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.

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.

Carbonic Anhydrases II, IX, and XII in Reflux Esophagitis.

BACKGROUND: The pathogenesis of gastroesophageal reflux disease (GERD) has not been resolved in detail. Esophageal epithelial cells provide resistance to acidic reflux via several mechanisms, many of which involve buffering acid with bicarbonate and transporting protons. Carbonic anhydrases (CAs) are enzymes that control the acid-base balance by catalyzing the reversible hydration of carbon dioxide to produce bicarbonate and hydrogen ions. AIMS: We aimed to determine the immunohistochemical expression patterns of CAII, CAIX, and CAXII in the normal esophageal squamous epithelium and in patients with GERD. METHODS: We evaluated 82 biopsy samples, including 26 with a histologically normal esophagus, 26 with histologically mild esophagitis, and 30 with severe esophagitis. Expression patterns of CAII, CAIX, and CAXII in the esophageal squamous epithelium were determined by immunohistochemical staining. RESULTS: Cytoplasmic CAII expression was predominantly detected in the upper luminal part of the squamous epithelium and was significantly (p < 0.01) increased in GERD. Expression of CAIX was essentially membranous. The isozyme was constantly present in the peripapillary cells. In the interpapillary areas, clustered expression was observed to emerge and increase significantly (p < 0.01) in esophagitis. CAXII expression was the most abundant of the isozymes and was mainly membranous. In the normal squamous epithelium, CAXII expression was confined to the basal layer; in severe esophagitis, CAXII expression increased significantly in both basal (p < 0.05) and superficial (p < 0.01) halves of the epithelium. CONCLUSIONS: We demonstrate upregulated expression of CAII, CAIX, and CAXII in GERD. The increase in expression likely contributes to esophageal epithelial resistance to acidic reflux.

TLR2 and TLR4 in colorectal cancer: relationship to tumor necrosis and markers of systemic inflammation.

In colorectal cancer (CRC), systemic inflammation is associated with poor prognosis, but the underlying mechanisms are not fully characterized. Tumor necrosis may contribute to systemic inflammation by inducing interleukin (IL)-6 signaling, and proinflammatory cytokines such as IL-6 and IL-8, and matrix metalloproteinase (MMP)-8 also are linked to adverse CRC outcomes. Because Toll-like receptors (TLRs) are important mediators of inflammatory responses, we investigated the roles of TLR2 and TLR4 in CRC-associated systemic inflammatory responses, especially tumor necrosis. In 118 patients with CRC, extensive tumor necrosis was associated with low TLR4 expression in tumor cells. Tumor cell TLR4 expression was inversely correlated with serum IL-6 and MMP-8 levels, blood total leukocyte and neutrophil counts, and serum C-reactive protein levels. Tumor cell TLR2 expression was not significantly associated with necrosis or systemic inflammation, but low expression in normal mucosa was linked to high serum MMP-8 and IL-8. These findings indicate that tumor necrosis is associated with low TLR4 expression in cancer cells and that low TLR4 expression correlates with a strong systemic inflammatory response. The low TLR2 expression in normal mucosa and its association with systemic inflammation suggest that the normal mucosa may reflect or contribute to the systemic inflammatory response.

Alterations in serum amino-acid profile in the progression of colorectal cancer: associations with systemic inflammation, tumour stage and patient survival.

BACKGROUND: Cancer cachexia is a complex wasting syndrome affecting patients with advanced cancer, with systemic inflammation as a key component in pathogenesis. Protein degradation and release of amino acids (AAs) in skeletal muscle are stimulated in cachexia. Here, we define factors contributing to serum AA levels in colorectal cancer (CRC). METHODS: Serum levels of nine AAs were characterised in 336 CRC patients and their relationships with 20 markers of systemic inflammatory reaction, clinicopathological features of cancers and patient survival were analysed. RESULTS: Low serum glutamine and histidine levels and high phenylalanine levels associated with indicators of systemic inflammation, including high modified Glasgow Prognostic Score, high blood neutrophil/lymphocyte ratio and high serum levels of CRP, IL-6 and IL-8. Low levels of serum glutamine, histidine, alanine and high glycine levels also associated with advanced cancer stage and with poor cancer-specific survival in univariate analysis. CONCLUSIONS: In CRC, serum AA levels are associated with systemic inflammation and disease stage. These findings may reflect muscle catabolism induced by systemic inflammation in CRC.

