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Barrett’s esophagus progresses to high-grade dysplasia or cancer along the well-established metaplasia-dysplasia-adenocarcinoma sequence. The aim of this study was to evaluate the value of p53, Ki67, and toll-like receptor 5 (TLR5) in prediction of malignant progression of Barrett’s metaplasia and low-grade dysplasia. This was a retrospective matched case–control study based on Northern and Central Finland population. Patients diagnosed with esophageal high-grade dysplasia or adenocarcinoma were included. From these patients, all previous endoscopy samples were obtained along with original diagnostic HE-slides and clinical data. Age- and sex-matched patients with non-progressing Barrett’s metaplasia and low-grade dysplasia confirmed with follow-up endoscopies were used as controls. Two gastrointestinal pathologist re-reviewed all original HE-slides, and newly made sections to confirm representative tissue material blinded from clinical data. p53, Ki67, and TLR5 were immunohistochemically stained. Final cohort included 45 patients with progressive Barrett’s metaplasia (n = 21) or low-grade dysplasia (n = 24), and 92 patients with non-progressive Barrett’s metaplasia (n = 52) or low-grade dysplasia (n = 40). In Barrett’s metaplasia, aberrant p53 expression was observed in 6% of samples in progressors and 0% in non-progressors. In low-grade dysplasia, aberrant p53 was seen in 56% of samples in progressors and 17% in non-progressors (Odd’s ratio 6.7, 95% CI 1.8–24.6). Ki67 or TLR5 showed no association with disease progression. In this matched case–control study, p53 expression associated with a high risk of malignant progression in Barrett’s low-grade dysplasia. Routine staining of p53 is indicated in expert confirmed low-grade dysplasia.

Anoikis refers to apoptosis induced by the loss of contact with the extracellular matrix. Anoikis resistance is essential for metastasis. We have recently shown that it is possible to quantitatively evaluate putative anoikis resistant (AR) subpopulations in colorectal carcinoma (CRC). Abundance of these multi-cell structures is an independent marker of adverse prognosis. Here, we have quantified putative AR subpopulations in lymph node (LN) metastases of CRC and evaluated their prognostic value and relationship with the characteristics of primary tumors. A case series included 137 unselected CRC patients, 54 with LN metastases. Areal densities (structures/mm²) of putative AR structures in primary tumors had been analyzed previously and now were determined from all nodal metastases (n = 183). Areal density of putative AR structures was higher in LN metastases than in primary tumors. Variation of the areal density within different LN metastases of a single patient was lower than between metastases of different patients. Abundance of putative AR structures in LN metastases was associated with shorter cancer specific survival (p = 0.013), and this association was independent of T and N stages. Abundance of putative AR structures in primary tumors and LN metastases had a cumulative adverse effect on prognosis. Enrichment of putative AR subpopulations in LN metastases suggest that in metastasis formation, there is a selection favoring cells capable of forming these structures. Higher intra-case constancy relative to inter-case variation suggests that such selection is stable in metastasis development. Our findings indirectly support the biological validity of our concept of putative AR structures.

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.

