Mihai Danciu

Array CGH identifies distinct DNA copy number profiles of oncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstable sporadic colorectal carcinomas

DNA copy number changes represent molecular fingerprints of solid tumors and are as such relevant for better understanding of tumor development and progression. In this study, we applied genome-wide array comparative genomic hybridization (aCGH) to identify gene-specific DNA copy number changes in chromosomal (CIN)- and microsatellite (MIN)-unstable sporadic colorectal cancers (sCRC). Genomic DNA was extracted from microdissected, matching normal colorectal epithelium and invasive tumor cells of formalin-fixed and paraffin-embedded tissues of 22 cases with colorectal cancer (CIN = 11, MIN = 11). DNA copy number changes were determined by aCGH for 287 target sequences in tumor cell DNAs, using pooled normal DNAs as reference. aCGH data of tumor cell DNAs was confirmed by fluorescence in situ hybridization (FISH) for three genes on serial tissues as those used for aCGH. aCGH revealed DNA copy number changes previously described by metaphase CGH (gains 7, 8q, 13q, and 20q; losses 8p, 15q, 18q, and 17p). However, chromosomal regions 20q, 13q, 7, and 17p were preferentially altered in CIN-type tumors and included DNA amplifications of eight genes on chromosome 20q (TOP1, AIB1, MYBL2, CAS, PTPN1, STK15, ZNF217, and CYP24), two genes on chromosome 13q (BRCA2 and D13S25), and three genes on chromosome 7 (IL6, CYLN2, and MET) as well as DNA deletions of two genes on chromosome 17p (HIC1 and LLGL1). Finally, additional CIN-tumor-associated DNA amplifications were identified for EXT1 (8q24.11) and MYC (8q24.12) as well as DNA deletions for MAP2K5 (15q23) and LAMA3 (18q11.2). In contrast, distinct MIN-tumor-associated DNA amplifications were detected for E2F5 (8p22–q21.3), GARP (11q13.5–q14), ATM (11q22.3), KAL (Xp22.3), and XIST (Xq13.2) as well as DNA deletions for RAF1 (3p25), DCC (18q21.3), and KEN (21q tel). aCGH revealed distinct DNA copy number changes of oncogenes and tumor suppressor genes in CIN- and MIN-type sporadic colorectal carcinomas. The identified candidate genes are likely to have distinct functional roles in the carcinogenesis and progression of CIN- and MIN-type sporadic CRCs and may be involved in the differential response of CIN- and MIN-type tumor cells to (adjuvant) therapy, such as 5-fluorouracil.

Prognostic and Predictive Values of the Immunoscore in Patients with Rectal Cancer

Purpose: To determine whether the tumor immune infiltrate, as recently evaluated with the Immunoscore methodology, could be a useful prognostic marker in patients with rectal cancers. Experimental design: The influence of the immune infiltrate on patients outcome was investigated in patients with or without preoperative chemoradiation therapy (pCRT). The density of total (CD3+) and cytotoxic (CD8+) T lymphocytes was evaluated by immunohistochemistry and quantified by a dedicated image analysis software in surgical specimens of patients with rectal cancer (n = 111) who did not receive pCRT and in tumor biopsies performed before pCRT from additional 55 patients. The results were correlated with tumor recurrence, patients survival, and response to pCRT. Results: The densities of CD3+ and CD8+ lymphocytes and the associated Immunoscore (from I0 to I4) were significantly correlated with differences in disease-free and overall survival (HR, 1.81 and 1.72, respectively; all P < 0.005). Cox multivariate analysis supports the advantage of the Immunoscore compared with the tumor–node–metastasis (TNM) staging in predicting recurrence and survival (all P < 0.001). Lymph node ratio added information in a prognostic model (all P < 0.05). In addition, high infiltration of CD3+ and CD8+ lymphocytes in tumor biopsies was associated with downstaging of the tumor after pCRT (CD3+ cells; Fisher exact test P = 0.01). Conclusions: The Immunoscore could be a useful prognostic marker in patients with rectal cancer treated by primary surgery. The determination of the immune infiltrate in biopsies before treatment could be a valuable information for the prediction of response to pCRT. Clin Cancer Res; 20(7); 1891–9. ©2014 AACR.