Platelet count, aspirin use, and characteristics of host inflammatory responses in colorectal cancer.

BACKGROUND: Platelets not only contribute to hemostasis but also to the regulation of inflammatory reactions and cancer pathogenesis. We hypothesized that blood platelet count would be associated with systemic inflammation, the densities of tumor infiltrating immune cells, and survival in colorectal cancer (CRC), and these relationships could be altered by aspirin use. METHODS: We measured blood platelet count in a cohort of 356 CRC patients and analyzed its relationships with tumor and patient characteristics including aspirin use, markers of systemic inflammation (modified Glasgow Prognostic Score, mGPS; serum levels of CRP, albumin, and 13 cytokines), blood hemoglobin levels, five types of tumor infiltrating immune cells (CD3, CD8, FoxP3, Neutrophil elastase, mast cell tryptase), and survival. RESULTS: Platelet count inversely correlated with blood hemoglobin levels (p < 0.001) and positively correlated with serum levels of CRP and multiple cytokines including IL-1RA, IL-4, IL-6, IL-7, IL-8, IL-12, IFNγ, and PDGF-BB (p < 0.001 for all), while aspirin use was not associated with the levels of systemic inflammatory markers. High platelet count was also associated with high mGPS (p < 0.001) but did not show statistically significant multivariable adjusted associations with the densities of tumor infiltrating immune cells. Higher platelet counts were observed in higher tumor stage (p < 0.001), but platelet count or aspirin use were not associated with patient survival. CONCLUSIONS: High platelet count is associated with systemic inflammation in CRC. This study could not demonstrate statistically significant associations between platelet count, aspirin use, and the densities of tumor infiltrating immune cells.

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

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.

Tumour-stroma ratio and prognosis in gastric adenocarcinoma.

BACKGROUND: Tumour microenvironment, including tumour-stroma ratio (TSR), might help identifying high-risk cancer patients. However, the significance of TSR in gastric cancer is unclear, especially in the intestinal and diffuse subtypes. The aim of this study was to investigate the tumour-stroma ratio in gastric adenocarcinoma, and its intestinal and diffuse histological subtypes, in relation to prognosis. METHODS: Five hundred and eighty-three gastric adenocarcinoma patients who underwent surgery in Oulu University hospital during years 1983-2016 were included in this retrospective cohort study. TSR was analysed from the slides that were originally used for diagnostic purposes. Patients were divided into stroma-poor (≤50% stroma) and stroma-rich (>50% stroma) groups and TSR was analysed in relation to 5-year mortality and overall mortality. RESULTS: Patients with stroma-rich tumours had worse 5-year prognosis (HR 1.80, 95% CI 1.41-2.28) compared to stroma-poor tumours. Stratified analysis showed that stroma-rich tumours had worse 5-year prognosis in both intestinal (HR 1.68, 95% CI 1.24-2.27) and diffuse histological types (HR 2.09, 95% CI 1.35-3.23) compared to stroma-poor tumours, respectively. CONCLUSIONS: High proportion of stroma is an independent prognostic factor in both intestinal and diffuse histological subtypes of gastric adenocarcinoma.

High-serum MMP-8 levels are associated with decreased survival and systemic inflammation in colorectal cancer.

