Clinical, Biological, Radiological Pathological and Immediate Post-Operative Remission of Sparsely and Densely Granulated Corticotroph Pituitary Tumors: A Retrospective Study of a Cohort of 277 Patients With Cushing's Disease.

Purpose: Cushing's disease is the most common cause of endogenous hypercortisolemia due to a corticotroph pituitary tumor. Up-to-date there is no reliable biomarker of invasiveness among corticotroph tumors, while it is well established in the literature that sparsely granulated somatotroph tumors are characterized by poorer prognosis. The aim of the study was to correlate multiple data including clinical, biochemical, radiological, and pathological findings (including granulation pattern) as well as immediate post-operative remission status among patients operated on due to corticotroph tumors. Methods: We enrolled all patients consecutively operated on for planned transsphenoidal neurosurgery due to corticotroph PitNETs in years 2010-2018. We excluded from analysis silent corticotroph tumors, plurihormonal PitNETs, and the Crooke's cell adenomas. Results: We recorded 348 hormonally active corticotroph PitNETs. The results of the analysis showed the female predominance 79.88% (n = 278), with the mean age of Cushing's disease occurrence 43.27 years of age. The mean time from the first signs and symptoms to the operation was 2 years. The women were diagnosed earlier (20-40 years of age vs. 50-60 years of age among men). We performed a detailed analysis of 277 cases classified by granularity pattern as DG or SG corticotroph PitNETs. Densely granulated tumors (DG) occurred four times more frequently than sparsely granulated (SG) (n = 225 vs. n = 52), at similar age (mean 42.94; median 40 vs. mean 45.46; median 45.5; p = 0.3896), but were characterized by lower Knosp's scale grades (p = 0.0147*), smaller preoperative tumors' volumes measured at MRI, and more commonly exhibited lower Ki-67 labeling index (<3%) (p = 0.0168*). What is more, DG adenomas more frequently achieved an immediate remission status (measured as postoperative cortisol concentration <2 µg/dl; p = 0.0180*), and the mean postoperative cortisol concentration in DG group was lower than in SG group (mean 5.375 µg/dl vs. 10.47 µg/dl; median 2.49 µg/dl vs. 6.52 µg/dl; p = 0.0028**). Conclusions: Our study indicates that DG corticotroph adenomas occurred at younger age, more commonly were microadenomas as compared to SG tumors, less frequently had invasive features in comparison to SG corticotroph adenomas (p = 0.0019**), and more commonly achieved an immediate postsurgical hormonal remission (p = 0.0180*). We highlight the need for an accurate differentiation of DG and SG subtypes in the pathomorphological diagnosis of corticotropic tumors, especially in invasive PitNETs.

The role of microRNAs in epithelial to mesenchymal transition and cancers; focusing on mir-200 family.

Epithelial to mesenchymal transition (EMT) is a process associated with cancer malignancy and metastases. Cells undergoing EMT lose their epithelial phenotype and acquire mesenchymal phenotype. This process is accompanied by several molecular changes such as decrease of E-cadherin and increase of N-cadherin which is called the "cadherin swich". MicroRNAs (miRNAs, miRs) are small non-coding RNAs having ability to regulate genes post-transcriptionally. Nowadays they are believed to take part in multiple physiological and pathological processes including cancer development. Comparison between TargetScan7 (www.targetscan.org) results for miR-200b and metanalysis of genes involved in EMT showed that miR-200b has a potential binding site in 60 genes that are involved in EMT (the majority of them were associated with mesenchymal phenotype). Our review summarizes literature findings contributing to experimentally proven interactions between miR-200b and genes involved in EMT process including cell receptors, signaling pathways, cell cycle or cell adhesion. The results of those interactions indicate that miR-200b may have an inhibitory impact on EMT or even in selected cases is able to restore epithelial phenotype.

Epithelial Cells of Deep Infiltrating Endometriosis Harbor Mutations in Cancer Driver Genes.

