Lars Gustav Lyckander

A characterization in childhood inflammatory bowel disease, a new population-based inception cohort from South-Eastern Norway, 2005–07, showing increased incidence in Crohn's disease

Objective. Owing to rising incidence rates in inflammatory bowel disease (IBD), there has been increased interest in causal relationships in pediatric disease. The present population-based inception cohort was recruited in the Oslo area from 2005 to 2007, with the aim of conducting a detailed characterization of treatment-naïve patients at diagnosis. Material and methods. After an invitation was extended to all general practitioners in the catchment area, patients aged <18 years with suspected IBD were diagnosed by proximal and distal endoscopy, MRI, demographic, clinical, and histological and molecular characteristics. Symptomatic non-IBD patients served as controls. Results. Of 100 pediatric patients, 62 had IBD (39 Crohns disease (CD), 19 ulcerative colitis (UC), 4 IBD unclassified (IBDU)) and 38 other diseases. Median age at diagnosis for IBD was 13.1 years (56.4% males), median symptom duration 6 months, and 69% L3 (Vienna classification). With 195,000 children aged <18 years in the catchment area, the incidence rate of IBD per 100,000/years inhabitants was 10.9 (6.8 for CD, 3.6 for UC, and 0.6 IBDU) and for those aged <16 years (178,500) the incidence rate was 10.6. The higher NOD2 allele frequency among children may partly contribute to the increase. Conclusions. The results indicate a marked rise in the incidence of CD in contrast to no increase in UC in South-Eastern Norway, compared with the figures from the last 15 years. Time from onset of symptoms to diagnosis still represents a challenge for early characterization in IBD.

Evaluation of disease activity in IBD at the time of diagnosis by the use of clinical, biochemical, and fecal markers

Abstract Objective. The present population based adult cohort was part of a new prospective study of patients with inflammatory bowel disease (IBD) in South-Eastern Norway, the Inflammatory Bowel South-Eastern Norway II study, investigating disease characteristics in an attempt to improve our knowledge regarding factors related to early clinical phenotype and disease activity. Material and methods. Patients suspected to have IBD on the basis of predefined symptoms, including abdominal pain, diarrhea, and/or blood in stool for more than 10 days were examined at the local hospital. Colonoscopy with biopsies was performed and blood and stool samples were taken. Results. In ulcerative colitis (UC) patients, the median Simple Clinical Colitis Activity Index (SCCAI) was 4 (range 0–10) in mild and 6 (range 0–14) in patients with moderate or severe endoscopic activity of inflammation (p = 0.002). The calprotectin concentration in feces was significantly related to the SCCAI (p = 0.034) and the Mayo endoscopic subscore (p = 0.031). There was a significant association between the C-reactive protein (CRP) value, leucocytes and thrombocytes and the SCCAI, but only leucocytes were significantly associated with the Mayo endoscopic subscore. In Crohns disease (CD) patients, there was no statistical significant association between the Harvey-Bradshaw Index (HBI) and the endoscopic grade of mucosal inflammation (p = 0.8). The calprotectin concentration in feces was significantly related to the endoscopic activity score (p = 0.004), but not to the HBI (p = 0.5). HBI was significantly related to the CRP value (p = 0.047) and thrombocytes (p = 0.03). Conclusions . In UC, both biochemical and fecal markers are related to disease activity and extent of disease, whereas in CD, the fecal calprotectin concentration is a reliable marker of mucosal affection, but not for systemic disease activity.

Defective Paneth Cell—Mediated Host Defense in Pediatric Ileal Crohn's Disease

OBJECTIVES:Adult ileal Crohns disease (CD) is characterized by a specific decrease in ileal Paneth cell α-defensins. In addition to NOD2, we previously identified a disturbance of the Wnt-signaling transcription factor TCF-4 as a major mechanism for this deficiency. The aim of this study was to evaluate human α-defensin-5 (HD-5) and TCF-4 in an independent cohort of pediatric CD patients.METHODS:Expression levels of HD-5 and TCF-4 mRNA were quantified by real-time PCR in biopsies from newly diagnosed untreated pediatric CD patients (<18 years, n=36) and age-matched symptomatic non-inflammatory bowel disease controls with a histologically normal gut (n=29). To assess the influence of current inflammation, mucosal interleukin-8 (IL-8) and fecal calprotectin levels were determined.RESULTS:Small intestinal HD-5 and TCF-4 mRNA were significantly reduced in pediatric ileal CD (L1+L3) (P=0.022 and P=0.0005, respectively) and were significantly correlated (r=0.499; P=0.0001). In ileal but not colonic CD, TCF-4 was also reduced in the colon (P=0.005). Importantly, both HD-5 and TCF-4 were independent of inflammation, as measured by IL-8 expression or fecal calprotectin. In contrast to the small intestine, colonic Paneth cell HD-5 mRNA was significantly elevated in colonic CD (L2) (P=0.026) and was correlated with fecal calprotectin levels (r=0.481; P=0.020).CONCLUSIONS:In this study, we describe a specific decrease in HD-5 and TCF-4 mRNA expression levels in children with ileal CD. In the small intestine, this decrease was independent of current inflammation, whereas inflammation seems to induce Paneth cell metaplasia in the colon. Our data extend the hypothesis of an important role of antimicrobial host defense in pediatric CD patients.

