miRNA Expression Profiling in G1 and G2 Pancreatic Neuroendocrine Tumors.

Pancreatic neuroendocrine neoplasms pose a growing clinical challenge due to their rising incidence and variable prognosis. The current study aims to investigate microRNAs (miRNA; miR) as potential biomarkers for distinguishing between grade 1 (G1) and grade 2 (G2) pancreatic neuroendocrine tumors (PanNET). A total of 33 formalin-fixed, paraffin-embedded samples were analyzed, comprising 17 G1 and 16 G2 tumors. Initially, literature-based miRNAs were validated via real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), confirming significant downregulation of miR-130b-3p and miR-106b in G2 samples. Through next-generation sequencing, we have identified and selected the top six miRNAs showing the highest difference between G1 and G2 tumors, which were further validated. RT-qPCR validation confirmed the downregulation of miR-30d-5p in G2 tumors. miRNA combinations were created to distinguish between the two PanNET grades. The highest diagnostic performance in distinguishing between G1 and G2 PanNETs by a machine learning algorithm was achieved when using the combination miR-106b + miR-130b-3p + miR-127-3p + miR-129-5p + miR-30d-5p. The ROC analysis resulted in a sensitivity of 83.33% and a specificity of 87.5%. The findings underscore the potential use of miRNAs as biomarkers for stratifying PanNET grades, though further research is warranted to enhance diagnostic accuracy and clinical utility.

Gastric-Type Expression Signature in Hepatocellular Carcinoma.

It is known that V-set and immunoglobulin domain containing 1 (VSIG1) is a cell-cell adhesion molecule that can serve as an indicator of better survival in patients with gastric cancer. Its interaction with cytoplasmic thyroid transcription factor 1 (TTF-1) has been hypothesized to characterize gastric-type HCC, but its clinical importance is far from understood. As VSIG1 has also been supposed to be involved in the epithelial-mesenchymal transition (EMT) phenomenon, we checked for the first time in the literature the supposed interaction between VSIG1, TTF-1, and Vimentin (VIM) in HCCs. Immunohistochemical (IHC) stains were performed on 217 paraffin-embedded tissue samples that included tumor cells and normal hepatocytes, which served as positive internal controls. VSIG1 positivity was seen in 113 cases (52.07%). In 71 out of 217 HCCs (32.71%), simultaneous positivity for VSIG1 and TTF-1 was seen, being more specific for G1/G2 carcinomas with a trabecular architecture and a longer OS (p = 0.004). A negative association with VIM was revealed (p < 0.0001). Scirrhous-type HCC proved negative for all three examined markers. The present paper validates the hypothesis of the existence of a gastric-type HCC, which shows a glandular-like architecture and is characterized by double positivity for VSIG1 and TTF-1, vimentin negativity, and a significant OS.

Insights into the Tumor Microenvironment-Components, Functions and Therapeutics.

Similarly to our healthy organs, the tumor tissue also constitutes an ecosystem. This implies that stromal cells acquire an altered phenotype in tandem with tumor cells, thereby promoting tumor survival. Cancer cells are fueled by abnormal blood vessels, allowing them to develop and proliferate. Tumor-associated fibroblasts adapt their cytokine and chemokine production to the needs of tumor cells and alter the peritumoral stroma by generating more collagen, thereby stiffening the matrix; these processes promote epithelial-mesenchymal transition and tumor cell invasion. Chronic inflammation and the mobilization of pro-tumorigenic inflammatory cells further facilitate tumor expansion. All of these events can impede the effective administration of tumor treatment; so, the successful inhibition of tumorous matrix remodeling could further enhance the success of antitumor therapy. Over the last decade, significant progress has been made with the introduction of novel immunotherapy that targets the inhibitory mechanisms of T cell activation. However, extensive research is also being conducted on the stromal components and other cell types of the tumor microenvironment (TME) that may serve as potential therapeutic targets.

Dynamic Interplay in Tumor Ecosystems: Communication between Hepatoma Cells and Fibroblasts.