Colorectal cancer (CRC) is the third most common cancer worldwide, and its incidence is increasing. Inflammation associates with the pathogenesis of cancer by several mechanisms. Toll-like receptors (TLRs) mediate and regulate the inflammatory response and their role in cancer progression has been established in many cancers. This study aimed to clarify the roles of TLR2 and TLR4 and their significance in the development, progression, and prognosis of CRC in relation to inflammation in a series of 149 CRC patients operated in Oulu University Hospital (2006–2010). We characterized the tissue expression in CRC tumors and serum levels of TLR2 and TLR4, and the associations of expression patterns with tumor features and the prognosis of cancer. We found downregulation of TLR4 and upregulation of TLR2 in CRC, and low expression of TLR4 in the invasive front of the tumor predicted poor prognosis and metastatic disease. Serum TLR2 levels were inversely associated with systemic inflammation in patients with CRC, and our findings suggested that serum TLR2 levels might depend more on normal colorectal mucosa contributions than on tumor tissue. Also, the mean serum TLR4 levels were lower in patients than in controls. We also found that tumor necrosis in CRC associated with low TLR4 expression in carcinoma epithelium, and low TLR4 expression associated with systemic inflammation. Tumoral TLR2 expression did not correlate with necrosis from systemic inflammation, but low expression of TLR2 in normal mucosa was linked to indicators of systemic inflammation, supporting the concept that the normal colon mucosa may contribute to the regulation of systemic inflammation. In conclusion, TLR2 and TLR4 showed divergent roles in CRC. TLR2 was upregulated and TLR4 downregulated in CRC. Downregulation of TLR4 was related to tumoral necrosis and adverse prognosis. High normal mucosa TLR2 expression associated with high serum TLR2 concentration and signs of lower systemic inflammatio, Tiivistelmä Paksu- ja peräsuolisyöpä on maailmanlaajuisesti kolmanneksi yleisin syöpä ja sen ilmaantuvuus on kasvussa. Tulehdus vaikuttaa syövän syntyyn ja kehitykseen useilla mekanismeilla. Kohonnut elimistön tulehdusvaste liittyy huonoon ennusteeseen, mutta paikallinen tulehdus kasvaimen leviämisvyöhykkeessä on yhdistetty parempaan selviytymiseen. Tollin kaltaiset reseptorit (TLR) välittävät ja säätelevät tulehdusvastetta, ja niiden merkitys syövän etenemisessä on osoitettu monessa syövässä. Tämän tutkimuksen tarkoitus oli selvittää TLR2:n ja TLR4:n rooleja ja merkitystä tulehduksen kannalta paksu- ja peräsuolisyövän kehittymisessä, etenemisessä ja ennusteessa 149:n Oulun yliopistollisessa sairaalassa vuosina 2006–2010 leikatun potilaan aineistossa. Määritimme TLR2:n ja TLR4:n ilmentymistasot syöpäkasvaimissa ja niiden seerumitasot sekä näiden yhteyksiä kasvaimen ominaisuuksiin ja syövän ennusteeseen. TLR4:n ilmentyminen oli vähentynyt ja TLR2:n lisääntynyt kasvaimissa, ja matala TLR4:n ilmentyminen kasvaimen leviämisvyöhykkeessä liittyi huonompaan ennusteeseen ja etäpesäkkeiseen tautiin. TLR2:n seerumitasot korreloivat käänteisesti elimistön tulehdusvasteeseen syöpäpotilailla, ja löydöksemme viittaa siihen, että TLR2:n seerumipitoisuuteen myötävaikuttaa enemmän normaalin paksusuolen limakalvo kuin kasvainkudos. TLR4:n seerumitasot olivat matalammat potilailla verrattuna verrokkeihin. Myös kasvaimen kuolio liittyi matalaan TLR4:n ilmentymiseen kasvaimessa, mikä puolestaan oli yhteydessä elimistön tulehdukseen. Kasvaimen TLR2 ilmentyminen ei liittynyt kasvaimen kuolioon tai tulehdukseen, mutta matala TLR2:n ilmentyminen normaalilla limakalvolla liittyi elimistön tulehdusta kuvaaviin muuttujiin, mikä tukee ajatusta normaalin limakalvon vaikutuksesta elimistön tulehduksen säätelyssä. Yhteenvetona voidaan todeta, että TLR2:n ja TLR4:n roolit paksu- ja peräsuolisyövässä ovat erilaiset: TLR2:n ilmentyminen lisääntyy ja TLR4:n vähenee. TLR4:n ilmentymisen väheneminen lii

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.

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.

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.

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.