Increase of regulatory T cells in ileal mucosa of untreated pediatric Crohn's disease patients

Abstract Background. Inflammatory bowel disease (IBD) of pediatric and adult onset differs in several aspects although little knowledge exists about pathogenic disparity. Regulatory T cells (Tregs) characterized as CD4+CD25+Foxp3+ are modulators of gut homeostasis, but their role in human IBD remains unclear. Objective. To evaluate the mucosal distribution of Foxp3+ and CD25+ cells in untreated pediatric IBD patients at the time of diagnosis. Material and methods. Untreated pediatric (n = 14) and adult (n = 12) Crohns disease (CD) patients were prospectively included together with age-matched symptomatic controls. Colonic and ileal mucosal biopsies collected at diagnosis were studied by immunohistochemistry for enumeration of T cells and for mucosal expression of Foxp3 and CD25. Multicolor immunofluorescence staining was performed in situ to phenotype Foxp3+ cells as Tregs and characterize the CD25+ cells. Results. The density of mucosal T cells displayed only small variations, while that of Foxp3+ cells and CD25+ cells was increased in CD patients. Multicolor immunofluorescence showed that most CD25+ cells were macrophages. Interestingly, in the ileum of pediatric CD patients the density of Foxp3+ cells was significantly higher than in adult CD patients. Co-expression of Foxp3 and CD25, as well as Foxp3 and CTLA-4, indicated that the Foxp3+ cells were Tregs. Conclusion. Mucosal numbers of Foxp3+ Tregs and activated (CD25+) macrophages are elevated in both pediatric and adult ileal CD. The greater increase of ileal Foxp3+ Tregs in pediatric CD than in adult CD might contribute to the relatively less frequent phenotype of isolated ileal enteritis in CD children.

Increased number and activation of colonic macrophages in pediatric patients with untreated Crohn's disease

Background: Pediatric inflammatory bowel disease (IBD) may be phenotypically different from adult IBD. In IBD lesions, macrophages are overactivated, suggesting involvement of innate immunity in the pathogenesis. Here, mucosal macrophages were studied in selected untreated pediatric patients compared with adults from a population‐based Norwegian cohort of IBD patients. Age‐matched non‐IBD controls were also included. Methods: Untreated children (<18 years) and adults (≥18 years) were included at diagnosis with colonic and ileal biopsies. Controls were symptomatic non‐IBD patients with histologically normal gut. Frozen mucosal sections were examined by immunohistochemistry for cellular expression of the pan‐macrophage marker CD68 and the costimulatory molecule CD40. Two‐color immunofluorescence staining in situ was performed to identify CD40+ macrophages. Results: Non‐IBD adults had significantly higher mucosal density of colonic CD68+ macrophages than non‐IBD children. In pediatric Crohns disease (CD), macrophages were significantly increased in the colon (but not in the ileum) compared with controls. Their mucosal density in pediatric CD was significantly higher than in pediatric ulcerative colitis. The number of CD40+ (activated) macrophages was significantly elevated in both histologically inflamed and uninflamed colon and ileum of IBD children. Conclusions: Histologically normal colon mucosa contains fewer macrophages in children than in adults. However, in colon of children with untreated CD the mucosal macrophage density is increased. Activated mucosal macrophages are increased in untreated pediatric IBD regardless of inflammatory grade. Such upregulated innate mucosal immune activation may contribute to the colonic phenotype of childhood CD. (Inflamm Bowel Dis 2009)

Abstract 4514: Low release of exosomal miR-663a from hypoxic tumor cells and poor tumor response to neoadjuvant radiotherapy in rectal cancer patients

