The putative pleiotropic functions of meprin ß in gastric cancer.

BACKGROUND: The gastric microbiome and inflammation play a key role in gastric cancer (GC) by regulating the immune response in a complex manner and by inflammatory events supporting carcinogenesis. Meprin ß is a zinc endopeptidase and participates in tissue homeostasis, intestinal barrier function and immunological processes. It influences local inflammatory processes, dysbiosis and the microbiome. Here, we tested the hypothesis that meprin ß is expressed in GC and of tumor biological significance. PATIENTS AND METHODS: Four hundred forty whole mount tissue sections of patients with therapy-naive GC were stained with an anti-meprin ß antibody. The histoscore and staining pattern were analyzed for each case. Following dichotomization at the median histoscore into a "low" and "high" group, the expression was correlated with numerous clinicopathological patient characteristics. RESULTS: Meprin ß was found intracellularly and at the cell membrane of GC. Cytoplasmic expression correlated with the phenotype according to Lauren, microsatellite instability and PD-L1 status. Membranous expression correlated with intestinal phenotype, mucin-1-, E-cadherin-, ß-catenin status, mucin typus, microsatellite instability, KRAS mutation and PD-L1-positivity. Patients with cytoplasmic expression of meprin ß showed a better overall and tumor-specific survival. CONCLUSIONS: Meprin ß is differentially expressed in GC and has potential tumor biological relevance. It might function as a tumor suppressor or promotor depending on histoanatomical site and context.

[Intratumoral heterogeneity of gastric cancer-impact on biomarker evaluation]. / Intratumorale Heterogenität des Magenkarzinoms ­ Einfluss auf die Biomarkeretablierung.

BACKGROUND: Gastric carcinomas often measure more than 5 cm at primary diagnosis. Predictive biomarker testing is usually carried out on tissue biopsies, which do not represent the entire tumor biology and intratumoral heterogeneity. OBJECTIVES: The aim of this study was to explore gastric cancer's intratumoral heterogeneity and its impact on the evaluation of predictive and prognostic biomarkers. MATERIALS AND METHODS: The study cohort consisted of approximately 500 patients with therapy-naive adenocarcinomas of the stomach or the esophagogastric junction. The following biomarkers were determined: HER2, MET, Ki67, PD-L1/PD­1, VISTA, EBV-status, and PIK3CA. RESULTS: All examined biomarkers were influenced by gastric cancer's intratumoral heterogeneity. Tissue biopsies might carry the risk of sampling errors, which may significantly hamper adequate tumor classification in a clinical setting. CONCLUSIONS: Our findings unravel issues of tumor heterogeneity in gastric cancer. Biomarker diagnostics on tissue biopsies should be carried out on at least five biopsies of different tumor areas. If possible, biomarker diagnostics should be repeated on resection specimens. Tissue microarrays should no longer be used for research studies of gastric cancer.

Paracrine Interaction of Cholangiocellular Carcinoma with Cancer-Associated Fibroblasts and Schwann Cells Impact Cell Migration.

Although the Mitogen-activated protein kinase (MAPK) pathway is enriched in cholangiocarcinoma (CCA), treatment with the multityrosine kinase-inhibitor Sorafenib is disappointing. While cancer-associated fibroblasts (CAF) are known to contribute to treatment resistance in CCA, knowledge is lacking for Schwann cells (SC). We investigated the impact of stromal cells on CCA cells and whether this is affected by Sorafenib. Immunohistochemistry revealed elevated expression of CAF and SC markers significantly correlating with reduced tumor-free survival. In co-culture with CAF, CCA cells mostly migrated, which could be diminished by Sorafenib, while in SC co-cultures, SC predominantly migrated towards CCA cells, unaffected by Sorafenib. Moreover, increased secretion of pro-inflammatory cytokines MCP-1, CXCL-1, IL-6 and IL-8 was determined in CAF mono- and co-cultures, which could be reduced by Sorafenib. Corresponding to migration results, an increased expression of phospho-AKT was measured in CAF co-cultured HuCCT-1 cells, although was unaffected by Sorafenib. Intriguingly, CAF co-cultured TFK-1 cells showed increased activation of STAT3, JNK, ERK and AKT pathways, which was partly reduced by Sorafenib. This study indicates that CAF and SC differentially impact CCA cells and Sorafenib partially reverts these stroma-mediated effects. These findings contribute to a better understanding of the paracrine interplay of CAF and SC with CCA cells.