Inflammatory and neoplastic diseases of the esophagus and duodenum cause a significant burden to healthcare and economies worldwide. Their etiology is multifactorial, but precursor lesions and risk factors are identifiable. Gastroesophageal reflux disease (GERD) is exceedingly common in western countries and causes the most medical health care expenses of all gastrointestinal diseases. GERD is the main risk factor for esophageal adenocarcinoma (EAC)––it can complicate into Barrett’s esophagus (BE) and further into esophageal adenocarcinoma. EAC has a poor overall prognosis still today, despite evolving management. High-quality prognostic biomarkers for EAC and progression of BE into cancer are lacking. Unlike esophageal diseases, nonampullary duodenal adenomas and adenocarcinoma (DAC) are rare neoplasias, although their incidence is slowly rising. In these rare diseases, the molecular pathological profiling is still incomplete, and the range of applicable prognostic biomarkers is very limited. Carbonic anhydrases (CA) are highly active, pH-controlling enzymes of the body. They participate in various physiological processes, but their primary function is to preserve optimal intra- and extracellular pH conditions in normal tissues. In cancer, CAII, CAIX, and CAXII seem to contribute to the development of malignant pH arrangement. The malignant pH gradient––slightly alkaline intracellular pH and extracellular acidosis––is in many ways critical to cancer progression, by benefiting cancer cell abilities to grow, migrate, invade, and metastasize. This study’s aim was to investigate the immunohistochemical expression patterns and potential roles of CAII, CAIX, and CAXII in the predisposing, premalignant and malignant conditions of distal esophagus and nonampullary duodenum. CA expression was evaluated in normal esophagus, GERD, Barrett’s esophagus, esophageal adenocarcinoma, normal duodenum, nonampullary duodenal adenomas and duodenal adenocarcinoma. The patient c, Tiivistelmä Ruokatorven ja pohjukaissuolen tulehdukselliset sairaudet ja kasvaimet aiheuttavat merkittäviä kustannuksia ja kuormittavat terveydenhuoltoa kaikkialla maailmassa. Näiden sairauksien etiologia on monitekijäinen, mutta hoidettavia esiasteita ja riskitekijöitä on tunnistettu ja niiden tutkiminen on potilaan hoidon ja ennusteen kannalta tärkeää. Ruokatorven refluksitauti on yleistynyt pitkään maailmanlaajuisesti. Sen hoito aiheuttaa nykyään suurimmat lääkehoidolliset kustannukset kaikista ruuansulatuskanavan sairauksista. Refluksitauti on myös ruokatorven adenokarsinooman merkittävin riskitekijä — Barrettin ruokatorvi on refluksitaudin komplikaatio ja ruokatorven adenokarsinooman esiaste. Ruokatorven adenokarsinooman ennuste on edelleen huono. Biomerkkiaineita ruokatorvisyövän ennusteen ja Barrettin ruokatorven muuntumisriskin selventämiseksi on etsitty pitkään, mutta käyttökelpoisia merkkiaineita ei ole juurikaan kliinisessä käytössä. Ei-ampullaarisen pohjukaissuolen adenoomat ja adenokarsinooma ovat sen sijaan hyvin harvinaisia kasvaimia. Tämän vuoksi niiden molekyylipatologisten ennustetekijöiden ja biomerkkiaineiden määritystyö on vielä epätäydellistä verrattuna yleisempiin syöpätyyppeihin. Hiilihappoanhydraasit (CA) ovat korkean aktiviteetin entsyymejä, jotka kontrolloivat pH:n muutoksia elimistössä. Ne osallistuvat lukuisiin kehon fysiologisiin prosesseihin, mutta niiden pääasiallinen tehtävä on säilyttää optimaaliset pH-olosuhteet soluissa ja niiden ulkopuolella. Syöpäsoluissa CAII, IX ja XII kuitenkin näyttävät olevan osallisina nk. pahanlaatuisen pH-gradientin syntymisessä. Tässä suhteessa niiden liiallinen tai liian vähäinen toiminta vaikuttaa olevan hyödyllistä syöpäsoluille. Tämän tutkimuksen tavoitteena oli selvittää CAII, IX ja XII:n immunohistokemiallinen ilmentyminen ja potentiaalinen merkitys ruokatorven ja pohjukaissuolen syövissä sekä niille altistavissa muutoksissa. Hiilihappoanhydraasien ilmentyminen määritettiin normaalissa ruokatorves

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.