BACKGROUND: Matrix metalloproteinase-8 (MMP-8) is a protease mainly expressed by neutrophils that cleaves numerous substrates, including collagens and cytokines. We have previously shown that serum MMP-8 levels increase in colorectal cancer (CRC) and correlate with distant metastasis. However, short follow-up in our prospective cohort did not enable survival analyses at the time of the first publication. METHODS: Preoperative serum MMP-8 levels were measured by immunofluorometric assay in 271 CRC patients and related to clinicopathological parameters, markers of systemic inflammation (modified Glasgow Prognostic Score, mGPS; serum levels of C-reactive protein (CRP), albumin and 13 cytokines), the density of six types of tumour-infiltrating immune cells and survival. RESULTS: Increased MMP-8 levels associated with higher mGPS and higher serum levels of CRP and several cytokines, including IL-1ra, IL-7 and IL-8 (p < 0.001 for all). Serum MMP-8 negatively correlated with tumour-infiltrating mast cells (invasive margin: p = 0.005, tumour centre: p = 0.010). The patients with high-serum MMP-8 levels (>100 ng/mL) had poor cancer-specific survival, independent of tumour stage, grade, lymphatic invasion, patient age, BRAF VE1 immunohistochemistry, mismatch repair deficiency, Immunoscore and mGPS (multivariate HR 2.12, 95% CI 1.21-3.71, p = 0.009). CONCLUSIONS: High-serum MMP-8 levels are associated with systemic inflammation and adverse outcome in CRC.

Weak HIF-1alpha expression indicates poor prognosis in resectable pancreatic ductal adenocarcinoma.

BACKGROUND: HIF-1alpha and CAIX proteins are commonly expressed under hypoxic conditions, but other regulatory factors have been described as well. Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxia and strong stromal reaction and has a dismal prognosis with the currently available treatment modalities. METHODS: We investigated the expression and prognostic role of HIF-1alpha and CAIX in PDAC series from Northern Finland (n = 69) using immunohistochemistry. RESULTS: In our PDAC cases, 95 and 85% showed HIF-1alpha and CAIX expression, respectively. Low HIF-1alpha expression correlated with poor prognosis, and multivariate analysis identified weak HIF-1alpha intensity as an independent prognostic factor for PDAC-specific deaths (HR 2.176, 95% CI 1.216-3.893; p = 0.009). There was no correlation between HIF-1alpha and CAIX expression levels, and the latter did not relate with survival. CONCLUSIONS: Our findings are in contrast with previous research by finding an association between low HIF-1alpha and poor prognosis. The biological mechanisms remain speculative, but such an unexpected relation with prognosis and absence of correlation between HIF-1alpha and CAIX suggests that the prognostic association of HIF-1alpha may not directly be linked with hypoxia. Accordingly, the role of HIF-1alpha might be more complex than previously thought and the use of this marker as a hypoxia-related prognostic factor should be addressed with caution.

The relationships between serum cytokine levels and tumor infiltrating immune cells and their clinical significance in colorectal cancer.

Increased inflammatory cell infiltration correlates to improved survival in colorectal cancer (CRC). Development and progression of CRC is associated with alterations in serum cytokine levels but their significance is not well defined. In this study, we investigated the relationships between the serum levels of 13 cytokines and the densities of eight types of tumor infiltrating inflammatory cells and their impact on disease-free survival (DFS), cancer-specific survival (CSS) and overall survival (OS) in a prospectively recruited group of 147 CRC patients. There were strong positive correlations between the serum concentrations of different cytokines, as well as between the different types of tumor infiltrating immune cells, whereas the associations between serum cytokines and tumor infiltrating immune cells were generally weak. High serum IL-12 levels associated with increased densities of peritumoral CD8(+) T cells, intraepithelial CD3(+) T cells and intratumoral neutrophils, while high serum CCL4 levels associated with increased densities of peritumoral CD68(+) cells. In multivariate survival models, increased infiltration of intraepithelial CD3(+) T cells and increased serum CCL4 associated with improved DFS, whereas higher intratumoral CD83(+) dendritic cell density and increased serum interferon gamma levels associated with improved CSS and OS. Also high density of peritumoral CD3(+) T cells associated with improved CSS. In conclusion, serum cytokines and tumor infiltrating immune cells in CRC represent entities with high intragroup correlations but relatively weak intergroup correlations. The results suggest that tumor infiltrating CD3(+) T cells, CD83(+) dendritic cells, serum CCL4 and serum interferon gamma represent relevant markers of disease outcome.

Clinical impact and network of determinants of tumour necrosis in colorectal cancer.