Tumors are intricate ecosystems where cancer cells and non-malignant stromal cells, including cancer-associated fibroblasts (CAFs), engage in complex communication. In this study, we investigated the interaction between poorly (HLE) and well-differentiated (HuH7) hepatoma cells and LX2 fibroblasts. We explored various communication channels, including soluble factors, metabolites, extracellular vesicles (EVs), and miRNAs. Co-culture with HLE cells induced LX2 to produce higher levels of laminin ß1, type IV collagen, and CD44, with pronounced syndecan-1 shedding. Conversely, in HuH7/LX2 co-culture, fibronectin, thrombospondin-1, type IV collagen, and cell surface syndecan-1 were dominant matrix components. Integrins α6ß4 and α6ß1 were upregulated in HLE, while α5ß1 and αVß1 were increased in HuH7. HLE-stimulated LX2 produced excess MMP-2 and 9, whereas HuH7-stimulated LX2 produced excess MMP-1. LX2 activated MAPK and Wnt signaling in hepatoma cells, and conversely, hepatoma-derived EVs upregulated MAPK and Wnt in LX2 cells. LX2-derived EVs induced over tenfold upregulation of SPOCK1/testican-1 in hepatoma EV cargo. We also identified liver cancer-specific miRNAs in hepatoma EVs, with potential implications for early diagnosis. In summary, our study reveals tumor type-dependent communication between hepatoma cells and fibroblasts, shedding light on potential implications for tumor progression. However, the clinical relevance of liver cancer-specific miRNAs requires further investigation.

Corrigendum: SPOCK1 promotes the development of hepatocellular carcinoma.

[This corrects the article DOI: 10.3389/fonc.2022.819883.].

Compositional Analysis of Glycosaminoglycans in Different Lung Cancer Types-A Pilot Study.

Lung cancer is one of the most commonly diagnosed cancer types. Studying the molecular changes that occur in lung cancer is important to understand tumor formation and identify new therapeutic targets and early markers of the disease to decrease mortality. Glycosaminoglycan chains play important roles in various signaling events in the tumor microenvironment. Therefore, we have determined the quantity and sulfation characteristics of chondroitin sulfate and heparan sulfate in formalin-fixed paraffin-embedded human lung tissue samples belonging to different lung cancer types as well as tumor adjacent normal areas. Glycosaminoglycan disaccharide analysis was performed using HPLC-MS following on-surface lyase digestion. Significant changes were identified predominantly in the case of chondroitin sulfate; for example, the total amount was higher in tumor tissue compared to the adjacent normal tissue. We also observed differences in the degree of sulfation and relative proportions of individual chondroitin sulfate disaccharides between lung cancer types and adjacent normal tissue. Furthermore, the differences in the 6-O-/4-O-sulfation ratio of chondroitin sulfate were different between the lung cancer types. Our pilot study revealed that further investigation of the role of chondroitin sulfate chains and enzymes involved in their biosynthesis is an important aspect of lung cancer research.

SPOCK1 Overexpression Suggests Poor Prognosis of Ovarian Cancer.

PURPOSE: Sparc/osteonectin, cwcv, and kazal-like domains proteoglycan 1 (SPOCK1) has been found in a variety of malignant tumors and is associated with a poor prognosis. We aimed to explore the role of SPOCK1 in ovarian cancer. METHODS: Ovarian cancer cell lines SKOV3 and SW626 were transfected with SPOCK1 overexpressing or empty vector using electroporation. Cells were studied by immunostaining and an automated Western blotting system. BrdU uptake and wound healing assays assessed cell proliferation and migration. SPOCK1 expression in human ovarian cancer tissues and in blood samples were studied by immunostaining and ELISA. Survival of patients with tumors exhibiting low and high SPOCK1 expression was analyzed using online tools. RESULTS: Both transfected cell lines synthesized different SPOCK1 variants; SKOV3 cells also secreted the proteoglycan. SPOCK1 overexpression stimulated DNA synthesis and cell migration involving p21CIP1. Ovarian cancer patients had increased SPOCK1 serum levels compared to healthy controls. Tumor cells of tissues also displayed abundant SPOCK1. Moreover, SPOCK1 levels were higher in untreated ovarian cancer serum and tissue samples and lower in recipients of chemotherapy. According to in silico analyses, high SPOCK1 expression was correlated with shorter survival. CONCLUSION: Our findings suggest SPOCK1 may be a viable anti-tumor therapeutic target and could be used for monitoring ovarian cancer.

Liver alterations and detection of SARS-CoV-2 RNA and proteins in COVID-19 autopsies.