Prognostic significance of high mobility group A2 (HMGA2) in pancreatic ductal adenocarcinoma: malignant functions of cytoplasmic HMGA2 expression.

PURPOSE: HMGA2 has frequently been found in benign as well as malignant tumors and a significant association between HMGA2 overexpression and poor survival in different malignancies was described. In pancreatic ductal adenocarcinoma (PDAC), nuclear HMGA2 expression is associated with tumor dedifferentiation and presence of lymph node metastasis. Nevertheless, the impact of HMGA2 occurrence in other cell compartments is unknown. METHODS: Intracellular distribution of HMGA2 was analyzed in PDAC (n = 106) and peritumoral, non-malignant ducts (n = 28) by immunohistochemistry. Findings were correlated with clinico-pathological data. Additionally, intracellular HMGA2 presence was studied by Western blotting of cytoplasmic and nuclear fractions of cultured cells. RESULTS: HMGA2 was found in the cytoplasm and in the nucleus of cultured cells. In human tumor tissue, HMGA2 was also frequently found in the cytoplasm and the nucleus of tumor cells, however, nuclear staining was generally stronger. Direct comparison from tumor tissue with corresponding non-neoplastic peritumoral tissue revealed significantly stronger expression in tumors (p = 0.003). Of note, the nuclear staining was significantly stronger in lymph node metastatic cell nuclei compared to primary tumor cell nuclei (p = 0.049). Interestingly, cytoplasmic staining positively correlated with lymph vessel (p = 0.004) and venous invasion (p = 0.046). CONCLUSION: HMGA2 is a prognostic marker in PDAC. Firstly, we found a positive correlation for cytoplasmic HMGA2 expression with lympho-vascular invasion and, secondly, we found a significantly stronger nuclear expression of HMGA2 in cancer-positive lymph node nuclei compared to primary tumor cell nuclei. So far, the role of cytoplasmic HMGA2 is nearly unknown, however, our data lend support to the hypothesis that cytoplasmic HMGA2 expression is involved in nodal spread.

Multiscale heterogeneity in gastric adenocarcinoma evolution is an obstacle to precision medicine.

BACKGROUND: Cancer is a somatic evolutionary disease and adenocarcinomas of the stomach and gastroesophageal junction (GC) may serve as a two-dimensional model of cancer expansion, in which tumor subclones are not evenly mixed during tumor progression but rather spatially separated and diversified. We hypothesize that precision medicine efforts are compromised when clinical decisions are based on a single-sample analysis, which ignores the mechanisms of cancer evolution and resulting intratumoral heterogeneity. Using multiregional whole-exome sequencing, we investigated the effect of somatic evolution on intratumoral heterogeneity aiming to shed light on the evolutionary biology of GC. METHODS: The study comprised a prospective discovery cohort of 9 and a validation cohort of 463 GCs. Multiregional whole-exome sequencing was performed using samples form 45 primary tumors and 3 lymph node metastases (range 3-10 tumor samples/patient) of the discovery cohort. RESULTS: In total, the discovery cohort harbored 16,537 non-synonymous mutations. Intratumoral heterogeneity of somatic mutations and copy number variants were present in all tumors of the discovery cohort. Of the non-synonymous mutations, 53-91% were not present in each patient's sample; 399 genes harbored 2-4 different non-synonymous mutations in the same patient; 175 genes showed copy number variations, the majority being heterogeneous, including CD274 (PD-L1). Multi-sample tree-based analyses provided evidence for branched evolution being most complex in a microsatellite instable GC. The analysis of the mode of evolution showed a high degree of heterogeneity in deviation from neutrality within each tumor. We found evidence of parallel evolution and evolutionary trajectories: different mutations of SMAD4 aligned with different subclones and were found only in TP53 mutant GCs. CONCLUSIONS: Neutral and non-neutral somatic evolution shape the mutational landscape in GC along its lateral expansions. It leads to complex spatial intratumoral heterogeneity, where lymph node metastases may stem from different areas of the primary tumor, synchronously. Our findings may have profound effects on future patient management. They illustrate the risk of mis-interpreting tumor genetics based on single-sample analysis and open new avenues for an evolutionary classification of GC, i.e., the discovery of distinct evolutionary trajectories which can be utilized for precision medicine.