BACKGROUND: The disease outcome in colorectal cancer (CRC) can vary in a wide range within the same tumour stage. The aim of this study was to clarify the prognostic value and the determinants of tumour necrosis in CRC. METHODS: The areal proportion (%) of tumour tissue showing coagulative necrosis was evaluated in a cohort of 147 CRC patients and correlated with basic clinicopathological characteristics, microvascular density (MVD), cell proliferation rate, KRAS and BRAF mutations, and survival. To validate the prognostic significance of tumour necrosis, an independent cohort of 418 CRC patients was analysed. RESULTS: Tumour necrosis positively correlated with tumour stage (P=8.5E-4)-especially with T class (4.0E-6)-and inversely correlated with serrated histology (P=0.014), but did not significantly associate with cell proliferation rate, MVD, and KRAS or BRAF mutation. Abundant (10% or more) tumour necrosis associated with worse disease-free survival independent of stage and other biological or clinicopathological characteristics in both cohorts, and the adverse effect was directly related to its extent. High CD105 MVD was also a stage independent marker for worse disease-free survival. CONCLUSIONS: Tumour necrosis percentage is a relevant histomorphological prognostic indicator in CRC. More studies are needed to disclose the mechanisms of tumour necrosis.

Gremlin1 expression associates with serrated pathway and favourable prognosis in colorectal cancer.

AIMS: Gremlin1 is a bone morphogenetic protein (BMP) antagonist with a suggested role in colorectal cancer (CRC) progression. We have analysed Gremlin1 protein expression in CRC and assessed its correlation with clinicopathological characteristics, including developmental pathway and prognosis. METHODS AND RESULTS: Material included a non-selected series of 148 surgically treated CRC cases. The tumour-node-metastasis (TNM) stage, histological grade and inflammatory infiltrate at the invasive margin were assessed, and tumours were classified to serrated or non-serrated types. Immunohistochemistry was conducted to evaluate Gremlin1 expression. Prognosis (60-month follow-up) was analysed by Kaplan-Meier methods and Cox regression analysis. Gremlin1 expression was detected in epithelial cells both in normal mucosa and in carcinomas. Abundant expression in carcinomas associated with low TNM stage (P = 0.044), low histological grade (P = 0.044), serrated histology (P = 0.033 or P = 0.053 depending on the classification cut-off) and intensive inflammatory infiltrate at the invasive margin (P = 0.044), and was a stage independent indicator of extended survival (P = 0.029). CONCLUSIONS: Gremlin1 protein expression in CRC associates with low tumour stage and extended survival independently of tumour stage, suggesting that it represents a relevant prognostic indicator in CRC. High expression in carcinomas with serrated histology suggests a potential role for Gremlin1 in the serrated pathway of CRC.

Toll-like receptor 9 mediates invasion and predicts prognosis in squamous cell carcinoma of the mobile tongue.

BACKGROUND: Toll-like receptor 9 (TLR9) is a cellular receptor, which recognizes bacterial and host-derived DNA. Stimulation of TLR9 induces cellular invasion via matrix metalloproteinase 13 (MMP-13). The aim of this study was to evaluate the role of TLR9 in invasion of oral tongue squamous cell carcinoma (OTSCC). METHODS: The effects of TLR9 ligands on oral squamous cell carcinoma cell lines were studied with invasion and migration assays, as well as in a myoma organotypic model. RESULTS: The TLR9 ligand, CpG-ODN, increased invasion and migration in OTSCC lines. These effects were reduced by TLR9 siRNA or inhibition with TLR9 antibodies. Immunohistochemical analysis of tissues from 195 patients with OTSCC revealed that TLR9 was expressed in 181/195 carcinomas. The expression of TLR9 was higher in the malignant cells than in the normal epithelium. High TLR9 expression was associated with high MMP-13 expression and poor differentiation. High TLR9 expression was also identified as an independent predictor of poor prognosis (HR 1.810, 95% CI [1.053-3.112]). CONCLUSION: Toll-like receptor 9 mediates OTSCC invasion and migration in vitro and is an independent prognostic factor of OTSCC. Inhibition of TLR9 may be a novel therapeutic opportunity in oral cancer.