Purpose: To examine and compare the prognostic value of immune cell score (ICS) and Klintrup–Mäkinen (KM) grade in gastric cancer. Methods: Gastric adenocarcinoma tissues from samples of 741 patients surgically treated in two hospitals in Finland were assessed for ICS and KM grade. Cox regression with adjustment for confounders provided hazard ratios (HRs) and 95% CIs. Subgroup analyses were performed in intestinal and diffuse type subgroups. The primary outcome was 5-year overall survival. Results: High ICS was associated to longer 5-year survival (adjusted HR 0.70, 95% CI 0.52–0.94), compared to low ICS. The difference was significant in intestinal type subgroup (adjusted HR 0.54, 95% CI 0.36–0.81) but not in diffuse type subgroup (adjusted HR 0.92, 95% CI 0.58–1.46). High KM grade was an independent prognostic factor for longer 5-year overall survival (adjusted HR 0.59, 95% CI 0.45–0.77) in both intestinal (adjusted HR 0.61, 95% CI 0.44–0.85) and diffuse subgroups (adjusted HR 0.52, 95% CI 0.31–0.86). ICS and KM grade were moderately correlated (ρ = 0.425). When both immune cell score and KM grade were included in the regression analysis, only KM grade remained prognostic. Conclusions: Both ICS and KM grade are prognostic factors in gastric adenocarcinoma, but immunohistochemistry-based ICS might not have additional prognostic value over hematoxylin–eosin-based KM grade.

Purpose: The Finnish National Esophago-Gastric Cancer Cohort (FINEGO) was established with the aim of identifying factors that could contribute to improved outcomes in oesophago-gastric cancer. The aim of this study is to describe the patients with gastric cancer included in FINEGO. Participants: A total of 10 457 patients with gastric cancer or tumour diagnosis in the Finnish Cancer Registry or the Finnish Patient Registry during 1987–2016 were included in the cohort, with follow-up from Causes of Death Registry until 31 December 2016. All of the participants were at least 18 years of age, and had undergone either resectional or endoscopic mucosal surgery with curative or palliative intent. Findings: to date Of the 10 457 patients, 90.1% were identified to have cancer in both cancer and patient registries. In all, the median age was 70 at the time of surgery, 54.5% of the patients were men and 64.4% had no comorbidities. Education data were available for 31.1% of the patients, of whom the majority had had <12 years of formal education. Of the 7798 with cancer staging data available, 41.1% had a local cancer. Adenocarcinoma was the most common (94.2%) histological type. Almost all patients underwent open gastrectomy and 214% in hospitals with annual volume of more than 30 gastrectomies per year. A total of 8561 deaths occurred during the study period, of which 6474 were due to oesophago-gastric cancers. The 5-year survival was 34.6% and 5-year cancer-specific survival was 39.7%. Future plans: The data in FINEGO can be currently used for registry-based research but is being expanded by data extraction from patient records and scanning of histological samples from the Finnish biobanks. Initially, we are planning on studies on the national trends in treatment and mortality, and studies on the demographic factors and their influence on survival.

