Łukasz Koperski

Matrix metalloproteinase-9 (MMP-9) in human intractable epilepsy caused by focal cortical dysplasia

Focal cortical dysplasia (FCD) is a developmental brain disorder characterized by localized abnormalities of cortical layering and neuronal morphology. It is associated with pharmacologically intractable forms of epilepsy in both children and adults. The mechanisms that underlie FCD-associated seizures and lead to the progression of the disease are unclear. Matrix metalloproteinases (MMPs) are enzymes that are able to influence neuronal function through extracellular proteolysis in various normal and pathological conditions. The results of experiments that have used rodent models showed that extracellular MMP-9 can play an important role in epileptogenesis. However, no studies have shown that MMP-9 is involved in the pathogenesis of human epilepsy. The aim of the present study was to determine whether MMP-9 plays a role in intractable epilepsy. Using an unbiased antibody microarray approach, we found that up regulation of MMP-9 is prominent and consistent in FCD tissue derived from epilepsy surgery, regardless of the patients age. Additionally, an up regulation of MMP-1, -2, -8, -10, and -13 was found but was either less pronounced or limited only to adult cases. In the dysplastic cortex, immunohistochemistry revealed that the highest MMP-9 immuno reactivity occurred in the cytoplasm of abnormal neurons and balloon cells. The neuronal over expression of MMP-9 also occurred in sclerotic hippocampi that were excised together with the dysplastic cortex, but sclerotic hippocampi were free of dysplastic features. In both locations, MMP-9 was also found in reactive astrocytes, albeit to a lesser extent. At the subcellular level, increased MMP-9 immunoreactivity was prominently upregulated at synapses. Thus, although upregulation of the enzyme in FCD is not causally linked to the developmental malformation, it may be a result of ongoing abnormal synaptic plasticity. The present findings support the hypothesis of the pathogenic role of MMP-9 in human epilepsy and may stimulate discussions about whether MMPs could be novel therapeutic targets for intractable epilepsy.

Bone regeneration potential of the new chitosan-based alloplastic biomaterial.

Over the last few years, alloplastic bone substitute materials are raising much interest as an alternative to autologic transplants and xenogenic materials especially in oral surgery. These non-immunogenic and completely resorbable biomaterials are becoming the basis for complete and predictable guided bone regeneration in many cases. The objective of our research was to evaluate the dynamics of bone formation in rats’ skulls after implantation of the new chitosan/tricalcium phosphate/alginate biomaterial in comparison to the commercially available alloplastic bone graft. A total of 45 adult male rats weighing 300–400 g were used for the study. The 85-mm-diameter defects in calvaria bone were prepared with a trephine bur, and then filled with the bone substitute materials: chitosan/tricalcium phosphate/alginate or easy-graft Classic (Degradable Solutions AG) (EA) or left just with the blood clot. Animals were sacrificed at 1 and 3 months for histological, histomorphometrical and micro-tomographic evaluations. Histological evaluation at 1 month showed early new bone formation, observed around the experimental biomaterial (CH/TCP/Alg). There were no features of purulent inflammation and necrosis or granulomatous inflammation. Microscopic examination after 3 months following the surgery revealed trabecular bone formation around chitosan-based bone graft with no significant inflammatory response. Less satisfactory and differing results were observed for the commercially available EA and control blood clot. The tested material (chitosan) showed a high degree of biocompatibility and osteoconductivity in comparison with the control groups. Additionally, it seemed to be a “user-friendly” material for oral surgeons.

Urethral distension as a novel method to simulate sphincter insufficiency in the porcine animal model

Objectives:  To describe a novel animal model of intrinsic sphincter deficiency.

Family of microRNA-146 Regulates RARβ in Papillary Thyroid Carcinoma

Retinoic acid is a promising tool in adjuvant cancer therapies, including refractory thyroid cancer, and its biological role is mediated by the retinoic acid receptor beta (RARβ). However, expression of RARβ is lowered in papillary thyroid carcinoma (PTC), contributing to promotion of tumor growth and inefficiency of retinoic acid and radioactive iodine treatment. The causes of aberrant RARB expression are largely unknown. We hypothesized that the culpable mechanisms include the action of microRNAs from the miR-146 family, previously identified as significantly upregulated in PTC tumors. To test this hypothesis, we assessed the expression of RARB as well as miR-146a-5p and miR-146b-5p in 48 PTC tumor/normal tissue pairs by Taqman assay to reveal that the expression of RARB was 3.28-fold decreased, and miR-146b-5p was 28.9-fold increased in PTC tumors. Direct interaction between miRs and RARB was determined in the luciferase assay and further confirmed in cell lines, where overexpression of miR-146a-5p and miR-146b-5p caused a 31% and 33% decrease in endogenous RARB mRNA levels. Inhibition of miR-146a and miR-146b resulted in 62.5% and 45.4% increase of RARB, respectively, and a concomitant decrease in proliferation rates of thyroid cancer cell lines, analyzed in xCELLigence system.We showed that two microRNAs of the miR-146 family directly regulate RARB. Inhibition of miRs resulted in restoration of RARB expression and decreased rates of proliferation of thyroid cancer cells. By restoring RARB levels, microRNA inhibitors may become part of an adjuvant therapy in thyroid cancer patients.