Endometriosis is an inflammatory condition manifested by the presence of endometrial-like tissue outside of the uterine cavity. The most common clinical presentations of endometriosis are dysmenorrhea, infertility, and severe pelvic pain. Few hypotheses attempt to explain the pathogenesis of endometriosis; however, none of the theories have been fully confirmed or considered universal. We examined somatic mutations in eutopic endometrium samples, deep endometriotic nodules and peripheral blood from 13 women with deep endometriosis of the rectovaginal space. Somatic variants were identified in laser microdissected samples using next-generation sequencing. A custom panel of 1296 cancer-related genes was employed, and selected genes representing cancer drivers and non-drivers for endometrial and ovarian cancer were thoroughly investigated. All 59 detected somatic variants were of low mutated allele frequency (<10%). In deep ectopic lesions, detected variants were significantly more often located in cancer driver genes, whereas in eutopic endometrium, there was no such distribution. Our results converge with other reports, where cancer-related mutations were found in endometriosis without cancer, particularly recurrent KRAS mutations. Genetic alterations located in ectopic endometriotic nodules could contribute to their formation; nevertheless, to better understand the pathogenesis of this disease, more research in this area must be performed.

The regulation of cyclin D promoters - review.

Cyclins are key regulators of cell cycle progression and survival. Particularly cyclins D (cyclin D1, D2, and D3) act in response to the mitogenic stimulation and are pivotal mediators between proliferative pathways and the nuclear cell cycle machinery. Dysregulation of cyclins expression results in impaired development, abnormal cell growth or tumorigenesis. In this review we summarize current knowledge about regulatory role of the cyclin D promoters, transcriptional factors: regulators, co-activators and adaptor proteins necessary to their activation. We focused on the intracellular signaling pathways vital to cell growth, differentiation and apoptosis including transcription factor families: activator protein 1 (AP1), nuclear factor (NFκB), signal transducer and activator of transcription (STAT), cAMP response element-binding protein (CREB) and Sp/NF-Y, with a special insight into the tissue specific cyclin representation.

Pituitary tumours - a large retrospective single-centre study of over 2300 cases. Experience of a tertiary reference centre.

INTRODUCTION: Pituitary adenomas (PAs), also known as a pituitary neuroendocrine tumours (PitNET), are usually benign tumours of the anterior lobe of the pituitary gland and account for the third most common intracranial neoplasm. The most common type of pituitary adenoma is lactotroph adenoma, in which dopamine agonists are the first-line treatment. Nevertheless, in selected cases surgery or even radiotherapy may be required. In the current study, we aimed to analyse all patients who underwent surgery due to intrasellar mass in order to evaluate frequency of particular pituitary tumours, clinical diagnosis, and pathology findings. MATERIAL AND METHODS: We retrospectively analysed all cases of patients consecutively operated due to intrasellar mass between 1st January 2010 and 31st December 2018 at the Department of Neurosurgery, Military Institute of Medicine in Warsaw, Poland. RESULTS: Our database included 2348 cases: 1390 women (59.2%) and 958 men (40.8%). The mean age for women was 48.4 years (SD ± 15.72; median 49) and for men 50.9 years (SD ± 14.94; median 53). In our cohort we found: 869 gonadotroph and null cell adenomas, 751 somatotroph and mammosomatotroph adenomas, 386 corticotroph adenomas, 71 plurihormonal adenomas, 59 craniopharyngiomas, 44 lactotroph adenomas, 18 purely thyrotroph adenomas, and other rare cases of pituitary tumours including one pituitary carcinoma metastasising to the liver (corticotroph origin). CONCLUSIONS: We provide a comprehensive analysis of both clinical and pathological findings of the largest cohort of patients operated on for pituitary adenomas in one tertiary reference centre. To the best of our knowledge, this is the largest up-to-date published analysis in our country.

Metalloproteinase 14 (MMP-14) and hsa-miR-410-3p expression in human inflamed dental pulp and odontoblasts.