Microscopic enteritis: Bucharest consensus

Microscopic enteritis (ME) is an inflammatory condition of the small bowel that leads to gastrointestinal symptoms, nutrient and micronutrient deficiency. It is characterised by microscopic or sub-microscopic abnormalities such as microvillus changes and enterocytic alterations in the absence of definite macroscopic changes using standard modern endoscopy. This work recognises a need to characterize disorders with microscopic and submicroscopic features, currently regarded as functional or non-specific entities, to obtain further understanding of their clinical relevance. The consensus working party reviewed statements about the aetiology, diagnosis and symptoms associated with ME and proposes an algorithm for its investigation and treatment. Following the 5(th) International Course in Digestive Pathology in Bucharest in November 2012, an international group of 21 interested pathologists and gastroenterologists formed a working party with a view to formulating a consensus statement on ME. A five-step agreement scale (from strong agreement to strong disagreement) was used to score 21 statements, independently. There was strong agreement on all statements about ME histology (95%-100%). Statements concerning diagnosis achieved 85% to 100% agreement. A statement on the management of ME elicited agreement from the lowest rate (60%) up to 100%. The remaining two categories showed general agreement between experts on clinical presentation (75%-95%) and pathogenesis (80%-90%) of ME. There was strong agreement on the histological definition of ME. Weaker agreement on management indicates a need for further investigations, better definitions and clinical trials to produce quality guidelines for management. This ME consensus is a step toward greater recognition of a significant entity affecting symptomatic patients previously labelled as non-specific or functional enteropathy.

New antimicrobial chitosan derivatives for wound dressing applications.

Chitosan is a non-toxic, biocompatible, biodegradable natural cationic polymer known for its low imunogenicity, antimicrobial, antioxidant effects and wound-healing activity. To improve its therapeutic potential, new chitosan-sulfonamide derivatives have been designed to develop new wound dressing biomaterials. The structural, morphological and physico-chemical properties of synthesized chitosan derivatives were analyzed by FT-IR, (1)H NMR spectroscopy, scanning electron microscopy, swelling ability and porosity. Antimicrobial, in vivo testing and biodegradation behavior have been also performed. The chitosan derivative membranes showed improved swelling and biodegradation rate, which are important characteristics required for the wound healing process. The antimicrobial assay evidenced that chitosan-based sulfadiazine, sulfadimethoxine and sulfamethoxazole derivatives were the most active. The MTT assay showed that some of chitosan derivatives are nontoxic. Furthermore, the in vivo study on burn wound model induced in Wistar rats demonstrated an improved healing effect and enhanced epithelialization of chitosan-sulfonamide derivatives compared to neat chitosan. The obtained results strongly recommend the use of some of the newly developed chitosan derivatives as antimicrobial wound dressing biomaterials.

Aurora A is differentially expressed and regulated in chromosomal and microsatellite instable sporadic colorectal cancers

The centrosome-associated kinase aurora A has been shown to be involved in genetic instability and to be (over)expressed in several human carcinomas. This study investigated aurora A gene copy numbers, mRNA and protein expression as well as tumour cell proliferation and aneuploidy in chromosomal and microsatellite instable sporadic colorectal cancers. Case-matched tissues of normal (n=71) and dysplastic (n=49) colorectal epithelium and invasive carcinomas (n=71) were included in this study. PCR-based microsatellite analysis classified 14/71 (20%) of carcinomas as microsatellite instable. A stepwise increase of aurora A mRNA expression (P<0.0001; quantitative RT-PCR) and aurora A protein expressing tumour cells (P=0.0141; immunohistochemistry) occurred in the adenoma-carcinoma sequence. Within invasive carcinomas, aurora A mRNA levels (P=0.0259) and aurora A positive tumour cells (P<0.0001) were closely associated with tumour cell proliferation (Ki-67 specific immunohistochemistry). Compared with chromosomal instable carcinomas, microsatellite instable carcinomas had significantly more aurora A positive tumour cells (P=0.0043) and a higher tumour cell proliferation (P=0.0335). In contrast, only chromosomal instable carcinomas exhibited marked tumour cell aneuploidy (P=0.0004, fluorescence in situ hybridization) and significantly higher aurora A gene copy numbers (P=0.0206) as compared with microsatellite instable carcinomas. This study further supports a role of aurora A in the carcinogenesis of sporadic colorectal cancers. Moreover, it demonstrates that in a minority of predominantly microsatellite instable carcinomas the presence of aurora A positive tumour cells is merely reflecting tumour cell proliferation. In contrast, the large majority of chromosomal instable carcinomas shows additional (de)regulation of aurora A by gene amplification and concomitant tumour cell aneuploidy. Thus, sporadic colorectal cancers exhibit different mechanisms of aurora A regulation and this may impact the efficacy of aurora-targeted therapies.