Characteristics and significance of colorectal cancer associated lymphoid reaction.

A subset of colorectal cancers (CRCs) exhibits so-called Crohn's like lymphoid reaction (CLR), an inflammatory reaction pattern that consists of numerous transmural lymphoid aggregates. However, the composition of these aggregates, their biological mechanisms and their prognostic significance are not well-defined. We analyzed two CRC cohorts (418 and 149 patients) and determined clinicopathological features including survival. A new method for evaluating CLR based on counting the areal density of the lymphoid follicles (CLR density) was adopted. Immune cell densities at intratumoral and peritumoral regions, as well as the composition of the lymphoid follicles, were studied by immunohistochemistry. We found that CLR comprised of lymphoid aggregates with no evidence of granuloma formation. High CLR density associated with lower tumor stage, lack of preoperative radiotherapy or chemoradiotherapy and deficient mismatch repair enzyme expression. CLR density had positive correlations with peritumoral and intratumoral densities of CD83(+) mature dendritic cells and T cells. High CLR density associated with better survival and had prognostic value that was independent of stage, Klintrup-Mäkinen score for peritumoral inflammation and the numbers of tumor infiltrating T cells. CLR density evaluation had excellent intraobserver and interobserver agreement. In conclusion, the results suggest that CLR contributes to the adaptive antitumor immunity. Quantitative evaluation of CLR density is a relevant prognostic indicator in CRC.

Toll-like receptor 9 expression in mucoepidermoid salivary gland carcinoma may associate with good prognosis.

BACKGROUND: Mucoepidermoid carcinoma (MEC) is the most common salivary gland malignancy. Although several biomarkers have been evaluated, histological grade remains the most valuable prognostic marker. Toll-like receptor 9 (TLR9) is an immune receptor recognizing microbial DNA. Its expression associates with prognosis or cancer properties in several cancers. This study examined the role of TLR9 in MEC. METHODS: Sixty patients with salivary gland MEC were collected from two Finnish university hospitals, and tumor samples were stained for TLR9. Salivary gland high-grade MEC cell line (UT-MUC-1) was cultured to assess TLR9 and MMP-13 expression. The function of TLR9 was studied in vitro using traditional Matrigel(®) invasion assay and novel human myoma organotypic model. RESULTS: Cancer-specific survival was related with tumor grade (P = 0.01), and there were no deaths in patients with low-grade MEC. TLR9 was expressed in 56 of 60 (93%) tumors. High TLR9 expression indicated better survival in the patient series (P = 0.002) and showed a trend for association with lower disease stage (P = 0.06) and higher differentiation grade (P = 0.068). In multivariate analysis, TLR9 expression was prognostically insignificant due to heavy correlation to disease stage and higher gradus. Treating UT-MUC-1 cells with TLR9 ligand CpG in vitro induced MMP-13 expression and invasion in Matrigel(®) invasion assay, whereas decreased invasion was seen in myoma organotypic model. CONCLUSION: Functional TLR9 is present in salivary MEC, and high level of expression may indicate good prognosis. However, more studies are needed to evaluate biological consequences of TLR9 interaction in tumor cells.

Interleukin 6 gene polymorphism -174 is associated with the diffuse type gastric carcinoma.

The aim of this study was to assess the significance of the interleukin 6 gene polymorphism -174 in gastric cancer risk. The interleukin 6 -174 G/C (rs1800795) gene polymorphisms was analyzed in gastric cancer, peptic ulcer, and nonulcer dyspepsia patients and in healthy control subjects and the data were correlated with the histopathological features of the patients' biopsies. The interleukin 6 -174 GG and GC genotypes have been previously associated with high interleukin 6 serum levels. We discovered that the interleukin 6 -174 GG and GC genotypes are associated with an increased risk of the diffuse histologic subtype of gastric carcinomas (OR: 6.809, P = 0.034), but absent in the intestinal type carcinomas (OR: 1.109, P = 0.908). No significant associations with peptic ulcer, gastric atrophy, or intestinal metaplasia were seen. Our results demonstrate that the interleukin 6 -174 GG and GC genotypes increase the risk of the diffuse type gastric carcinoma, but not the intestinal type gastric carcinoma or its precursor conditions, including atrophy or intestinal metaplasia. Thus, interleukin 6 seems to be an important carcinogenetic factor in the diffuse type gastric adenocarcinoma and its carcinogenetic effect could be noninflammatory.