The objective of this study is to evaluate MMP-14 expression in odontoblasts and in the bulk of dental pulp of teeth with pulpitis; to determine the expression of microRNA-410 (miR-410) in pulp tissue, since sequence analysis suggests that miR-410 has potential binding site on MMP-14's 3'UTR, and hence, can regulate expression of the latter one. Tissue samples of dental pulp from teeth with pulpitis and healthy (control) were formalin fixed and paraffin embedded (FFPE). Samples were examined using immunohistochemical staining for MMP-14 and the expression of miR-410 was evaluated using qRT-PCR. In both, healthy and inflamed pulp odontoblasts stained more intensively than remaining pulp tissue, but this difference was not statistically significant. More positive staining was observed in inflamed pulps compared to healthy pulps. Expression of miR-410 was found significantly lower in inflamed pulps than in healthy ones. In the two examined zones, odontoblasts and remaining pulp, miR-410 was expressed on a similar level. No statistically significant correlation of miR-410 and MMP-14 expression was found. We showed that inflammation changes the MMP-14 expression in pulp tissue and odontoblasts. This study demonstrates for the first time miR-410 expression in human dental pulp and that expression of this microRNA was downregulated in inflamed dental pulp and odontoblasts.

Lineage-dependent role of miR-410-3p as oncomiR in gonadotroph and corticotroph pituitary adenomas or tumor suppressor miR in somatotroph adenomas via MAPK, PTEN/AKT, and STAT3 signaling pathways.

PURPOSE: miR-410-3p plays opposite roles in different cancers and may act as an oncomiR or tumor suppressor miR. The purpose of this study was to assess the role of miR-410-3p in somatotroph, gonadotroph, and corticotroph pituitary adenomas. METHODS: Tissue samples were obtained from 75 patients with pituitary adenoma. miR-410-3p expression was assessed using qRT-PCR performed on RNA isolated from fresh frozen samples. In vitro experiments were performed on cell lines derived from somatotroph (GH3), gonadotroph (RC-4B/C), and corticotroph (AtT-20) pituitary tumors. Cells were transfected with synthetic mimic of miR-410-3p or non-targeting scrambled-miR control. Subsequently, proliferation assays and transwell invasion assays were performed. The expression of cyclin D1, E1, and B1 in cells after transfection was determined using qRT-PCR. The activation of MAPK, PTEN/AKT and STAT3 signaling pathways were assessed using western blot. RESULTS: We have found that the level of expression of miR-410-3p differs in particular types of pituitary adenomas. miR-410-3p significantly upregulates proliferation and invasiveness of RC-4B/C and AtT-20 cells, while inhibiting GH3 cells. We observed that the levels of cyclin B1 upon transfection with miR-410-3p mimic were increased in RC-4B/C and AtT-20, yet decreased in GH3 cells. We have shown that miR-410-3p promoted the activation of MAPK, PTEN/AKT, and STAT3 signaling pathways in RC-4B/C and AtT-20 cells, but suppressed their activity in GH3 cells. CONCLUSIONS: miR-410-3p acts as an oncomiR in gonadotroph and corticotroph adenoma cells, while as a tumor suppressor miR in somatotroph adenoma cells.

Location of Mutation in BRCA2 Gene and Survival in Patients with Ovarian Cancer.

Purpose: BRCA2 plays a central role in homologous recombination by loading RAD51 on DNA breaks. The objective of this study is to determine whether the location of mutations in the RAD51-binding domain (RAD51-BD; exon 11) of BRCA2 gene affects the clinical outcome of ovarian cancer patients.Experimental Design: A study cohort of 353 women with ovarian cancer who underwent genetic germline testing for BRCA1 and BRCA2 genes was identified. Progression-free survival (PFS), platinum-free interval (PFI), and overall survival (OS) were analyzed. The Cancer Genome Atlas (TCGA) cohort of ovarian cancer (n = 316) was used as a validation cohort.Results: In the study cohort, 78 patients were carriers of germline mutations of BRCA2 After adjustment for FIGO stage and macroscopic residual disease, BRCA2 carriers with truncating mutations in the RAD51-BD have significantly prolonged 5-year PFS [58%; adjusted HR, 0.36; 95% confidence interval (CI), 0.20-0.64; P = 0.001] and prolonged PFI (29.7 vs. 15.5 months, P = 0.011), compared with noncarriers. BRCA2 carriers with mutations located in other domains of the gene do not have prolonged 5-year PFS (28%, adjusted HR, 0.67; 95% CI, 0.42-1.07; P = 0.094) or PFI (19 vs. 15.5 months, P = 0.146). In the TCGA cohort, only BRCA2 carriers harboring germline or somatic mutations in the RAD51-BD have prolonged 5-year PFS (46%; adjusted HR, 0.30; 95% CI, 0.13-0.68; P = 0.004) and 5-year OS (78%; adjusted HR, 0.09; 95% CI, 0.02-0.38; P = 0.001).Conclusions: Among ovarian cancer patients, BRCA2 carriers with mutations located in the RAD51-BD (exon 11) have prolonged PFS, PFI, and OS. Clin Cancer Res; 24(2); 326-33. ©2017 AACR.