Next-generation sequencing reveals microRNA markers of adrenocortical tumors malignancy

Background Adrenocortical carcinoma is a rare finding among common adrenocortical tumors, but it is highly aggressive and requires early detection and treatment. Still, the differential diagnosis between benign and malignant lesions is difficult even for experienced pathologists and there is a significant need for novel diagnostic methods. In this study we aimed to reveal a complete set of microRNAs expressed in the adrenal gland and to identify easily detectable, stable and objective biomarkers of adrenocortical malignancy. Methods We employed next-generation sequencing to analyze microRNA profiles in a unique set of 51 samples, assigned to either a learning dataset including 7 adrenocortical carcinomas (ACCs), 8 adrenocortical adenomas (AAs) and 8 control samples (NAs), or a validation dataset including 8 ACCs, 10 AAs and 10 NAs. The results were validated in real-time Q-PCR. Results We detected 411 miRNAs expressed in 1763 length isoforms in the examined samples. Fifteen miRNAs differentiate between malignant (ACC) and non-malignant (AA + NA) tissue in the test set of independent samples. Expression levels of 6 microRNAs, miR-503-5p, miR-483-3p, miR-450a-5p, miR-210, miR-483-5p, miR-421, predict sample status (malignancy/non-malignancy) with at least 95% accuracy in both datasets. The best single-gene malignancy marker, miR-483-3p, has been validated by real-time RT PCR. Conclusions As a result of the study we propose clinically valid and easily detectable biomarkers of adrenocortical malignancy that may significantly facilitate morphological examination. Since microRNAs can be detected in blood, the study brings tools for development of non-invasive diagnostics of adrenocortical carcinomas.

Radioguided surgery in patient with pancreatic neuroendocrine tumour followed by PET/CT scan as a new approach of complete resection evaluation — case report

Radioguided surgery using 68-gallium labelled somatostatin analogues is a promising method for detection of small, intra-abdominal, neuroendocrine neoplasm (NEN). However, due to high background activity (physiological uptake in e.g. spleen, adrenal glands and kidneys) tumours of the tail and body of pancreas might be impossible to detect with hand-held gamma probe. Therefore a new concept of intraoperative PET/CT scan of the resected tissue can be helpful in determining whether the tumour has been excised within the margins of healthy tissue. A 71-year-old woman with primary, non-metastatic NEN of the tail of pancreas is described. The tumour was diagnosed using MRI and PET/CT scan. Before operation patient was administered intravenously 80 MBq of ⁶⁸Ga-DOTATATE. The surgery procedure was performed 60-180 minutes post injection. During the procedure pancreas was visualized, but the tumour could not be localized neither with palpation nor gamma probe. The tail of the pancreas was resected en bloc with spleen and adjacent lymph nodes. PET/CT scan of the tissue specimen was performed immediately followed by pathological examination. PET/CT scan of the resected tissue showed moderate activity in the tail of pancreas, and a small focus of high activity in the tail. Area of high SSTR expression in the tail corresponded with preoperative findings in MRI and whole-body PET/CT. Histopathological examination of the specimen confirmed the presence of neuroendocrine tumour grade 1. Immediate PET/CT scan of the surgical specimen can bring new quality to intraoperative assessment of completeness of resection of neuroendocrine tumours.

High cytoplasmic HuR expression is associated with advanced pT stage, high grade and increased microvessel density in urothelial bladder carcinoma

PURPOSE HuR (human antigen R) protein is a RNA binding protein that stabilizes the mRNA and controls the translation of genes involved in cell proliferation, differentiation, and carcinogenesis. Overexpression of HuR was reported in a variety of cancers, however its clinical significance in urothelial bladder cancer (UBC) is still unknown. Our aim is to investigate the association between HuR expression and selected histopathological factors, such as tumor grade, pT stage, regional lymph nodes status and microvessel density (MVD). METHODS We studied expression of HuR protein in 119 patients with UBC in stages pTis and pTa-pT4 using immunohistochemistry (IHC). Tumor MVD was evaluated immunohistochemically using anti-CD31 antibody. RESULTS We observed no association between nuclear HuR immunoreactivity and tumor grade, stage or MVD. We found a significant association between cytoplasmic HuR positivity and high tumor grade, pT stage and MVD (p<0,001). We also observed significantly higher MVD values in cases with positive cytoplasmic HuR expression (p<0,001). No association between HuR immunoreactivity and lymph nodes status was found. CONCLUSIONS Our results may suggest that HuR is involved in the process of acquiring malignant histopathological features and ability to invade the muscularis propria by UBC cells. Considering frequent difficulties in diagnosing UBC in specimens obtained from transurethral tumor resection and the risk of understaging, cytoplasmic HuR expression would suggest an advanced disease and necessitate serial sectioning of the specimen in search of muscle invasion. Association between HuR expression and MVD could suggest HuR involvement in the process of angiogenesis in UBC.

