Risk of progression in Barrett’s esophagus based on diagnoses of general and gastrointestinal pathologists:a retrospective case-control study from Northern and Central Finland

Abstract Background: Esophageal adenocarcinoma (EAC) is the sixth leading cause of cancer-related death worldwide. It develops through Barrett’s metaplasia — dysplasia sequence. However, the effectiveness of endoscopic surveillance is limited, since diagnosis of low-grade dysplasia (LGD) is known to be challenging for pathologists. Our aim was to compare the risk of Barrett’s progression based on diagnoses of general and expert gastrointestinal (GI) pathologists in a population-based cohort. Methods: A total of 60 patients with non-dysplastic metaplasia (BE) or LGD progressing to high grade dysplasia (HGD) or EAC during follow-up could be identified in the population. For comparison, series representing non-progressive BE (n = 56) and LGD cases (n = 54), matched for age, gender, and length of follow-up were collected. All available original HE stained slides (n = 292) were blindly re-evaluated by two experienced GI pathologists and patient groups of progressive non-progressive BE and LGD were formed according to revised diagnoses. Results: Original diagnosis for each sample was changed in 25% of BE, 59% of LGD, and 33% of HGD diagnoses. Of the original LGD diagnoses, 53% were downgraded to BE or indefinite for dysplasia (ID). Of LGD diagnoses made by an expert GI pathologist, 61% were in the progressive LGD group, whereas only 42% of general pathologists’ LGD diagnoses were in the progressive LGD group. Conclusion: Based on this retrospective case-control study, LGD is strongly over-diagnosed among general pathologists. LGD diagnosed by expert GI pathologists predicts progressive disease. Recommendation for consensus diagnosis by expert GI pathologists is justified also in the Finnish population-based setting

A straightforward method for adipocyte size and count analysis using open-source software QuPath

Abstract Changes in adipose tissue morphology, depicted by cell morphology alterations such as enlargement of fat cells, always accompany over-weight and obesity. The variables related to cell size have been shown to associate with low-grade inflammation of adipose tissue and common obesity-related comorbidities including metabolic syndrome and type 2 diabetes. Quantifying fat cell morphology from images of histological specimens can be tedious. Here, we present a straightforward method for the task using the free open-source software QuPath with its inbuilt tools only. Measurements of human adipose tissue samples with the described protocol showed an excellent correlation with those obtained with ImageJ software with Adipocyte Tools plugin combined with manual correction of misdetections. Intraclass correlation between the two methods was at good to excellent level. The method described here can be applied to considerably large tissue areas, even whole-slide analysis

Tumor budding and prognosis in gastric adenocarcinoma

Abstract Tumor budding has been associated with poor prognosis in several cancer types, but its significance in gastric cancer is unknown. The aim of this study was to assess the prognostic significance of tumor budding in gastric adenocarcinoma, and its main histologic types. Some 583 gastric adenocarcinoma patients who underwent surgery in Oulu University Hospital during the years 1983–2016 were included in this retrospective cohort study. Tumor budding was counted per 0.785 mm² fields from the slides originally used for diagnostic purposes. Patients were divided into low-budding (<10 buds) and high-budding (≥10 buds) groups. Tumor budding was analyzed in relation to 5-year survival and overall survival. Cox regression was used to calculate hazard ratios (HR) with 95% confidence intervals (CI), adjusted for confounders. Determining tumor budding was difficult in diffuse-type cancer due to the uncohesive growth pattern of these tumors. Patients with high tumor budding had worse 5-year survival compared with patients with low tumor budding (adjusted HR, 1.55; 95% CI, 1.20–2.01). In intestinal-type adenocarcinomas, the high-budding group had significantly poorer 5-year survival compared with the low-budding group (adjusted HR, 1.57; 95% CI, 1.14–2.15). There were no differences in 5-year survival between the budding groups in the diffuse type adenocarcinoma. In conclusion, high tumor budding is an independent prognostic factor in gastric adenocarcinoma, but its value is limited to the intestinal type of gastric adenocarcinoma. In diffuse type gastric adenocarcinoma, the assessment of tumor budding is hardly feasible, and it does not have prognostic relevance

KRAS and BRAF mutations induce anoikis resistance and characteristic 3D phenotypes in Caco‑2 cells

