the relationship between EGFR gene copy numbers, activation of the EGFR pathway, expression and mutation of E-cadherin, V600E BRAF mutation and clinical outcome of patients with gastric and OGJ cancer treated with cetuximab combined with FUFOX. Methods Patients included in this correlative study ( n = 39) were a subset of patients from the clinical phase II study. The association between EGFR gene copy number, activation of the EGFR pathway, abundance and mutation of E-cadherin which plays an important role in these disorders, BRAF mutation and clinical outcome of patients was studied. EGFR gene copy number was assessed by FISH. Expression of the phosphorylated forms of EGFR and its downstream effectors Akt and MAPK, in addition to E-cadherin was analysed by immunohistochemistry. The frequency of mutant V600E BRAF was evaluated by allele-specific PCR and the mutation profile of the E-cadherin gene CDH1 was examined by DHPLC followed by direct sequence analysis. Correlations with overall survival (OS), time to progression (TTP) and overall response rate (ORR) were assessed. Results Our study showed a significant association between increased EGFR gene copy number (≥ 4.0) and OS in gastric and OGJ cancer, indicating the possibility that patients may be selected for treatment on a genetic basis. Furthermore, a significant correlation was shown between activated EGFR and shorter TTP and ORR, but not between activated EGFR and OS. No V600E BRAF mutations were identified. On the other hand, an interesting trend between high E-cadherin expression levels and better OS was observed and two CDH1 exon 9 missense mutations (A408V and D402H) were detected. Conclusion Our finding that increased EGFR gene copy numbers, activated EGFR and the E-cadherin status are potentially interesting biomarkers needs to be confirmed in larger randomized clinical trials. Trial registration Multicentre clinical study with the European Clinical Trials Database number 2004-004024-12.

ObjectivePOLE-mutant, microsatellite-instable (MSI), p53-mutant and non-specific molecular profile (NSMP) are TCGA-defined molecular subgroups of endometrial cancer (EC). Hypothesizing that morphology and tumor immunology might differ depending on molecular background concerning composition and prognostic impact, we aimed to comprehensively interconnect morphologic, immunologic and molecular data.MethodsTCGA-defined molecular groups were determined by immunohistochemistry and sequencing in n = 142 endometrioid EC. WHO-defined histopathological grading was performed. The immunologic microenvironment (iTME) was characterised by the quantification of intraepithelial and stromal populations of tumor-infiltrating lymphocytes (TIL: overall T-cells; T-Killer cells; regulatory T-cells (Treg)). Immunologic parameters were correlated with WHO-grading, TCGA-subgroups and prognosis.ResultsHigh density TIL were significantly more frequent in high-grade (G3) compared to low-grade (G1/2) EC in the whole cohort and in the subgroup of POLE-wildtype-/microsatellite-stable-EC. MSI was associated with high-level TIL-infiltration when taking into account the type of mismatch repair defect (MLH1/PMS2; MSH2/MSH6). Prognostic impact of biomarkers depended on molecular subgroups: In p53-mutant EC, Treg were independently prognostic, in NSMP, the unique independently prognostic biomarker was WHO-grading.ConclusionsEC morphology and immunology differ depending on genetics. Our study delineated two molecularly distinct subgroups of immunogenic EC characterized by high-density TIL-infiltration: MSI EC and high-grade POLE-wildtype/microsatellite-stable-EC. Prognostic impact of TIL-populations relied on TCGA-subgroups indicating specific roles for TIL depending on molecular background. In NSMP, histopathological grading was the only prognostic biomarker demonstrating the relevance of WHO-grading in an era of molecular subtyping.

Background Computational pathology uses deep learning (DL) to extract biomarkers from routine pathology slides. Large multicentric datasets improve performance, but such datasets are scarce for gastric cancer. This limitation could be overcome by Swarm Learning (SL). Methods Here, we report the results of a multicentric retrospective study of SL for prediction of molecular biomarkers in gastric cancer. We collected tissue samples with known microsatellite instability (MSI) and Epstein–Barr Virus (EBV) status from four patient cohorts from Switzerland, Germany, the UK and the USA, storing each dataset on a physically separate computer. Results On an external validation cohort, the SL-based classifier reached an area under the receiver operating curve (AUROC) of 0.8092 (± 0.0132) for MSI prediction and 0.8372 (± 0.0179) for EBV prediction. The centralized model, which was trained on all datasets on a single computer, reached a similar performance. Conclusions Our findings demonstrate the feasibility of SL-based molecular biomarkers in gastric cancer. In the future, SL could be used for collaborative training and, thus, improve the performance of these biomarkers. This may ultimately result in clinical-grade performance and generalizability.