Functional analysis of a novel, thyroglobulin-embedded microRNA gene deregulated in papillary thyroid carcinoma.

MicroRNAs, non-coding regulators of gene expression, are known culprits of thyroid cancer. Using next-generation sequencing, we identified a novel microRNA gene, encoded within an important thyroid regulator – thyroglobulin, and analyzed its functionality in the thyroid gland. In vitro and in silico analyses proved that the novel miR-TG is processed from the precursor, and co-expressed with thyroglobulin. Both genes are specific for thyroid tissue and downregulated in papillary thyroid carcinoma by 44% (p = 0.04) and 48% (p = 0.001), respectively. Putative target genes for miR-TG were identified using in silico tools, which pinpointed MAP4K4, an oncogene upregulated in thyroid cancer. Analysis of transcriptome by RNA-seq revealed that overexpression of miR-TG in PTC-derived cell line led to downregulation of several genes, including MAP4K4 (fold change 0,82; p = 0.036). The finding was confirmed by SQ-PCR (fold change 071; p = 0.004). Direct interaction between miR-TG and MAP4K4 was confirmed in the luciferase assay (p = 0.0006). Functional studies showed increase proliferation in K1 cell line transfected with miR-TG. We propose that in normal thyroid miR-TG plays a fine-tuning effect on the maintenance of MAPK pathway, inhibiting the expression of miR’s target MAP4K4. This regulation is disturbed in cancer due to downregulation of the novel, thyroglobulin-embedded microRNA, characterized in this study.

Clinicopathological and immunohistochemical analysis of epithelial-lined (true) cysts of the adrenal gland with proposal of a new histogenetic categorization

Epithelial- lined (true) cysts are rare lesions and until now the only information we had about their histogenesis was based on the analysis of a few cases. We retrospectively reviewed 8 cases of cysts with a true epithelial lining (confirmed immunohistochemically). The pathological findings and immunohistochemical analysis of the epithelial linings allowed for categorization of the cysts into 3 groups. Five cysts had pure mesothelial lining, which was flattened to cuboidal, and demonstrated a positive reaction for mesothelial markers (eg. calretinin, WT1), and a negative reaction for EpCAM, EMA, PAX8 and ER. Two cysts had cuboidal to flattened lining, the cells of which were diffusely or focally positive for mesothelial markers, for some epithelial markers (eg. EpCAM and EMA) and despite a lack of müllerian-type epithelium demonstrated a positive reaction for PAX8 and focally for ER. A cyst derived from adreno-hepatic fusion (AHF)-related intra-adrenal bile ductules was diagnosed in a right adrenal gland which was directly adherent to the liver, microscopically features of AHF were visible with intermingling of adrenal and liver parenchymal cells. The immunoreactivity pattern was similar among the preserved cells of the cyst-lining, the intra-adrenal bile ductules and the normal bile ductules in the adjoining liver parenchyma. On the basis of this case series from a single institution (8 presented now and 1 reported before) we propose a new histogenetic categorization of adrenal epithelial cysts into: 1. pure mesothelial cysts (the most common type), 2. mesothelial cysts with incomplete or complete müllerian metaplasia 3. AHF-related cysts.

Adrenal cyst with both Müllerian and mesothelial differentiation: a clinicopathological and immunohistochemical study with implications for histogenesis

True epithelial-lined cysts are rare forms of adrenal cystic lesions, the pathogenesis of which is still not fully understood. In this report we present a case of an adrenal cyst diagnosed incidentally on imaging in a 31-year-old, previously healthy, obese woman. Due to non-specific hormonal disorders and enlargement of the lesion, a right-sided laparoscopic adrenalectomy was performed. The cyst was lined predominantly by ciliated cuboidal-to-columnar, Müllerian-type epithelium, and focally by flat-to-cuboidal, mesothelium-like lining. Immunohistochemistry demonstrated a strong positive reaction in the cells of both types of lining for CKAE1+E3, CK19, CK7 and WT1, and both had a negative reaction for CK20, CD34, Melan-A, SF1, TTF1, SMA and CDX2. The cells of the ciliated cuboidal-to-columnar epithelium were strongly positive for PAX8, ER, Ep-CAM and EMA, focally positive for PR, and were negative for calretinin, whereas the cells of the flat-to-cuboidal lining were positive for calretinin and podoplanin and showed only a weak positive response in individual cells for PAX8, EMA and Ep-CAM, but were negative for ER and PR. This is the first reported case of an adrenal ciliated epithelial cyst with Müllerian differentiation (confirmed immunohistochemically) in the English literature. The differences in morphology and immunophenotype of the two types of lining (epithelial Müllerian phenotype versus mesothelial phenotype), suggest that some adrenal epithelial cysts probably form due to metaplasia of mesothelium-derived lining. A similar mechanism may also be involved in the pathogenesis of at least some of the so-called Müllerian cysts (or inclusions) in other locations.