Gastric cancer is among the most common cancer types in the world and the third most common cause of death by cancer. The prognosis of individual patients treated with surgery varies greatly. New prognostic factors are needed to complement the prognostic information provided by the currently used tumor-node-metastasis (TNM) staging system. Several novel prognostic factors that can be assessed from routinely stained hematoxylin-eosin (HE) slides have been discovered in different cancer types. They include tumor-stroma ratio (TSR), tumor budding, stromal maturity and Klintrup-Mäkinen (KM) grade. TSR is based on estimating the amount of intratumoral stroma. Tumor budding is assessed by counting tumor buds, which are single tumor cells or clusters of up to four cells detached from the main tumor mass. Assessment of stromal maturity is based on presence of thick eosinophilic collagen bundles in the immature stroma. KM-grade is a method of semiquantitatively classifying the lymphocytic infiltrate present at the border of the tumor. All these factors have prognostic value in different cancer types, but their relevance in gastric cancer is unknown. In this thesis, prognostic value of TSR, tumor budding, stromal maturity and KM-grade in gastric cancer was studied, mainly in a large patient cohort from Oulu University Hospital. TSR, tumor budding, stromal maturity and KM-grade were all independent prognostic factors in the patient cohort. TSR and tumor budding were assessed with high interobserver agreement, while stromal maturity and KM-grade were assessed with moderate interobserver agreement. Based on our results, TSR, tumor budding, stromal maturity and KM-grade have prognostic value in gastric cancer. They could potentially be used to improve accuracy of estimating patients’ prognosis after surgery for gastric cancer., Tiivistelmä Mahasyöpä on yksi maailman yleisimmistä syöpätyypeistä ja maailman kolmanneksi yleisin syöpäkuoleman aiheuttaja. Kirurgisesti hoidettujen potilaiden ennuste vaihtelee suuresti. Uudet ennustetekijät olisivat hyödyllisiä nykyään käytetyn tumor-node-metastasis (TNM) -luokituksen antaman ennustetiedon täydentämiseksi. Useita uusia rutiinisti tehtävistä hematoksyliini-eosiini (HE) -värjätyistä laseista analysoitavissa olevia ennustetekijöitä on löydetty monista eri syöpätyypeistä. Niihin lukeutuvat kasvain-stroomasuhde (tumor-stroma ratio (TSR)), kasvaimen silmikointi, strooman kypsyys ja Klintrup-Mäkinen (KM) -aste. TSR perustuu tuumorin sisäisen strooman määrän arvioimiseen. Kasvaimen silmikointi analysoidaan laskemalla tuumorisilmujen määrä. Tuumorisilmut ovat yksittäisiä kasvainsoluja tai korkeintaan neljän solun ryhmiä, jotka ovat irronneet kasvaimen pääosasta. Strooman kypsyyden arviointi perustuu paksujen eosinofiilisten kollageenikimppujen esiintymiseen epäkypsässä stroomassa. KM-aste on semikvantitatiivinen menetelmä kasvaimen reunalla olevan lymfosyytti-infiltraatin luokitteluun. Kaikilla näistä tekijöistä on aiemmin osoitettu olevan ennustearvoa eri syöpätyypeissä, mutta niiden merkitys mahasyövässä tunnetaan puutteellisesti. Tässä väitöskirjassa tutkittiin TSR:n, kasvaimen silmikoinnin, strooman kypsyyden ja KM-asteen ennustearvoa, pääasiassa suuressa Oulun yliopistollisen sairaalan potilaskohortissa. TSR, kasvaimen silmikointi, strooman kypsyys ja KM-aste olivat kaikki itsenäisiä ennustetekijöitä. TSR:n ja kasvaimen silmikoinnin arvioinnin toistettavuus oli hyvä, ja strooman kypsyyden ja KM-asteen kohtalainen. Tuloksiemme perusteella TSR:llä, kasvaimen silmikoinnilla, strooman kypsyydellä ja KM-asteella on ennustearvoa mahasyövässä. Niitä voitaisiin mahdollisesti tulevaisuudessa hyödyntää leikattujen mahasyöpäpotilaiden ennustearvioiden parantamisessa.

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.

Toll‐like receptors (TLRs) are involved in colorectal cancer (CRC) pathogenesis. However, the significance of serum TLR concentrations in CRC is unknown. We analyzed serum TLR2 and TLR4 concentrations with ELISA in preoperative samples from 118 patients with CRC and 88 matched controls. We also assessed tissue TLR expression with immunohistochemistry and by detecting serum determinants of systemic inflammation. Most participants (>70%) had undetectable serum TLR2. The mean serum TLR4 levels were lower in patients than in controls (1.1 vs 1.8 ng/mL; p = 0.015). Undetectable TLR4 was more common in stage I (39%) than in stages II–IV (11%, p < 0.001). TLR2 or TLR4 expression in tumor cells did not correlate with serum levels, but abundant TLR2 expression in normal colon epithelium was associated with detectable serum TLR2 (p = 0.034). Undetectable serum TLR2 was linked to high modified Glasgow prognostic scores (p = 0.010), high CRP levels (p = 0.013), blood vessel invasion (p = 0.013), and tended to be associated with worse 5‐year survival (p = 0.052). In conclusion, serum TLR2 levels were inversely associated with systemic inflammation in patients with CRC. Moreover, serum TLR2 levels might depend more on normal colorectal mucosa contributions than on tumor tissue contributions. Further studies are required to assess the prognostic value of serum TLR2.