Therapy Resistance in Neoadjuvantly Treated Gastric Cancer and Cancer of the Gastroesophageal Junction is Associated with an Increased Expression of Immune Checkpoint Inhibitors-Comparison Against a Therapy Naïve Cohort.

With recent studies uncovering the complex landscape of immune checkpoint regulators in gastric cancer (GC), we aimed to characterize the expression of the checkpoint proteins V-domain Ig suppressor of T-cell activation (VISTA), programmed cell death 1 ligand 1 (PD-L1), and programmed cell death protein-1 (PD-1) in a cohort of GCs following platinum-based neoadjuvant chemotherapy. A total of 141 GC samples, 93 lymph node metastases, and 15 distant metastases were assessed using immunohistochemistry. Staining results were correlated with clinicopathological patient characteristics, genetic alterations, and survival. The expression of VISTA was detected in tumor cells of 38 (30.9%) GCs and immune cells of 139 (98.6%) GCs. The expression of PD-L1 was detected in tumor cells of 27 (22.7%) GCs and immune cells of 134 (96.4%) GCs. The expression of PD-1 was only observed in lymphocyte aggregates/intratumoral lymphoid follicles of 123 (87.2%) GCs. VISTA and PD-L1 correlated in their expression and were associated with poor tumor regression. Compared with an ancient cohort of therapy naïve GCs, we observed a major increase in overall immune cell density accompanied by an over proportional increase in PD-1 and VISTA-positive immune cells. The frequency of VISTA expression in tumor cells was also found to be substantially increased. To the contrary, expression of PD-L1 was decreased in immune cells and tumor cells of neoadjuvantly treated GCs. As a result, a subset of GCs using a single (only VISTA or PD-L1) or combined (VISTA and PD-L1) immune evasion mechanisms might benefit from an anti-PD-L1/anti-VISTA-targeted therapy.

HER2 gene (ERBB2) amplification is a low-frequency driver with potential predictive value in gallbladder carcinoma.

Gallbladder carcinoma (GBC) is an aggressive type of cancer with a dismal prognosis. Recent case reports have highlighted the human epidermal growth factor receptor 2 (HER2) as a promising target for individualized therapy in biliary tract cancer; however, current data on HER2 positivity in GBC is contradictory. This study aimed to assess the proportion of HER2 positivity and its clinical implications in a large and well-characterized European GBC cohort. HER2 status was determined in 186 cases of surgically resected gallbladder adenocarcinoma and a subset of coexistent high-grade biliary intraepithelial neoplasia (BilIN, n = 74) in accordance with the up-to-date consensus for HER2 testing in gastric cancer by immunohistochemistry and dual-color chromogenic in situ hybridization. Positivity for HER2 was observed in 5.4% of all cases (n = 10). In those patients with concomitant high-grade BilIN, two of four positive samples also showed amplification in the precursor lesion, while in the two remaining cases, positivity was either confined to invasive tumor or high-grade BilIN, exclusively. Equivocal staining found in eleven cases was not accompanied by gene amplification. Staging of the HER2-positive group was significantly different from the HER2-negative group with most cases presenting at stage IV, paralleled by a trend towards decreased survival. One patient who received dual HER2 inhibition almost went into full clinical remission despite treatment initiation in a metastasized state. Our results reveal a low prevalence of HER2 positivity and highlight HER2 gene amplification as an early, potentially driving event in gallbladder carcinogenesis. Prospective standardized HER2 testing and randomized control studies are needed to prove clinical efficacy of targeted HER2 inhibition in GBC.

Expression of the potential therapeutic target claudin-18.2 is frequently decreased in gastric cancer: results from a large Caucasian cohort study.