The most severe alterations in Coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infection are seen in the lung. However, other organs also are affected. Here, we report histopathologic findings in the liver and detection of viral proteins and RNA in COVID-19 autopsies performed at the Semmelweis University (Budapest, Hungary). Between March 2020 through March 2022, 150 autopsies on patients who died of COVID-19 were analyzed. Cause-of-death categories were formed based on the association with SARS-CoV-2 as strong, contributive, or weak. Samples for histopathologic study were obtained from all organs, fixed in formalin, and embedded in paraffin (FFPE). Immunohistochemical study (IHC) to detect SARS-CoV-2 spike protein and nucleocapsid protein (NP), CD31, claudin-5, factor VIII, macrosialin (CD68), and cytokeratin 7, with reverse transcriptase polymerase chain reaction (RT-PCR), and in situ hybridization (ISH, RNAscope®) for SARS-CoV-2 RNA were conducted using FFPE samples of livers taken from 20 autopsies performed ≤ 2 days postmortem. All glass slides were scanned; the digital images were evaluated by semiquantitative scoring and scores were analyzed statistically. Steatosis, single-cell and focal/zonal hepatocyte necrosis, portal fibrosis, and chronic inflammation were found in varying percentages. Sinusoidal ectasia, endothelial cell disruption, and fibrin-filled sinusoids were seen in all cases; these were assessed semiquantitatively for severity (SEF scored). SEF scores did not correlate with cause-of-death categories (p = 0.92) or with severity of lung alterations (p = 0.96). SARS-CoV-2 RNA was detected in 13/20 cases by PCR and in 9/20 by ISH, with IHC demonstration of spike protein in 4/20 cases and NP in 15/20. Viral RNA and proteins were located in endothelial and Kupffer cells, and in portal macrophages, but not in hepatocytes and cholangiocytes. In conclusion, endothelial damage (SEF scores) was the most common alteration in the liver and was a characteristic, but not specific alteration in COVID-19, suggesting an important role in the pathogenesis of COVID-19-associated liver disease. Detection of SARS-CoV-2 RNA and viral proteins in liver non-parenchymal cells suggests that while the most extended primary viral cytotoxic effect occurs in the lung, viral components are present in other organs too, as in the liver. The necrosis/apoptosis and endothelial damage associated with viral infection in COVID-19 suggest that those patients who survive more severe COVID-19 may face prolonged liver repair and accordingly should be followed regularly in the post-COVID period.

Glycosaminoglycan Analysis of FFPE Tissues from Prostate Cancer and Benign Prostate Hyperplasia Patients Reveals Altered Regulatory Functions and Independent Markers for Survival.

Prostate cancer is one of the most frequent cancer types among men. Several biomarkers and risk assessment methods are already available; however, enhancing their selectivity and sensitivity is still necessary. For improving therapeutic decisions, both basic and clinical research studies are still ongoing for a better understanding of the underlying molecular mechanisms. The enzymatic digests of heparan sulfate (HS) and chondroitin sulfate (CS) chains were investigated in tissue samples taken from patients with prostate cancer (PCa) and benign prostate hyperplasia (BPH) with the HPLC-MS methodology. None of the HS species analyzed showed correlating alterations with currently used markers such as clinical stage, Gleason score, or prostate-specific antigen (PSA) level. The total quantity and sulfation motifs of CS were both significantly different among BPH and different risk groups of PCa. Furthermore, the cancer-specific survival of patients can be predicted based on the levels of non-sulfated and doubly sulfated CS disaccharides as well as the total HS content and the doubly and triply sulfated HS disaccharide ratios. These disaccharide ratios proved to be independent markers from clinical parameters. Further investigations of glycosaminoglycan motifs were proposed for the validation of the results on independent patient cohorts as well.

Syndecan-1 in liver pathophysiology.

Syndecan-1 (SDC-1) is a heparan sulfate (HS)/chondroitin sulfate proteoglycan (PG) of the cell surface and the extracellular matrix (ECM), which regulates a broad spectrum of physiological and pathological processes such as cell proliferation, migration, inflammation, matrix remodeling, wound healing, and tumorigenesis. Syndecan-1 represents the major PG of the liver, expressed by hepatocytes and cholangiocytes, and its elevated expression is a characteristic feature of liver diseases. The highest syndecan-1 expression is found in liver cirrhosis and in hepatocellular carcinoma (HCC) developed in cirrhotic livers. In addition, as being a hepatitis C receptor, hepatitis C virus (HCV)-infected livers produce extremely large amounts of syndecan-1. The serum levels of the cleaved (shedded) extracellular domain have clinical significance, as their increased concentration reflects on poor prognosis in cirrhosis as well as in cancer. In vivo experiments confirmed that syndecan-1 protects against early stages of fibrogenesis mainly by enhanced clearance of transforming growth factor ß1 (TGFß1) and thrombospondin-1 (THBS1) via circulation, and against hepatocarcinogenesis by interfering with several signaling pathways and enhancing cell cycle blockade. In addition, syndecan-1 is capable to hinder lipid metabolism and ribosomal biogenesis in induced cancer models. These observations together with its participation in the uptake of viruses (e.g., HCV and SARS-CoV-2) indicate that syndecan-1 is a central player in liver pathologies.