Abstract In a number of types of cancer, anoikis, a form of apoptosis induced by loss of extracellular matrix (ECM) attachment, is disturbed. Anoikis resistance is essential in the formation of metastases. A recent study identified carcinoma cell subpopulations surviving without ECM contact in pathological specimens of colorectal cancer. The occurrence of these subpopulations indicated anoikis resistance. In the present study, it is demonstrated that KRAS and BRAF mutations induce anoikis resistance in colon cancer (Caco‑2) cells. In 3D cultures, Caco‑2 cells transfected with mutated KRAS or BRAF formed multicellular structures analogous to anoikis‑resistant subpopulations in actual carcinomas, and serve as an in vitro model for anoikis resistance. Caco‑2 cell lines were constructed, with KRAS or BRAF mutations, using retroviral delivery. The current study investigated anoikis resistance using an Annexin V apoptosis test from suspension cultures. 3D in vitro cultures, which were generated in collagen‑matrigel mixtures, were assessed using confocal microscopy. 3D cultures embedded in paraffin were analyzed using conventional histopathology. In suspension cultures, Caco‑2 cells with KRAS or BRAF mutations indicated a significantly lower proportion of Annexin positivity than the native Caco‑2 cells, indicating that these mutations induce anoikis resistance in Caco‑2 cells. 3D cultures displayed native Caco‑2 cells forming polarized cysts with a single layer thick epithelium, whereas Caco‑2 cells with KRAS or BRAF mutations formed partially filled cystic structures or solid round structures where only the outermost layer was in contact with the ECM. Additionally, KRAS mutations induced reversed polarity to Caco‑2 cells along with the emergence of solid growth. The present study demonstrated that KRAS and BRAF mutations induce anoikis resistance in Caco‑2 colorectal cancer cells. The growth patterns generated from the KRAS and BRAF mutated cells in 3D cultures revealed a resemblance to the putative anoikis‑resistant subpopulations in actual carcinomas, including micropapillary structures and solid tumor cell islands. Additionally, KRAS mutation induced the emergence of inverted polarity. In conclusion, 3D cultures with modified Caco‑2 cells serve as a valid in vitro model for anoikis resistance and inverted polarity

Dynamics of adipose tissue macrophage populations after gastric bypass surgery

Abstract Objective: This case–control study aimed to analyze the dynamics of macrophage infiltration in subcutaneous adipose tissue following bariatric surgery or conservative treatment of obesity and to clarify whether these features predict the weight loss outcome after the surgery. Methods: Subcutaneous tissue samples taken before and 12 months after laparoscopic Roux-en-Y gastric bypass surgery (n = 39) or conservative (n = 43) treatment for obesity were analyzed. Fat cell size was determined, and with CD68 immunohistochemistry, crown-like structures (CLS) were counted and single macrophages were quantitated. Results: A major decline in CLS density from 4.1 (SD 3.5) to 1.1 (SD 0.8) per 1000 fat cells (p < 0.000) was found, regardless of the degree of weight loss after the surgery. Surgery had no effect on the fraction of infiltrating single-cell macrophages in subcutaneous adipose tissue. The abundance of these macrophage populations before the intervention did not predict the degree of postsurgery weight loss or suboptimal response to the surgery. Conclusions: The effect of gastric bypass on adipose tissue inflammatory status associates closely with CLS density even in subjects with suboptimal weight loss. The study suggests that factors related to bypass surgery other than weight loss modify the inflammatory response in adipose tissue

Gut microbiota-host interactions and juvenile idiopathic arthritis

Abstract Background: Juvenile idiopathic arthritis is the most common form of chronic arthritis in children. There is mounting evidence that the microbiota may influence the disease. Main body: Recent observations in several systemic inflammatory diseases including JIA have indicated that abnormalities in the contents of the microbiota may be factors in disease pathogenesis, while other studies in turn have shown that environmental factors impacting the composition of the microbiota, such as delivery mode and early exposure to antibiotics, affect the risk of chronic inflammatory diseases including JIA. Microbial alterations may predispose to JIA through a variety of mechanisms, including impaired immunologic development, alterations in the balances of pro- versus anti-inflammatory bacteria, and low-grade mucosal inflammation. Additional confirmatory studies of microbiota aberrations and their risk factors are needed, as well as additional mechanistic studies linking these alterations to the disease itself. Conclusions: The microbiota may influence the risk of JIA and other systemic inflammatory conditions through a variety of mechanisms. Additional research is required to improve our understanding of the links between the microbiota and arthritis, and the treatment implications thereof

Pathophysiology of reflux oesophagitis:role of Toll-like receptors 2 and 4 and Farnesoid X receptor

Abstract The pathogenesis of gastroesophageal reflux disease (GERD) is not fully understood. It involves the activation of mucosal immune-mediated and inflammatory responses. Toll-like receptors (TLR) 2 and TLR4 are pattern-recognition receptors of the innate immune system; they recognize microbial and endogenous ligands. Farnesoid X receptor (FXR) is a bile acid receptor that regulates the inflammatory response. We aimed to evaluate TLR2, TLR4 and FXR expression patterns in GERD. We re-evaluated 84 oesophageal biopsy samples according to the global severity (GS) score, including 26 cases with histologically normal oesophagus, 28 with histologically mild oesophagitis and 30 with severe oesophagitis. We used immunohistochemistry and in situ hybridization to assess the expression patterns of TLR2, TLR4 and FXR in oesophageal squamous cells. Immunohistochemistry showed that nuclear and cytoplasmic TLR2 was expressed predominantly in the basal layer of normal oesophageal epithelium. In oesophagitis, TLR2 expression increased throughout the epithelium, and the superficial expression was significantly more intensive compared to normal epithelium, p <0.01. Nuclear and cytoplasmic TLR4 was expressed throughout the thickness of squamous epithelium, with no change in oesophagitis. FXR was expressed in the nuclei of squamous cells, and the intensity of the expression increased significantly in oesophagitis (p <0.05). FXR expression correlated with basal TLR2. In situ hybridization confirmed the immunohistochemical expression patterns of TLR2 and TLR4. In GERD, TLR2, but not TLR4, expression was upregulated which indicates that innate immunity is activated according to a specific pattern in GERD. FXR expression was increased in GERD and might have a regulatory connection to TLR2