Gastric cancer (GC) is frequently diagnosed and treated in advanced tumour stages with poor prognosis. Recent studies have identified isoform 2 of the tight junction protein claudin-18 (CLDN18.2) as a promising target in GC therapy. In this study, we aimed to outline the expression of CLDN18.2 and its correlation with clinico-pathological patient characteristics in a large and well-characterized cohort of GC patients. The expression of CLDN18.2 was studied in 481 GCs by immunohistochemistry on whole tissue sections. Immunostained GCs were evaluated using the histoscore (H-score) and subsequently divided into two groups: tumours showing any or no expression. CLDN18.2 expression was investigated for correlation with various clinico-pathological patient characteristics, including survival. CLDN18.2 expression was found in 203 GCs (42.2%). Of these tumours, 71 (14.8%) showed solely weak immunostaining. CLDN18.2 expression correlated with mucin phenotype, EBV status, the integrin αvß5, the EpCAM extracellular domain EpEX, and lysozyme. We found no correlation with survival, Laurén phenotype, or any other clinico-pathological patient characteristic. In conclusion, we demonstrate frequently decreased expression of CLDN18.2 in a GC cohort of appropriate size. Correlating CLDN18.2 expression with clinico-pathological patient characteristics reveals new linkages to αvß5, EpEX, and lysozyme, which may pave the way for further investigations regarding the role of tight junction proteins in GC progression. Though CLDN18.2 continues to pose an attractive target candidate, we conclude that a rather low overall expression rate challenges its significance in advanced GC therapy and indicates the need for further investigations across different populations.

Intratumoral heterogeneity and loss of ARID1A expression in gastric cancer correlates with increased PD-L1 expression in Western patients.

Recent whole-genome sequencing showed frequent mutations of ARID1A in gastric cancer (GC). In this study of a large independent Central European cohort, we evaluated the expression of ARID1A in whole tissue sections (WTS) of GC testing the following hypotheses: ARID1A shows intratumoral heterogeneity, and ARID1A expression and/or heterogeneity correlates with clinicopathological patient characteristics. ARID1A expression was studied by immunohistochemistry in 450 primary GCs and 143 corresponding lymph node metastases. The expression pattern was correlated with clinicopathological characteristics and patient survival. ARID1A genotype and CpG methylation status were additionally analyzed in 7 GCs with a heterogeneous "black-and-white" expression pattern. ARID1A was expressed heterogeneously in 23 (5.1%) GCs, depicting a black-and-white pattern of negative and positive tumor areas. Complete loss of expression was found in 43 (9.6%) GCs. ARID1A status correlated significantly with tumor type according to Laurén, Epstein-Barr virus status, microsatellite instability, PD-L1 status, and nodal spread. There was no correlation with patient survival. In 4 cases with heterogeneous ARID1A expression, frame shift variants were detected. Summing up, heterogeneous or complete loss of ARID1A expression occurred in 14.7% of GCs and correlated with PD-L1 status, indicating potential for future combined anti-PD-L1/ARID1A therapy. In a subgroup of cases, ARID1A loss was heterogeneous, which suggests that ARID1A mutations might be a later event in gastric carcinogenesis.

Expression of LGR5, FZD7, TROY, and MIST1 in Perioperatively Treated Gastric Carcinomas and Correlation with Therapy Response.

The cancer stem cell model is considered as a putative cause of resistance to chemotherapy and disease recurrence in malignant tumors. In this study, we tested the hypothesis that the response to neoadjuvant/perioperative chemotherapy correlates with the expression of four different putative cancer stem cell markers of gastric cancer (GC), i.e., LGR5, FZD7, TROY, and MIST1. The expression of LGR5, FZD7, TROY, and MIST1 was assessed by immunohistochemistry in 119 perioperatively treated GCs including pretherapeutic biopsies, resected primary GCs, and corresponding nodal and distant metastases. All four markers were detected in our cohort with variable prevalence and histoanatomical distributions. Few tumor cells expressed TROY. LGR5, FZD7, and MIST1 were coexpressed in 41.2% and completely absent in 6.2%. The prevalence of LGR5- and FZD7-positive GCs was higher and of TROY-positive GCs lower in perioperatively treated GCs compared with treatment-naïve tumors. LGR5, FZD7, and MIST1 in the primary tumors correlated significantly with their expression in the corresponding lymph node metastasis. An increased expression of LGR5 in primary GC correlated significantly with tumor regression. The expression of MIST1 in lymph node metastases correlated significantly with the number of lymph node metastases as well as overall and tumor-specific survival. FZD7 did not correlate with any clinicopathological patient characteristic. Our study on clinical patient samples shows that GCs may coexpress independently different stem cell markers; that neoadjuvant/perioperative treatment of GC significantly impacts on the expression of stem cell markers, which cannot be predicted by the analysis of pretherapeutic biopsies; and that their expression and tumor biological effect are heterogeneous and have to be viewed as a function of histoanatomical distribution.