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 Even though the post-operative outcome varies greatly among patients with nodal positive colon cancer (UICC stage III), personalized prediction of systemic disease recurrence is currently insufficient. We investigated in a retrospective setting whether genetic and immunological biomarkers can be applied for stratification of distant metastasis occurrence risk. Methods Eighty four patients with complete resection (R0) of stage III colon cancer from two clinical centres were analysed for genetic biomarkers: microsatellite instability, oncogenic mutations in KRAS exon2 and BRAF exon15, expression of osteopontin and the metastasis-associated genes SASH1 and MACC1. Tumor-infiltrating CD3 and CD8 positive T-cells were quantified by immunocytochemistry. Results were correlated with outcome and response to 5-FU based adjuvant chemotherapy, using Cox’s proportional hazard models and integrative two-step cluster analysis. Results Distant metastasis risk was significantly correlated with oncogenic KRAS mutations ( p  = 0.015), expression of SASH1 ( p  = 0.016), and the density of CD8-positive T-cells ( p  = 0.007) in Kaplan-Meier analysis. Upon multivariate Cox-regression analysis, KRAS mutation ( p  = 0.008) and density of CD8-positive TILs ( p  = 0.009) were retained as prognostic parameters for metachronous distant metastasis. Integrative two-step cluster analysis was used to combine all genetic markers, allowing stratification of patient subgroups. Post-operative distant metastasis risk ranged from 31% (low-risk) to 41% (intermediate), and 57% (high-risk) ( p  = 0.032). Increased expression of osteopontin ( p  = 0.019) and low density of CD8-positive T-cells ( p  = 0.043) were significantly associated with unfavourable response to 5-FU. Conclusions Integrative biomarker analysis allows stratification of stage III colon cancer patients for the risk of metastatic disease recurrence and may indicate response to 5-FU. Thus, biomarker analysis might facilitate the use of adjuvant therapy for high risk patients.

The molecular carcinogenesis of intraductal tubulopapillary neoplasms (ITPN), recently described as rare neoplasms in the pancreato-biliary tract with a favorable prognosis despite a high incidence of associated pancreato-biliary adenocarcinoma, is still poorly understood. To identify driver genes, chromosomal gains and losses, mutational signatures, key signaling pathways, and potential therapeutic targets, the molecular profile of 11 biliary and 6 pancreatic ITPNs, associated with invasive adenocarcinoma in 14/17 cases, are studied by whole exome sequencing (WES). The WES of 17 ITPNs reveals common copy number variants (CNVs) broadly distributed across the genome, with recurrent chromosomal deletions primarily in 1p36 and 9p21 affecting the tumor suppressors CHD5 and CDKN2A, respectively, and gains in 1q affecting the prominent oncogene AKT3. The identified somatic nucleotide variants (SNVs) involve few core signaling pathways despite high genetic heterogeneity with diverse mutational spectra: Chromatin remodeling, the cell cycle, and DNA damage/repair. An OncoKB search identifies putative actionable genomic targets in 35% of the cases (6/17), including recurrent missense mutations of the FGFR2 gene in biliary ITPNs (2/11, 18%). Our results show that somatic SNV in classical cancer genes, typically associated with pancreato-biliary carcinogenesis, were absent (KRAS, IDH1/2, GNAS, and others) to rare (TP53 and SMAD4, 6%, respectively) in ITPNs. Mutational signature pattern analysis reveals a predominance of an age-related pattern. Our findings highlight that biliary ITPN and classical cholangiocarcinoma display commonalities, in particular mutations in genes of the chromatin remodeling pathway, and appear, therefore, more closely related than pancreatic ITPN and classical pancreatic ductal adenocarcinoma.

