The Oxysterol Synthesising Enzyme CH25H Contributes to the Development of Intestinal Fibrosis

Intestinal fibrosis and stenosis are common complications of Crohn's disease [CD], frequently requiring surgery. Anti-inflammatory strategies can only partially prevent fibrosis; hence, anti-fibrotic therapies remain an unmet clinical need. Oxysterols are oxidised cholesterol derivatives with important roles in various biological processes. The enzyme cholesterol 25-hydroxylase [CH25H] converts cholesterol to 25-hydroxycholesterol [25-HC], which modulates immune responses and oxidative stress. In human intestinal samples from CD patients, we found a strong correlation of CH25H mRNA expression with the expression of fibrosis markers. We demonstrate reduced intestinal fibrosis in mice deficient for the CH25H enzyme, using the sodium dextran sulphate [DSS]-induced chronic colitis model. Additionally, using a heterotopic transplantation model of intestinal fibrosis, we demonstrate reduced collagen deposition and lower concentrations of hydroxyproline in CH25H knockouts. In the heterotopic transplant model, CH25H was expressed in fibroblasts. Taken together, our findings indicate an involvement of oxysterol synthesis in the pathogenesis of intestinal fibrosis

Protein tyrosine phosphatase non-receptor type 2 controls colorectal cancer development

Protein tyrosine phosphatase non-receptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC) as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. Particularly, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T-cells and increased PTPN2 levels negatively correlated with PD1, CTLA4, STAT1 and granzyme A. In vivo, T-cell and dendritic cell-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44+ effector/memory T-cells, as well as CD8+ T-cell infiltration and cytotoxicity into the tumor. In direct relevance to CRC treatment, T-cell-specific PTPN2 deletion potentiated anti-PD-1 efficacy and induced anti-tumor memory formation upon tumor re-challenge in vivo. Our data suggest a role for PTPN2 in suppressing anti-tumor immunity and promoting tumor development in CRC patients. Our in vivo results uncover PTPN2 as a key player in controlling immunogenicity of CRC, with the strong potential to be exploited to promote cancer immunotherapy

Expression of C-KIT, CD24, CD44s, and COX2 in benign and non-invasive apocrine lesions of the breast

Benign apocrine metaplasia (AM) of the adult breast is a very common, but enigmatic lesion. It has been speculated that AM might be a precursor of malignancy or an indicator of a susceptibility of the breast tissue to develop neoplasia, mainly based on comparing the frequency of AM in breast cancer and non-breast cancer patients [1]. Studies using comparative genomic hybridization have supported this by showing similar molecular alterations in benign and malignant apocrine lesions [2]. Few studies, however, have compared expression of biomarkers involved in tumor progression in AM and progressively more advanced atypical apocrine lesions. The expression of C-KIT, COX2, CD24, and CD44s was evaluated by immunohistochemistry in formalin-fixed, paraffin-embedded material of 9 AM, 20 apocrine ductal intraepithelial neoplasia (DIN1c-3) and 40 atypical apocrine lesions (not qualifying for DIN1c-3) and compared to expression of the same biomarkers in adjacent normal ductal epithelium. Of the 66 apocrine lesions, 62 (94 %) did not express C-KIT compared to 4/63 (6 %) of the normal glands (Fisher's exact, p < 0.001). COX2 was expressed in a significantly higher proportion of apocrine lesions than of normal glands (49 vs. 14 %, p < 0.001), and the number of apocrine lesions positive for CD24 was found to be higher with increasing aggressiveness of the lesions (Spearman, p < 0.001). In conclusion, benign and non-invasive proliferative apocrine lesions of the breast display immuno-phenotypical characteristics previously ascribed mainly to malignant transformation. This could lend support to the theory that AM is an early step towards malignant transformation, albeit associated with slow progression to carcinoma

Maturation of tertiary lymphoid structures and recurrence of stage II and III colorectal cancer

Tertiary lymphoid structures (TLS) are associated with favorable outcome in non-metastatic colorectal carcinoma (nmCRC), but the dynamics of TLS maturation and its association with effective anti-tumor immune surveillance in nmCRC are unclear. Here, we hypothesized that not only the number of TLS but also their composition harbors information on recurrence risk in nmCRC. In a comprehensive molecular, tissue, laboratory, and clinical analysis of 109 patients with stage II/III nmCRC, we assessed TLS numbers and degree of maturation in surgical specimens by multi-parameter immunofluorescence of follicular dendritic cell (FDC) and germinal center (GC) markers. TLS formed in most tumors and were significantly more prevalent in highly-microsatellite-instable (MSI-H) and/or BRAF-mutant nmCRC. We could distinguish three sequential TLS maturation stages which were characterized by increasing prevalence of FDCs and mature B-cells: [1] Early TLS, composed of dense lymphocytic aggregates without FDCs, [2] Primary follicle-like TLS, having FDCs but no GC reaction, and [3] Secondary follicle-like TLS, having an active GC reaction. A simple integrated TLS immunoscore reflecting these parameters identified a large subgroup of nmCRC patients with a very low risk of recurrence independently of clinical co-variables such as ECOG performance status, age, stage, and adjuvant chemotherapy. We conclude that (1) mismatch repair and BRAF mutation status are associated with the formation of TLS in nmCRC, (2) TLS formation in nmCRC follows sequential maturation steps, culminating in germinal center formation, and (3) this maturation process harbors important prognostic information on the risk of disease recurrence

Evaluation of multifunctional imaging parameters in gastro-oesophageal cancer using F-18-FDG-PET/CT with integrated perfusion CT