Proteomic Analysis of Lung Cancer Types-A Pilot Study.

Lung cancer is the leading cause of tumor-related mortality, therefore significant effort is directed towards understanding molecular alterations occurring at the origin of the disease to improve current treatment options. The aim of our pilot-scale study was to carry out a detailed proteomic analysis of formalin-fixed paraffin-embedded tissue sections from patients with small cell or non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma). Tissue surface digestion was performed on relatively small cancerous and tumor-adjacent normal regions and differentially expressed proteins were identified using label-free quantitative mass spectrometry and subsequent statistical analysis. Principal component analysis clearly distinguished cancerous and cancer adjacent normal samples, while the four lung cancer types investigated had distinct molecular profiles and gene set enrichment analysis revealed specific dysregulated biological processes as well. Furthermore, proteins with altered expression unique to a specific lung cancer type were identified and could be the targets of future studies.

Proteoglycans: Systems-Level Insight into Their Expression in Healthy and Diseased Placentas.

Proteoglycan macromolecules play key roles in several physiological processes (e.g., adhesion, proliferation, migration, invasion, angiogenesis, and apoptosis), all of which are important for placentation and healthy pregnancy. However, their precise roles in human reproduction have not been clarified. To fill this gap, herein, we provide an overview of the proteoglycans' expression and role in the placenta, in trophoblast development, and in pregnancy complications (pre-eclampsia, fetal growth restriction), highlighting one of the most important members of this family, syndecan-1 (SDC1). Microarray data analysis showed that of 34 placentally expressed proteoglycans, SDC1 production is markedly the highest in the placenta and that SDC1 is the most upregulated gene during trophoblast differentiation into the syncytiotrophoblast. Furthermore, placental transcriptomic data identified dysregulated proteoglycan genes in pre-eclampsia and in fetal growth restriction, including SDC1, which is supported by the lower concentration of syndecan-1 in maternal blood in these syndromes. Overall, our clinical and in vitro studies, data analyses, and literature search pointed out that proteoglycans, as important components of the placenta, may regulate various stages of placental development and participate in the maintenance of a healthy pregnancy. Moreover, syndecan-1 may serve as a useful marker of syncytialization and a prognostic marker of adverse pregnancy outcomes. Further studies are warranted to explore the role of proteoglycans in healthy and complicated pregnancies, which may help in diagnostic or therapeutic developments.

SPOCK1 with unexpected function. The start of a new career.

SPOCK1, 2, and 3 are considered matricellular proteoglycans without a structural role. Their functions are only partly elucidated. SPOCK1 was detected in the brain as a member of the neural synapses, then in the neuromuscular junctions. It plays a role in the regulation of the blood-brain barrier. Its best-characterized activity was its oncogenic potential discovered in 2012. Its deleterious effect on tumor progression was detected on 36 different types of tumors by the end of 2020. However, its mode of action is still not completely understood. Furthermore, even less was discovered about its physiological function. The fact that it was found to localize in the mitochondria and interfered with the lipid metabolism indicated that the full discovery of SPOCK1 is still waiting for us.

Expression of glycosaminoglycans in cirrhotic liver and hepatocellular carcinoma-a pilot study including etiology.

Chronic liver diseases have both high incidence and mortality rates; therefore, a deeper understanding of the underlying molecular mechanisms is essential. We have determined the content and sulfation pattern of chondroitin sulfate (CS) and heparan sulfate (HS) in human hepatocellular carcinoma and cirrhotic liver tissues, considering the etiology of the diseases. A variety of pathological conditions such as alcoholic liver disease, hepatitis B and C virus infections, and primary sclerosing cholangitis were studied. Major differences were observed in the total abundance and sulfation pattern of CS and HS chains. For example, the 6-O-sulfation of CS is fundamentally different regarding etiologies of cirrhosis, and a 2-threefold increase in HS N-sulfation/O-sulfation ratio was observed in hepatocellular carcinoma compared to cirrhotic tissues.