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

Introduction: Surgery for oesophageal and gastric cancers is associated with high morbidity, mortality and poor quality of life postoperatively. The Finnish National Esophago-Gastric Cancer Cohort has been established with the aim of identifying factors that could contribute to improved outcomes in oesophago-gastric cancer. Methods and analysis: All patients with oesophageal and gastric cancer diagnosed in Finland between 1987 and 2015 will be identified from the Finnish national registries. The Finnish Cancer Registry and Finnish Patient Registry will be used to identify patients that fulfil the inclusion criteria for the study: (1) diagnosis of oesophageal, gastro-oesophageal junction, or gastric cancer, (2) any surgical treatment for the diagnosed cancer and (3) age of 18 or over at the time of diagnosis. Clinical variables and complication information will be retrieved in extensive data collection from the medical records of the relevant Finnish hospitals and complete follow-up for vital status from Statistics Finland. Primary endpoint is overall all-cause mortality and secondary endpoints include complications, reoperations, medication use and sick leaves. Sub-studies will be implemented within the cohort to investigate specific populations undergoing oesophageal and gastric cancer surgery. The initial estimated sample size is 1800 patients with surgically treated oesophageal cancer and 7500 patients with surgically treated gastric cancer. Ethics and dissemination: The study has been approved by the Ethical Committee in Northern Ostrobothnia, Finland and The National Institute for Health and Welfare, Finland. Study findings will be disseminated via presentations at conferences and publications in peer-reviewed journals. Introduction Surgery for oesophageal and gastric cancers is associated with high morbidity, mortality and poor quality of life postoperatively. The Finnish National Esophago-Gastric Cancer Cohort has been established with the aim of ide

Objectives: Pancreatic ductal adenocarcinoma (PDAC) is characterized by abundant stroma with increased expression of tenascin C and fibronectin. Their role and tumor-stroma ratio in PDAC are not well known. The aim of this study was to evaluate tenascin C and fibronectin expression and tumor-stroma ratio and their prognostic relevance in PDAC. Methods: Ninety-five resected PDACs were immunohistochemically stained for tenascin C and fibronectin, and the expression was separately assessed in tumor bulk and front. Tumor-stroma ratio was determined with sections stained with hematoxylin-eosin. Results: Tenascin C and fibronectin were abundantly expressed in the stroma of PDAC, but absent in adjacent normal pancreatic tissue. Fibronectin expression of the bulk was associated with high T class (P = 0.045). In the main analysis, tenascin C and fibronectin expression and tumor-stroma ratio were not associated with patient survival. In a subgroup analysis of early-stage PDAC (T1–T2 tumors), high tenascin C expression in the tumor bulk was associated with poor prognosis (hazard ratio, 8.23; 95% confidence interval, 2.71–24.96). Conclusions: Tenascin C and fibronectin are abundantly expressed in PDAC, but they seem to have no major association with patient survival. However, in early-stage PDAC, tenascin C expression of the tumor bulk may have prognostic impact. Tumor-stroma ratio has no prognostic value in PDAC.