Post-transcriptional Regulation of MMP16 and TIMP2 Expression via miR-382, miR-410 and miR-200b in Endometrial Cancer.

BACKGROUND/AIM: The post-transcriptional regulation of matrix metalloproteinases (MMPs) via microRNAs (miRNAs) has been recently described in numerous human malignancies. However, the exact mechanisms of miRNA-mediated MMPs deregulation in endometrial cancer (EC) remain unclear. Herein, we aimed to analyze the expression of MMP2, MMP16 and TIMP2 and identify miRNAs that modulate their expression. MATERIALS AND METHODS: Protein expression was assessed by immunohistochemistry in formalin-fixed paraffin-embedded EC samples. Target prediction algorithms were applied to select miRNAs binding the 3'UTRs of MMP16 (miR-377, miR-382, miR-410, miR-200b) or TIMP2 (miR-200b), and their levels were measured by qPCR in laser capture-microdissected tissue fragments. Luciferase assays and western blotting were used to indicate individual miRNA- mRNA interactions. RESULTS: Overexpression of MMP2 and MMP16 in cancerous tissues corresponded to down-regulation of miR-377, miR-382 and miR-410, while decreased expression of TIMP2 was associated with miR-200b up-regulation. In vitro experiments confirmed direct regulation of MMP16 by miR-382 and miR-410, and TIMP2 by miR-200b in EC Ishikawa cells. CONCLUSION: We demonstrated novel mechanisms of miRNA-mediated regulation of MMPs activity in EC.

Increased expression of the microRNA 106b~25 cluster and its host gene MCM7 in corticotroph pituitary adenomas is associated with tumor invasion and Crooke's cell morphology.

PURPOSE: MCM7 (minichromosome maintenance complex component 7), a DNA replication licensing factor, is a host gene for the oncogenic miR-106b~25 cluster. It has been recently revealed as a relevant prognostic biomarker in a variety of cancers, including pituitary adenomas. The purpose of this study was to assess whether miR-106b~25 and MCM7 levels correlate with tumor invasiveness in a cohort of ACTH-immunopositive adenomas. METHODS: Tissue samples were obtained intraoperatively from 25 patients with pituitary adenoma. Tumor invasiveness was assessed according to the Knosp grading scale. MCM7, Ki-67 and TP53 levels were assessed by immunohistochemical staining, while the expression of miR-106b-5p, miR-93-5p, miR-93-3p and miR-25-3p were measured using quantitative real-time PCR performed on RNA isolated from FFPE tissues. RESULTS: We have found a significant increase in MCM7 and Ki-67 labeling indices in invasive ACTHomas. Moreover, MCM7 was ubiquitously overexpressed in Crooke's cell adenomas. The expression of miR-93-5p was significantly elevated in invasive compared to noninvasive tumors. In addition, all four microRNAs from the miR-106b~25 cluster displayed marked upregulation in Crooke's cell adenomas. Remarkably, MCM7 and miR-106b-5p both strongly correlated with Knosp grade. A combination of MCM7 LI and miR-106b~25 cluster expression was able to accurately differentiate invasive from noninvasive tumors and had a significant discriminatory ability to predict postoperative tumor recurrence/progression. CONCLUSIONS: miR-106b~25 and its host gene MCM7 are potential novel biomarkers for invasive ACTH-immunopositive pituitary adenomas. Additionally, they are both significantly upregulated in rare Crooke's cell adenomas and might therefore contribute to their aggressive phenotype.