SPOCK1 Promotes the Development of Hepatocellular Carcinoma.

The extracellular matrix proteoglycan SPOCK1 is increasingly recognized as a contributor to the development and progression of cancers. Here, we study how SPOCK1, which is present in non-tumorous hepatocytes at low concentrations, promotes the development and progression of malignant hepatocellular tumors. Although SPOCK1 is an extracellular matrix proteoglycan, its concentration increases in the cytoplasm of hepatocytes starting with very low expression in the normal cells and then appearing in much higher quantities in cells of cirrhotic human liver and hepatocellular carcinoma. This observation is similar to that observed after diethylnitrosamine induction of mouse hepatocarcinogenesis. Furthermore, syndecan-1, the major proteoglycan of the liver, and SPOCK1 are in inverse correlation in the course of these events. In hepatoma cell lines, the cytoplasmic SPOCK1 colocalized with mitochondrial markers, such as MitoTracker and TOMM20, a characteristic protein of the outer membrane of the mitochondrion and could be detected in the cell nucleus. SPOCK1 downregulation of hepatoma cell lines by siRNA inhibited cell proliferation, upregulated p21 and p27, and interfered with pAkt and CDK4 expression. A tyrosine kinase array revealed that inhibition of SPOCK1 in the liver cancer cells altered MAPK signaling and downregulated several members of the Sarc family, all related to the aggressivity of the hepatoma cell lines. These studies support the idea that SPOCK1 enhancement in the liver is an active contributor to human and rodent hepatocarcinogenesis and cancer progression. However, its mitochondrial localization raises the possibility that it has a currently unidentified physiological function in normal hepatocytes.

The effects of sulfated hyaluronan in breast, lung and colorectal carcinoma and monocytes/macrophages cells: Its role in angiogenesis and tumor progression.

Hyaluronan (HA) is a component of the extracellular matrix (ECM) it is the main non-sulfated glycosaminoglycan able to modulate cell behavior in the healthy and tumor context. Sulfated hyaluronan (sHA) is a biomaterial derived from chemical modifications of HA, since this molecule is not naturally sulfated. The HA sulfation modifies several properties of the native molecule, acquiring antitumor properties in different cancers. In this study, we evaluated the action of sHA of ~30-60 kDa with different degrees of sulfation (0.7 sHA1 and 2.5 sHA3) on tumor cells of a breast, lung, and colorectal cancer model and its action on other cells of the tumor microenvironment, such as endothelial and monocytes/macrophage cells. Our data showed that in breast and lung tumor cells, sHA3 is able to modulate cell viability, cytotoxicity, and proliferation, but no effects were observed on colorectal cancer cells. In 3D cultures of breast and lung cancer cells, sHA3 diminished the size of the tumorsphere and modulated total HA levels. In these tumor models, treatment of monocytes/macrophages with sHA3 showed a downregulation of the expression of angiogenic factors. We also observed a decrease in endothelial cell migration and modulation of the hyaluronan-binding protein TSG-6. In the breast in vivo xenograft model, monocytes/macrophages preincubated with sHA1 or sHA3 decreased tumor vasculature, TSG-6 and HA levels. Besides, in silico analysis showed an association of TSG-6, HAS2, and IL-8 with biological processes implicated in the progression of the tumor. Taken together, our data indicate that sHA in a breast and lung tumor context is able to induce an antiangiogenic action on tumor cells as well as in monocytes/macrophages (Mo/MØ) by modulation of endothelial migration, angiogenic factors, and vessel formation.

Overexpression of Human Syndecan-1 Protects against the Diethylnitrosamine-Induced Hepatocarcinogenesis in Mice.