BACKGROUND Positron emission tomography (PET) / computed tomography (CT) is among the most frequently used imaging modalities for initial staging of gastro-oesophageal (GE) cancer, whereas CT-perfusion (CTP) provides different multiparametric information. This proof of concept study compares CTP- and PET-parameters in patients with GE cancer to evaluate correlations and a possible prognostic value of a combined PET/CTP imaging procedure. METHODS A total of 31 patients with F-18-FDG-PET/CT and CTP studies were prospectively analysed. Patients had adenocarcinoma (n = 22) and oesophageal squamous cell carcinoma (SCC, n = 9). Imaging was performed before start of treatment. CTP parameters [blood flow (BF), blood volume (BV), mean transit time (MTT)] and metabolic parameters [(maximum and mean standardised uptake values and standard deviation (SUVmax, SUVmean, SUVsd), metabolic tumour volume (MTV) and tumour lesion glycolysis (TLG)], as well as flow metabolic product [FMP (BF × SUVmax)] were determined and their relationship was compared. Additionally their association to clinical parameters (differentiation grading, staging, HER2-status, follow-up status) and to histopathological regression (post-neoadjuvant regression grading) was evaluated. RESULTS Correlation between parameters of both modalities was significant between MTT and MTV (r = 0.375, p = 0.038); no other significant correlation was found. Patients with complete histopathological regression showed significantly lower BF and BV than patients with nearly complete or partial response. TLG and regression grading showed significant correlation with staging. All other quantitative parameters for CTP and PET data did not correlate significantly with histopathological regression grading, differentiation or staging. CONCLUSIONS The combination of PET and CTP parameters (FMP) showed no significant prognostic value. Significant correlations were only found between MTT and MTV, which indicates a possible perfusional/metabolic coupling. Therefore, pre-therapeutic CTP and PET- parameters provide complementary information about the pre-therapeutic tumour status and are not interchangeable. Only CTP parameters might be able to predict complete histopathological regression. On the other hand, only PET parameters are correlated with staging

Role of intravoxel incoherent motion parameters in gastro-esophageal cancer: relationship with 18F-FDG-PET, CT perfusion and MR perfusion imaging parameters

BACKGROUND Identification of pre-therapeutic predictive markers in gastro-esophageal cancer is essential for individual-oriented treatment. This study evaluated the relationship of multimodality parameters derived from intravoxel incoherent motion method (IVIM), 18F- FDG-PET, CT perfusion and dynamic contrast enhanced MRI in patients with gastro- esophageal cancer and investigated their histopathological correlation. METHODS Thirty-one consecutive patients (28 male; median age 63.9 years; range 37-84 years) with gastro-esophageal adenocarcinoma (n=22) and esophageal squamous cell carcinoma (n=9) were analyzed. IVIM parameters: pseudodiffusion (D*), perfusion fraction (fp), true diffusion (D) and the threshold b-value (bval); PET-parameters: SUVmax, metabolic tumor volume (MTV) and total lesion glycolysis (TLG); CT perfusion parameters: blood flow (BF), blood volume (BV) and mean transit time (MTT); and MR perfusion parameters: time to enhance, positive enhancement integral, time-to-peak (TTP), maximum- slope-of-increase, and maximum-slope-of-decrease were determined, and correlated to each other and to histopathology. RESULTS IVIM and PET parameters showed significant negative correlations: MTV & bval (rs = -0.643, p = 0.002), TLG & bval (rs = -0.699, p < 0.01) and TLG & fp (rs = -0.577, p = 0.006). Positive correlation was found for TLG & D (rs = 0.705, p = 0.000). Negative correlation was found for bval & staging (rs = 0.590, p = 0.005). Positive correlation was found for positive enhancement interval & BV (rs = 0.547, p = 0.007), BF & regression index (rs = 0.753, p = 0.005) and for time-to-peak & staging (rs = 0.557, p = 0.005). CONCLUSIONS IVIM parameters (bval, fp, D) provide quantitative information and correlate with PET parameters (MTV, TLG) and staging. IVIM might be a useful tool for additional characterization of gastro-esophageal cancer

Transplantation of Human Intestine Into the Mouse: A Novel Platform for Study of Inflammatory Enterocutaneous Fistulas

BACKGROUND AND AIMS Enteric fistulas represent a severe and medically challenging comorbidity commonly affecting Crohn's disease [CD] patients. Gut fistulas do not develop in animal models of the disease. We have used transplantation of the human fetal gut into mice as a novel platform for studying inflammatory enterocutaneous fistulas. METHODS Human fetal gut segments were transplanted subcutaneously into mature SCID mice, where they grew and fully developed over the course of several months. We first analysed the resident immune cells and inflammatory response elicited by systemic lipopolysaccharide [LPS] in normal, fully developed human gut xenografts. Thereafter, we used immunostaining to analyse fully developed xenografts that spontaneously developed enterocutaneous fistulas. RESULTS Resident human innate and adaptive immune cells were demonstrated in gut xenografts during steady state and inflammation. The expression of human IL-8, IL-1β, IL-6, TNF-α, A20, and IkBα was significantly elevated in response to LPS, with no change in IL-10 gene expression. Approximately 17% [19/110] of fully developed subcutaneous human gut xenografts spontaneously developed enterocutaneous fistulas, revealing striking histopathological similarities with CD fistula specimens. Immunohistochemical analyses of fistulating xenografts revealed transmural lymphocytic enteritis associated with massive expansion of resident human CD4+ lymphocytes and their migration into the intraepithelial compartment. Regionally, mucosal epithelial cells assumed spindle-shaped mesenchymal morphology and formed fistulous tracts towards chronic non-healing wounds in the host mouse skin overlying the transplants. CONCLUSIONS Inflammation and fistulas developed in human gut xenografts lacking IL-10 gene response. This novel model system will enable systematic studies of the inflamed and fistulating human gut in live animals