Introduction: Hypoxia is an important hallmark of the tumor microenvironment and contributes significantly to radiotherapy resistance in solid tumors. Exosomes are nanosized vesicles actively released from tumor cells, and recent studies support their central role in the aggravated biology caused by tumor hypoxia through their miRNA cargo. In this study, we aimed to characterize miRNAs of exosomes from hypoxic colorectal cancer (CRC) cell lines and investigate these miRNAs in circulating exosomes from rectal cancer patients with poor response to neoadjuvant radiotherapy. Methods: Five CRC cell lines were cultured in RPMI-1640 medium supplemented with 1% bovine serum albumin under normoxia (21% O2) or hypoxia (0.2% O2) for 24 hours. Exosomes were isolated from conditioned media by differential ultracentrifugation, and integrity and size were determined by cryo-electron microscopy and NanoSight tracking analysis and further characterized by Western blot and flow cytometry analyses. In a prospective biomarker study, plasma samples were collected from 29 patients with rectal cancer at the time of diagnosis, and histologic tumor response (tumor regression grade, TRG) to neoadjuvant radiotherapy was evaluated. Exosomes were isolated from plasma using the miRCURY™ Exosome Isolation Kit (Exiqon). Expression profiling of exosomal miRNAs of the CRC cell lines and the patients’ plasma samples was conducted using the miRCURY LNA™ Universal RT microRNA PCR Human panel I (Exiqon). Data normalization was performed based on global array mean. Results: Normoxic and hypoxic CRC cells released vesicles, 30-150 nm in size, that expressed proteins known to be enriched in exosomes (CD9, CD63, CD81, Alix) and with absence of markers of the endoplasmic reticulum (GRP78, Calnexin) and the Golgi apparatus (GM130). Exosomes from the CRC cell lines harbored strong cell line-specific miRNA profiles; however, when comparing the hypoxic cell lines collectively with the normoxic counterparts, four exosomal miRNAs (miR-195-5p, miR-423-3p, miR-622, miR-663a) were significantly down-regulated by hypoxia. Of these, circulating exosomal miR-663a was lower (p = 0.033) in rectal cancer patients with poor tumor response (TRG 2-3) to neoadjuvant radiotherapy. Conclusion: Vesicles released from CRC cell lines were characterized as exosomes carrying miRNAs that were retrieved in the circulation of rectal cancer patients, where low level of exosomal miR-663a at the time of diagnosis was associated with poor tumor response to neoadjuvant radiotherapy. These findings suggest that repressed release of miR-663a from tumor cells into the circulation may be a marker of radiotherapy resistance caused by tumor hypoxia. The results are currently under validation in an independent patient cohort. Trial registration: NCT01816607 Citation Format: Tonje Bjornetro, Karianne R. Handeland, Sebastian Meltzer, Rampradeep Samiappan, Lars G. Lyckander, Caroline Jegerschold, Linda Sonstevold, Nirujah S. Thusyanthan, Kathrine R. Redalen, Anne H. Ree. Low release of exosomal miR-663a from hypoxic tumor cells and poor tumor response to neoadjuvant radiotherapy in rectal cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4514. doi:10.1158/1538-7445.AM2017-4514

Abstract 1782: Reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage in tumor hypoxia, poor radiotherapy response, and metastatic progression of rectal cancer

Background: Hypoxia is an important feature of the tumor microenvironment and correlates with radiotherapy resistance and metastatic progression. Additionally, as the malignant phenotype promotes aerobic glycolysis and contributes to the microenvironmental acidification, an underlying mechanism in radiation resistance and metastasis is likely linked to metabolic changes caused by mitochondrial reprogramming. We hypothesized that ROS and mtDNA damage might be indicators of poor therapy response and dissemination of rectal cancer. Methods: Three colorectal cancer (CRC) cell lines (HCT116, HT29, LoVo) were cultured under hypoxia (0.2% O2) or normoxia (21% O2) for 24 hours. In a prospective rectal cancer trial, serum was sampled from patients at the time of diagnosis. Levels of ROS were determined by the ability to oxidize dihydrochlorofluoroscein into a fluorescent derivative. mtDNA damage was quantified by the ability of modification on template DNA to inhibit restriction enzyme cleavage. For the study patients, histologic tumor response to neoadjuvant radiotherapy was evaluated according to standard ypTN staging and tumor regression grade (TRG) scoring. Groups were compared using Student’s t-test. Survival differences were assessed by the log-rank test. Results: Compared to normoxic conditions, hypoxia significantly reduced ROS levels in all CRC cell lines. Moreover, in serum samples, ROS levels were lower for T4 cases than for patients with T2-3 disease (p = 0.037), and accordingly, low circulating ROS was associated with adverse metastasis-free survival (MFS; p = 0.004) and overall survival (p Conclusion: We showed that culturing of CRC cells under hypoxia caused decrease in ROS levels but increase in mtDNA damage. Interestingly, in serum samples from rectal cancer patients, low ROS levels were associated with T4 disease, representing a bulky and often hypoxic tumor, and thus adverse survival. Moreover, low ratio of ROS to mtDNA damage was correlated with poor histologic tumor response to neoadjuvant radiotherapy. These parameters may reflect an aggressive biological phenotype instigated by a hostile tumor microenvironment. The results are currently under validation in an independent patient cohort. Trial registration: NCT00278694 Citation Format: Paula A. Bousquet, Sebastian Meltzer, Linda Sonstevold, Ying Esbensen, Lars G. Lyckander, Svein Dueland, Kjersti Flatmark, Kathrine R. Redalen, Lars Eide, Anne H. Ree. Reactive oxygen species (ROS) and mitochondrial DNA (mtDNA) damage in tumor hypoxia, poor radiotherapy response, and metastatic progression of rectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1782. doi:10.1158/1538-7445.AM2017-1782