TIMP4 expression is regulated by miR-200b-3p in prostate cancer cells.

In prostate cancer TIMP4 expression level fluctuates with tumor progression. The mechanism and factors influencing its expression remain unclear. The aim of the study was to test the hypothesis on regulation of TIMP4 by microRNA-200b-3p. The levels of TIMP4 and miR-200b-3p expression were determined by real time PCR in 27 prostate carcinomas and eight benign prostatic hyperplasia samples. We found that miR-200b-3p positively correlated with TIMP4 expression in cancer samples (r = 0.46; p < 0.02). Moreover, mean miR-200b-3p level and TIMP4 expression were both higher in cancer tissues compared to benign prostatic hyperplasia samples (p > 0.05). Next, to test probable mechanisms of the regulation androgen-sensitive human prostate adenocarcinoma cells (LNCaP) were transfected with synthetic-miR-200b-3p or its synthetic antagonist. Modulation of miR-200b-3p in LNCaP cells had an impact on TIMP4 expression confirming the observation made in analyzed clinical samples. Two targets of miR-200b-3p: ZEB1 and ETS1 were investigated subsequently as potential regulators of TIMP4, however, no effect of their modulation on TIMP4 expression in LNCaP cells was found. Concluding, miR-200b-3p mediates regulation of TIMP4 expression in prostate cancer but exact mechanism needs to be investigated.

The expression of MMP-14 and microRNA-410 in FFPE tissues of human endometrial adenocarcinoma.

Endometrial cancer (EC) is the most common gynecological malignancy in Europe and North America. It is classified into two types exhibiting different characteristics and prognosis. Type I is an estrogen-dependent tumor, histologically classified as low grade and low stage, usually with an excellent prognosis. Type II EC is unrelated to estrogen stimulation and is characterized by a poor prognosis. MicroRNAs (miRNAs, miRs) are small non-coding RNA polynucleotides that regulate gene expression post-transcriptionally. Various dysregulations in microRNA expression are often considered to have an impact on the diagnosis, prognosis and overall survival in patients diagnosed with different types of cancers. Recent data suggest that microRNAs play an important role in the pathogenesis of EC. The aim of the study was to evaluate the involvement of matrix metaloprotease 14 (MMP-14) and microRNA-410 in formation of the EC tumor. To this end expression of MMP-14 and microRNA-410 was assessed within the cancer, transient and healthy zones in the histological sections of tumours using immunohistochemical staining and laser capture microdissection (LCM) followed by a quantitative real-time PCR. The results revealed significantly higher expression of MMP-14 in the cancer tissue zone in comparison to the healthy tissue zone, as well as a lower expression of microRNA-410 in the cancer zone compared with the healthy zone. This reverse correlation may suggest a regulatory role of miRNA-410 in modulating levels of MMP-14 in EC. This is the first report on such regulation in human endometrial cancer.

Upregulation of mitogen-activated protein kinase in ganglioglioma.

Ganglioglioma (GG) is a low-grade neoplasm, often associated with intractable epilepsy in pediatric patients. Available data suggest a relationship between GG and other glioneuronal lesions. So far, little is known about activation of kinases belonging to the mTor (mammalian target of rapamycin) pathway, although its upregulation is often found in brain tumors. Involvement of mTor kinase is responsible for excessive proliferation. In the current study we focused on the possible role of the Erk/Mapk (extracellular-signal-regulated kinase/mitogen-activated protein kinase) pathway in GG development. Eight GG tumors were resected from pediatric patients. Collected data reveal activation of proteins from the Erk pathway: Mek (extracellular regulated protein kinase kinase/mitogen-activated protein kinase kinase), Erk, Rsk1 (ribosomal S6 kinase 1), Rheb (Ras homolog enriched in brain) and Msk1 (mitogen- and stress-activated protein kinase 1), as detected by the western blot method. Moreover, activation of other proteins upstream of mTor - IGF-1R ß (insulin-like growth factor 1ß receptor), INR ß (insulin receptor ß), Akt/PKB (protein kinase B) - or downstream - 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) and rpS6 (ribosomal protein S6) - was also confirmed.