Although syndecan-1 (SDC1) is known to be dysregulated in various cancer types, its implication in tumorigenesis is poorly understood. Its effect may be detrimental or protective depending on the type of cancer. Our previous data suggest that SDC1 is protective against hepatocarcinogenesis. To further verify this notion, human SDC1 transgenic (hSDC1+/+) mice were generated that expressed hSDC1 specifically in the liver under the control of the albumin promoter. Hepatocarcinogenesis was induced by a single dose of diethylnitrosamine (DEN) at an age of 15 days after birth, which resulted in tumors without cirrhosis in wild-type and hSDC1+/+ mice. At the experimental endpoint, livers were examined macroscopically and histologically, as well as by immunohistochemistry, Western blot, receptor tyrosine kinase array, phosphoprotein array, and proteomic analysis. Liver-specific overexpression of hSDC1 resulted in an approximately six month delay in tumor formation via the promotion of SDC1 shedding, downregulation of lipid metabolism, inhibition of the mTOR and the ß-catenin pathways, and activation of the Foxo1 and p53 transcription factors that lead to the upregulation of the cell cycle inhibitors p21 and p27. Furthermore, both of them are implicated in the regulation of intermediary metabolism. Proteomic analysis showed enhanced lipid metabolism, activation of motor proteins, and loss of mitochondrial electron transport proteins as promoters of cancer in wild-type tumors, inhibited in the hSDC1+/+ livers. These complex mechanisms mimic the characteristics of nonalcoholic steatohepatitis (NASH) induced human liver cancer successfully delayed by syndecan-1.

EGFR variant allele frequency predicts EGFR-TKI efficacy in lung adenocarcinoma: a multicenter study.

BACKGROUND: Although lung adenocarcinoma (LADC) with sensitizing mutations of the epidermal growth factor receptor (EGFR) is highly sensitive to EGFR tyrosine kinase inhibitors (EGFR-TKIs), in most cases disease progression inevitably occurs. Our aim was to investigate the predictive and prognostic significance of adjusted tumoral EGFR variant allele frequency (EGFR-aVAF) in the above setting. METHODS: Eighty-nine Caucasian advanced-stage LADC patients with known exon-specific EGFR mutations undergoing EGFR-TKI treatment were included. The correlations of EGFR-aVAF with clinicopathological variables including progression-free and overall survival (PFS and OS, respectively) were retrospectively analyzed. RESULTS: Of 89 EGFR-mutant LADC patients, 46 (51.7%) had exon 19 deletion, while 41 (46.1%) and 2 (2.2%) patients had exon 21- and exon 18-point mutations, respectively. Tumoral EGFR-aVAF was significantly higher in patients harboring EGFR exon 19 mutations than in those with exon 21-mutant tumors (P<0.001). Notably, patients with EGFR exon 19 mutant tumors demonstrated significantly improved PFS (P=0.003) and OS (P=0.02) compared to patients with exon 21 mutations. Irrespective of specific exon mutations, a statistically significant positive linear correlation was found between EGFR-aVAF of tumoral tissue and PFS (r=0.319; P=0.002). High (≥70%) EGFR-aVAF was an independent predictor of longer PFS [vs. low (<70%) EGFR-aVAF; median PFSs were 52 vs. 26 weeks, respectively; P<0.001]. Additionally, patients with high EGFR-aVAF also had significantly improved OS than those with low EGFR-aVAF (P=0.011). CONCLUSIONS: Our study suggests that high (≥70%) EGFR-aVAF of tumoral tissue predicts benefit from EGFR-TKI treatment in advanced LADC and, moreover, that exon 19 EGFR mutation is associated with high EGFR-aVAF and improved survival outcomes.

Cross-testing of major molecular markers indicates distinct pathways of tumorigenesis in gastric adenocarcinomas and synchronous gastrointestinal stromal tumors.

Small subtype of the gastrointestinal stromal tumor (micro-GIST, MG) is usually asymptomatic and is frequently found incidentally in association with gastric adenocarcinoma (GAC). The background of this coincidence is still an open question. This study comprehensively characterized nine MGs and GACs present in the same surgical specimen by cross-testing the markers of the major pathogenetic pathways of both tumor types. All of the MGs were immunohistochemically positive for CD117/KIT, CD34, and DOG1. DOG1 was also detected in four GACs. Four MGs carried mutations in c-KIT (exons 9, 11, and 13) and two cases in PDGFRα (exon 18). None of the GACs carried activating mutations in c-KIT or PDGFRα. MMR immunopanel identified one GAC as microsatellite unstable tumor. No EBV-positive tumor was found. According to the TCGA molecular classification, one GAC was categorized in the MSI subgroup, three GACs in the genomically stable subgroup, and the rest into the chromosomal instability subgroup. Although a common carcinogenic effect cannot be ruled out, our data suggest a distinct molecular background in the evolvement of the synchronous MGs and GACs. The presence of a MG in gastric resection specimens may be indicative of the development of synchronous malignant tumors in or outside the stomach.