Dynamics of adipose tissue macrophage populations after gastric bypass surgery.

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.

Serum and tissue CD23, IL-15, and FasL in cow's-milk protein-sensitive enteropathy and in coeliac disease.

OBJECTIVES: The aim of the study was to explore pathogenesis and find new serum markers for cow's-milk-sensitive enteropathy (CMSE) and coeliac disease (CD). We assessed the intestinal expression and serum concentration of CD23, IL-15, and FasL. We hypothesised that the serum levels of CD23, a protein expressed in the lymphoid follicles, would be associated with lymphonodular hyperplasia (LNH), a feature characteristic of CMSE. We also presumed that interleukin (IL)-15 and FasL, functionally connected with proliferation and apoptosis of the intraepithelial lymphocytes (IELs), would relate with the increased numbers of IELs present in both CMSE and CD. METHODS: Twenty-three children with CMSE, 20 with untreated CD, and 14 controls were studied for CD3, α/ß- and γ/δ-expressing IELs, and for duodenal and ileal expression of CD23, FasL, and IL-15 by immunohistochemistry, and for serum concentration of sCD23, sFasL, and sIL-15 by enzyme-linked immunosorbent assay. RESULTS: There was a trend for increase in sCD23 serum levels in untreated CMSE and in CD (P = 0.074; P = 0.077). CD23 was expressed in the mucosal germinal centres, but sCD23 was not related to presence of LNH. In CMSE, there was a trend for increase in serum sFasL (P = 0.07) and high levels associated with LNH (P = 0.025) and correlated with the IEL numbers (P < 0.05). Mucosal high endothelial venules adjacent to lymphoid follicles showed an intensive FasL expression. CONCLUSIONS: Serum sCD23 shows a trend of increment in CMSE and CD, and in the latter, sCD23 level may provide information about the severity of villous atrophy. In CMSE, high serum sFasL indicates both LNH and an increase of IELs, suggesting importance of FasL-mediated mechanisms in the pathogenesis of these features characteristic of CMSE. Further studies are necessary to evaluate whether intensive FasL expression in mucosal high endothelial venules presents a regulatory element in mucosal immunity.

Monocarboxylate Transporters 1 and 4 and Prognosis in Small Bowel Neuroendocrine Tumors.

Monocarboxylate transporters (MCTs) are cell membrane proteins transporting lactate, pyruvate, and ketone bodies across the plasma membrane. The prognostic role of MCTs in neuroendocrine tumors is unknown. We aimed to analyze MCT1 and MCT4 expression in small bowel neuroendocrine tumors (SB-NETs). The cohort included 109 SB-NETs and 61 SB-NET lymph node metastases from two Finnish hospitals. Tumor samples were immunohistochemically stained with MCT1 and MCT4 monoclonal antibodies. The staining intensity, percentage of positive cells, and stromal staining were assessed. MCT1 and MCT4 scores (0, 1 or 2) were composed based on the staining intensity and the percentage of positive cells. Survival analyses were performed with the Kaplan-Meier method and Cox regression, adjusted for confounders. The primary outcome was disease-specific survival (DSS). A high MCT4 intensity in SB-NETs was associated with better DSS when compared to low intensity (85.7 vs. 56.6%, p = 0.020). A high MCT4 percentage of positive cells resulted in better DSS when compared to a low percentage (77.4 vs. 49.1%, p = 0.059). MCT4 scores 0, 1, and 2 showed DSS of 52.8 vs. 58.8 vs. 100% (p = 0.025), respectively. After adjusting for confounders, the mortality hazard was lowest in the patients with a high MCT4 score. MCT1 showed no association with survival. According to our study, a high MCT4 expression is associated with an improved prognosis in SB-NETs.

A Straightforward Method for Adipocyte Size and Count Analysis Using Open-source Software QuPath.

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.