Histological assessment of stromal maturity as a prognostic factor in surgically treated gastric adenocarcinoma

Abstract Aims: Histological assessment of stromal maturity is a potential prognostic factor in colorectal cancer, but its applicability in gastric adenocarcinoma is completely unknown. The aim of this study was to evaluate the feasibility and prognostic significance of assessing stromal maturity in gastric adenocarcinoma. Methods and results: This study was conducted retrospectively in a cohort of 583 gastric adenocarcinoma patients treated surgically in Oulu University Hospital, Finland between 1983 and 2016. The original diagnostic slides were used for assessment of stromal maturity. Patients were divided into mature stroma and immature stroma groups, and stromal maturity was analysed in relation to 5‐year and overall survival (OS). The primary outcome of the study was 5‐year survival, and the secondary outcome was OS. The kappa‐coefficient for interobserver agreement was 0.609. Patients with immature stroma had worse 5‐year survival compared to patients with mature stroma [adjusted hazard ratio (HR) = 1.32, 95% confidence interval (CI) = 1.06–1.64]. Stromal maturity was significantly associated with 5‐year survival in intestinal‐type subgroup (adjusted HR = 0.63, 95% CI = 1.20–2.21), but not in the diffuse‐type subgroup (adjusted HR = 1.21, 95% CI = 0.87–1.70). Conclusions: Stromal maturity is an independent prognostic factor in gastric adenocarcinoma, and it can be analysed with moderate reproducibility

Brain tight junction protein expression in sepsis in an autopsy series

Abstract Background: Neuroinflammation often develops in sepsis along with increasing permeability of the blood-brain barrier (BBB), which leads to septic encephalopathy. The barrier is formed by tight junction structures between the cerebral endothelial cells. We investigated the expression of tight junction proteins related to endothelial permeability in brain autopsy specimens in critically ill patients deceased with sepsis and analyzed the relationship of BBB damage with measures of systemic inflammation and systemic organ dysfunction. Methods: The case series included all (385) adult patients deceased due to sepsis in the years 2007–2015 with available brain specimens taken at autopsy. Specimens were categorized according to anatomical location (cerebrum, cerebellum). The immunohistochemical stainings were performed for occludin, ZO-1, and claudin. Patients were categorized as having BBB damage if there was no expression of occludin in the endothelium of cerebral microvessels. Results: Brain tissue samples were available in 47 autopsies, of which 38% (18/47) had no expression of occludin in the endothelium of cerebral microvessels, 34% (16/47) developed multiple organ failure before death, and 74.5% (35/47) had septic shock. The deceased with BBB damage had higher maximum SOFA scores (16 vs. 14, p = 0.04) and more often had procalcitonin levels above 10 μg/L (56% vs. 28%, p = 0.045) during their ICU stay. BBB damage in the cerebellum was more common in cases with C-reactive protein (CRP) above 100 mg/L as compared with CRP less than 100 (69% vs. 25%, p = 0.025). Conclusions: In fatal sepsis, damaged BBB defined as a loss of cerebral endothelial expression of occludin is related with severe organ dysfunction and systemic inflammation

Localization of nucleic acid-sensing toll-like receptors in human and mouse pancreas

Abstract Nucleic acid‐sensing toll‐like receptors (TLRs) (3, 7, 8, 9) have a role both in antiviral innate immunity and in autoimmune disorders. We assessed the expression of TLR3, 7, 8 and 9 in human and mouse pancreas focusing on the subpopulations of cells in the Langerhans islets. We studied eight human samples with normal pancreatic islets and two samples from patients with type 1 diabetes. Additionally, 10 CD‐1 mouse pancreases were analysed. Immunohistochemical double‐stainings for the TLRs and insulin, glucagon or somatostatin, respectively, were performed along with appropriate controls. In human pancreas, strong immunoreaction of TLR7 and TLR8 was observed in the insulin‐positive beta cells, whereas glucagon‐ or somatostatin‐expressing cells of the islets were weakly stained or negative. In type 1 diabetes, the expression in islets was weak or lost (TLR7: p = 0.014, TLR8: p = 0.053), correlating with loss of beta cells. TLR3 and 9 were expressed only weakly with no correlation with specific cell types. In mouse pancreas, only TLR9 was detected. Intra‐pancreatic nerve ganglia strongly expressed TLR7. The strong expression of TLR7 and TLR8 in the beta cells of normal human islets could be an important piece in the puzzle of type 1 diabetes pathogenesis, and be linked with destruction of this particular subpopulation of the islet cells. In normal mice, only TLR9 can be constantly detected in the islets, highlighting differences between the species