Class I and III HDACs and loss of active chromatin features contribute to epigenetic silencing of CDX1 and EPHB tumor suppressor genes in colorectal cancer

Aberrant Wnt/β-catenin signaling is a driving force during initiation and progression of colorectal cancer. Yet, the Wnt/β-catenin targets CDX1, EPHB2, EPHB3 and EPHB4 (EPHB2-4) act as tumor suppressors in intestinal epithelial cells and frequently appear to be transcriptionally silenced in carcinomas. The molecular mechanisms which underlie the apparent loss of expression of a subset of Wnt/β-catenin targets in a background of persistent pathway activity are largely unknown. To gain insight into this, we quantified expression of CDX1 and EPHB2-4 in human tissue specimens of case-matched colorectal normal mucosa, adenoma and invasive carcinoma. In particular EPHB2-4 display biphasic, albeit not strictly coincident, expression profiles with elevated levels in adenomas and decreased transcription in approximately 30% of the corresponding carcinomas. Consistent with their divergent and variable expression we observed considerable heterogeneity among the epigenetic landscapes at CDX1 and EPHB2-4 in a model of colorectal carcinoma cell lines. Unlike the inactive CDX1 locus, EPHB2-4 maintain DNA hypomethylation of their promoter regions in the silent state. A strong reduction of active histone modifications consistently parallels reduced expression of CDX1 and EPHB3 and to some extent of EPHB2. Accordingly, treatment with inhibitors for DNA methyltransferases (DNMTs) and histone deacetylases(HDACs) restored CDX1 and EPHB2-4 expression depending upon epigenetic features at their promoters but also upon cellular background. Overall our findings show that downregulation of CDX1 and EphB receptor genes occurs independently and that different branches of epigenetic control systems including class I and III HDACs contribute to epigenetic silencing of Wnt/β-catenin targets during colorectal tumorigenesis.

ROC-king onwards: intraepithelial lymphocyte counts, distribution & role in coeliac disease mucosal interpretation

Objectives Counting intraepithelial lymphocytes (IEL) is central to the histological diagnosis of coeliac disease (CD), but no definitive ‘normal’ IEL range has ever been published. In this multicentre study, receiver operating characteristic (ROC) curve analysis was used to determine the optimal cut-off between normal and CD (Marsh III lesion) duodenal mucosa, based on IEL counts on >400 mucosal biopsy specimens. Design The study was designed at the International Meeting on Digestive Pathology, Bucharest 2015. Investigators from 19 centres, eight countries of three continents, recruited 198 patients with Marsh III histology and 203 controls and used one agreed protocol to count IEL/100 enterocytes in well-oriented duodenal biopsies. Demographic and serological data were also collected. Results The mean ages of CD and control groups were 45.5 (neonate to 82) and 38.3 (2–88) years. Mean IEL count was 54±18/100 enterocytes in CD and 13±8 in normal controls (p=0.0001). ROC analysis indicated an optimal cut-off point of 25 IEL/100 enterocytes, with 99% sensitivity, 92% specificity and 99.5% area under the curve. Other cut-offs between 20 and 40 IEL were less discriminatory. Additionally, there was a sufficiently high number of biopsies to explore IEL counts across the subclassification of the Marsh III lesion. Conclusion Our ROC curve analyses demonstrate that for Marsh III lesions, a cut-off of 25 IEL/100 enterocytes optimises discrimination between normal control and CD biopsies. No differences in IEL counts were found between Marsh III a, b and c lesions. There was an indication of a continuously graded dose–response by IEL to environmental (gluten) antigenic influence.