Colorectal carcinoma (CRC) is an important cause of cancer-associated deaths. About 30–50% of CRCs show KRAS or BRAF mutation. In many cancers, anoikis, i.e. apoptosis induced by loss of extracellular matrix (ECM) contact, is disturbed. Anoikis resistance is essential for the formation of metastases, and since anoikis resistance assessment is based on in vitro cell cultures, the prognostic value of anoikis resistance is largely unknown. We aimed to identify the histopathological features indicating anoikis resistance in CRC and analyze their prognostic value. The roles of BRAF and KRAS mutations and survivin in anoikis resistance were analyzed and 3-D cell culture was used to model the histopathology of anoikis resistant (AR) structures. The two cohorts of CRC cases used in the study consisted of 62 (series 1) and 137 patients (series 2). Immunohistochemistry for ECM proteins enabled identification of tumor cells with and without ECM contact, and in both populations, apoptosis was determined with staining for caspase-cleaved keratin 18. Based on absence of ECM contact and decreased apoptosis rate, we identified micropapillary (MIP), cribriform and solid structures to represent the putative AR populations. High areal density of AR structures associated independently with short survival and was an independent prognostic factor. MIPs showed lower survivin expression, proliferation and apoptosis rates than non-MIP cells, and low apoptosis rate was associated with poor prognosis in stage I and II cases. For 3-D in vitro model of AR structures, we transfected Caco-2 cells with mutated KRAS or BRAF genes; both induced anoikis resistance as measured with Annexin V test in suspension culture. In 3-D cultures, native Caco-2 cells formed polarized cysts. In contrast, mutated cell lines formed partially filled cysts or solid structures, and inverted polarity in KRAS mutant cells. In conclusion, it is possible to identify putative AR structures by conventional histopath, Tiivistelmä Paksu- ja peräsuolisyöpä on yleinen syöpäkuoleman aiheuttaja. KRAS- tai BRAF-geenien mutaatio todetaan 30–50 prosentissa suolisyövistä. Anoikis tarkoittaa apoptoosia, jonka käynnistää solun irtoaminen soluväliaineesta. Anoikisresistenssi on etäpesäkkeen synnyn edellytys. Anoikisresistenssiä voidaan todeta vain soluviljelyssä, joten sen merkitystä syövässä in vivo ei ole aiemmin arvioitu. Tässä työssä pyrittiin tunnistamaan anoikisresistenssiin viittaavat muutokset histopatologisista suolisyöpänäytteistä ja selvittämään niiden vaikutusta potilaan ennusteeseen. Lisäksi tutkittiin BRAF- ja KRAS-mutaatioiden yhteyttä anoikisresistenssiin ja mallinnettiin anoikisresistenttejä (AR) rakenteita kolmiulotteisessa soluviljelmässä. Potilasaineisto koostui 199 suolisyöpäpotilaasta. Kudosleikkeistä värjättiin soluväliaineen komponentteja sekä määritettiin apoptoottiset ja jakautuvat solut (M30- ja Ki-67-värjäykset). AR-solupopulaatioiden tunnistamisessa käytettiin kriteereinä soluväliainekontaktin puuttumista ja vähentynyttä apoptoositiheyttä. AR-populaatioiksi osoittautuivat mikropapillaariset (MIP), seulamaiset ja solidit rakenteet. Näiden rakenteiden korkea kokonaisesiintyvyys osoittautui itsenäiseksi huonon ennusteen tekijäksi. MIP-rakenteissa surviviinin ilmentyminen ja apoptoosi- ja proliferaatiotiheys olivat vähentyneet muihin kasvainsoluihin verrattuna. Lisäksi apoptoottisten solujen pieni määrä MIP-rakenteissa liittyi huonoon ennusteeseen paikallisessa syövässä. Mallinnusta varten Caco-2 solut transfektoitiin mutatoiduilla KRAS- tai BRAF-geeneillä. Onkogeenien transfektion todettiin indusoivan anoikisresistenssiä. Kolmiulotteisessa soluviljelyssä polarisoituneet Caco-2 solut muodostivat säännöllisiä rauhasmaisia rakenteita. Onkogeeneillä transfektoidut solut muodostivat puolestaan osittain tai kokonaan täyttyneitä rakenteita ja KRAS-transfektio aiheutti solujen polariteetin kääntymistä. Havainnot osoittavat, että anoikisresistenssiä edustavat rakenteet voida