Cancer stem cell (CSC) based gene expression signatures are associated with prognosis in various tumour types and CSCs are suggested to be particularly drug resistant. The aim of our study was first, to determine the prognostic significance of CSC-related gene expression in residual tumour cells of neoadjuvant-treated gastric cancer (GC) patients. Second, we wished to examine, whether expression alterations between pre- and post-therapeutic tumour samples exist, consistent with an enrichment of drug resistant tumour cells. The expression of 44 genes was analysed in 63 formalin-fixed, paraffin embedded tumour specimens with partial tumour regression (10-50% residual tumour) after neoadjuvant chemotherapy by quantitative real time PCR low-density arrays. A signature of combined GSK3B(high), β-catenin (CTNNB1)(high) and NOTCH2(low) expression was strongly correlated with better patient survival (p<0.001). A prognostic relevance of these genes was also found analysing publically available gene expression data. The expression of 9 genes was compared between pre-therapeutic biopsies and post-therapeutic resected specimens. A significant post-therapeutic increase in NOTCH2, LGR5 and POU5F1 expression was found in tumours with different tumour regression grades. No significant alterations were observed for GSK3B and CTNNB1. Immunohistochemical analysis demonstrated a chemotherapy-associated increase in the intensity of NOTCH2 staining, but not in the percentage of NOTCH2. Taken together, the GSK3B, CTNNB1 and NOTCH2 expression signature is a novel, promising prognostic parameter for GC. The results of the differential expression analysis indicate a prominent role for NOTCH2 and chemotherapy resistance in GC, which seems to be related to an effect of the drugs on NOTCH2 expression rather than to an enrichment of NOTCH2 expressing tumour cells.

Tumour budding (TB) has been associated with adverse clinicopathological factors and poor survival in a plethora of therapy‐naïve carcinoma entities including gastric adenocarcinoma (GC). As conventional histopathological grading is usually omitted in the post‐neoadjuvant setting of GC, our study aimed to investigate the prognostic impact of TB in GCs resected after neoadjuvant therapy. We evaluated TB according to the criteria from the International Tumour Budding Consensus Conference (ITBCC) in 167 post‐neoadjuvant resections of intestinal‐type GC and correlated the results with overall survival (OS) and clinicopathological parameters. GCs were categorised into Bd1 (0–4 buds, low TB), Bd2 (5–9 buds, intermediate TB), and Bd3 (≥10 buds, high TB). Carcinomas with intermediate and high TB were significantly enriched in higher ypTNM stages and strongly associated with reduced 5‐year OS in univariable analyses (p < 0.001). In multivariable analyses including sex, age, resection status, UICC stage, and tumour regression grading, TB remained a stage‐independent predictor of survival (p < 0.001, hazard ratio Bd2: 2.60, Bd3: 4.74). The assessment of TB according to the ITBCC criteria provides valuable prognostic information in the post‐neoadjuvant setting of intestinal‐type GC and may be a considerable substitute for the conventional grading system in GCs after neoadjuvant therapy.

Background Histone deacetylases (HDACs) modulate chromatin and may influence the effect of DNA-damaging drugs. We investigated HDAC1 and -2 expression in gastric carcinomas (GCs) for an association of patient outcome with conventional neoadjuvant chemotherapy. In vitro, HDAC inhibitors were evaluated as alternative treatment options. Methods HDAC1/2 expression was analyzed immunohistochemically in 127 pretherapeutic biopsy samples of neoadjuvant (platinum/5-fluorouracil) chemotherapy-treated GC patients and correlated with response and overall survival (OS). Chemosensitivity of four GC cell lines to cisplatin and the HDAC inhibitors suberoylanilide hydroxamic acid (SAHA) and valproic acid was determined by XTT assays. Efficiencies of combined drug schedules were analyzed. Results High expression of HDAC1/2 was found in 69 (54%) of 127 and 108 (85%) of 127 carcinomas, respectively, and was not associated with response or OS. In patients whose disease responded to therapy, high HDAC1 expression was associated with worse OS ( P  = 0.005). In cell lines, sequential treatment with SAHA and cisplatin showed synergistic effects irrespective of the initial cisplatin sensitivity. Conclusions HDAC1 and -2 expression is not suitable to predict response or survival for neoadjuvant-treated GC patients, but HDAC1 expression may be used for risk stratification in patients whose disease responds to therapy. Sequential treatment with SAHA and cisplatin may represent an alternative treatment option for GC patients.