Microscopic enteritis and pathomechanism of malabsorption.

Microscopic enteritis (ME) is the stage of microscopic and sub-microscopic changes (microenteropathy) associated with the symptoms of gluten sensitive enteropathy leading to micronutrient deficiencies. It is characterized by subtle mucosal abnormalities without prominent inflammation, villous effacement, erosions or ulcerations on conventional light microscopy. The intraepithelial lymphocytes are usually in normal range <25/100 enterocytes (microenteropathy) or increased (lymphocytic enteritis). ME is the entity behind atypical forms of CD previously known as potential and latent CD. Systemic inflammation predominantly is found to be engaged in pathophysiology of micro-nutrient deficiency even in absence of macroscopic mucosal changes.

The Effects of Adipose-Derived Stem Cell-Differentiated Adipocytes on Skin Burn Wound Healing in Rats.

Both adipose-derived stem cells (ADSCs) and fat grafting promote burn wound healing, but whether adipogen-derived cells using various inducers such as 3-isobutyl-1-methylxanthine (IBMX) and insulin affect wound healing is unknown. Herein, ADSC-differentiated adipogenic lineages were used in rat burn wounds to evaluate wound healing potential. ADSCs were cultivated using six different adipogenic differentiation conditions (IBMX ± insulin, IBMX for 5 days, high and low Dulbecco’s modified Eagle’s medium) and in vitro morphological changes and cell proliferations during adipogenic differentiation were recorded. Intermediate burn wounds were inflicted in 15 Wistar male rats. Afterwards, the rats were divided into five groups for subcutaneous injections under the wounds: control; ADSCs; differentiated adipocytes (−IBMX+INSULIN and +IBMX[D1–5]+INSULIN) and fat prepared by Coleman technique. Macroscopic changes and histology were documented for 3 weeks. Repeated measures analysis of variance was performed to analyze cell growth and wound healing with a statistical level set of P < .05. Induction cocktails significantly reduced proliferation and induced lipid droplet accumulation. Conditioning without insulin induced the least lipid accumulation, while discontinuing IBMX generated larger adipocytes (P < .001). Adipogenic differentiated ADSCs had similar wound healing abilities with ADSC and fat injections, but differentiated adipocytes (+IBMX[D1–5]+INSULIN) and fat grafting accelerated the early healing process relative to ADSC (P < .001). Reduced fibrosis and mild inflammatory infiltration limited to superficial dermis were observed in +IBMX(D1–5)+INSULIN and fat injection groups, while those reactions were mild to moderate in ADSC group. Differentiated adipocytes achieve similar wound healing results compared with ADSC and fat injections, but differentiated adipocytes (+IBMX[D1–5]+INSULIN) and fat grafting accelerate early healing relative to ADSC.

Macroporous structures based on biodegradable polymers—candidates for biomedical application

New hybrid cryogels comprising natural polymers (free atelocollagen or atelocollagen mixed with a hyaluronic acid derivative) and a synthetic polyester--poly(ε-caprolactone)--were successfully developed by a cryogenic treatment and a subsequent freeze-drying step. Systematic studies on the effect of preparation conditions (reaction mixture composition, total concentration of the feed dispersion, and freezing regime) on cryogelation efficiency were conducted. The degree of cross-linking and the morphology of the obtained materials were analyzed using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and (environmental) scanning electron microscopy (ESEM/SEM) techniques. Considering their possible biomedical application, the developed macroporous hydrogels were also investigated in terms of swelling behavior and hemo/biocompatibility. The produced hydrogels had an uniform interconnected open porous structure with a porosity of up to 95% and pores size in the range of 83-260 μm. All obtained cryogels were elastic, mechanically stable, with a superfast swelling kinetics. In vitro hemocompatibility assay gave hemolysis ratios (HRs) lower than 0.5%, which is below the permissible limit of 5%. The in vivo tolerance tests performed by implantation of cryogel specimens into Wistar rats proved their biocompatibility.