Decreased expression of miR-23b is associated with poor survival of endometrial cancer patients.

Endometrial cancer (EC) is one of the most common types of cancer of the female reproductive system. EC is classified into two types (EC1 and EC2). MiRNAs are single-stranded RNA molecules that regulate gene expression posttranscriptionally. They have aberrant expression profiles in cancer, including EC. This study aimed to assess the level of expression of a panel of 16 miRNAs in both types of EC and healthy endometrium (HE). A total of 45 patients were enrolled into the study, 18 patients diagnosed with EC1, 12 diagnosed with EC2, and 15 HE controls. Tumor tissues or healthy endometrial tissues were dissected from archival formalin-fixed paraffin-embedded (FFPE) using laser capture microdissection (LCM). RNA was isolated from collected material and the expression of selected miRNAs was determined using the real-time qPCR. We found that miR-23b, miR-125b-5p, miR-199a-3p, miR-221-3p, and miR-451a were downregulated in EC in comparison to HE. Moreover, the expression of miR-34a-5p and miR-146-5p was higher in EC1 compared to EC2. Analysis of The Cancer Genome Atlas (TCGA) database confirmed decreased levels of miR-23b, miR-125b-5p, and miR-199a-3p in EC. Decreased miR-23b expression was associated with worse survival of EC patients.

Evaluation of spatial PD1 and PD-L1 expression in inflammatory bowel disease samples - a pilot study.

Alterations of PD1/PD-L1 pathway may be associated with an excessive inflammatory response in the intestinal wall in inflammatory bowel diseases (IBD). To evaluate the expression of PD-1 and PD-L1 in 4 compartments of intestinal wall (mucosa, submucosa, muscularis propria and lymphatic follicles), high-resolution immunohistochemically stained slides were obtained from formalin-fixed paraffin-embedded samples of 10 Crohn's disease (CD), 9 ulcerative colitis (UC) and 10 unaffected individuals cases. The levels of expression were quantified using the QuPath software. PD-1 was detected in lymphatic follicles in affected and unaffected tissue samples and in inflammatory infiltration in IBD. There was no difference between groups neither in PD-1 overall expression nor in individual compartments, with the exception of the mucosal expression. It was higher in the mucosa of CD patients comparing to controls, however this difference was marginal (p = 0.0461). PD-L1 was expressed in endothelium and mesenteric nervous plexi, consistently in each group. There were no significant differences in PD-L1 immunoreactivity in context of histologic compartment nor clinical diagnosis. The results suggest that PD-1 and PD-L1 expression in intestinal tissue is heterogeneous in the analysed groups, thus it may be dependent on individual characteristics.

Single Dose of N-Acetylcysteine in Local Anesthesia Increases Expression of HIF1α, MAPK1, TGFß1 and Growth Factors in Rat Wound Healing.

In this study, we aimed to investigate the influence of N-acetylcysteine (NAC) on the gene expression profile, neoangiogenesis, neutrophils and macrophages in a rat model of incisional wounds. Before creating wounds on the backs of 24 Sprague-Dawley rats, intradermal injections were made. Lidocaine-epinephrin solutions were supplemented with 0.015%, 0.03% or 0.045% solutions of NAC, or nothing (control group). Scars were harvested on the 3rd, 7th, 14th and 60th day post-surgery. We performed immunohistochemical staining in order to visualize macrophages (anti-CD68), neutrophils (anti-MPO) and newly formed blood vessels (anti-CD31). Additionally, RT-qPCR was used to measure the relative expression of 88 genes involved in the wound healing process. On the 14th day, the number of cells stained with anti-CD68 and anti-CD31 antibodies was significantly larger in the tissues treated with 0.03% NAC compared with the control. Among the selected genes, 52 were upregulated and six were downregulated at different time points. Interestingly, NAC exerted a significant effect on the expression of 45 genes 60 days after its administration. In summation, a 0.03% NAC addition to the pre-incisional anesthetic solution improves neovasculature and increases the macrophages' concentration at the wound site on the 14th day, as well as altering the expression of numerous genes that are responsible for the regenerative processes.

N-Acetylcysteine Added to Local Anesthesia Reduces Scar Area and Width in Early Wound Healing-An Animal Model Study.

The aim of the study was to evaluate if a pre-incisional N-acetylcysteine (NAC) treatment altered the process of wound healing in a rat model. The dorsal skin of 24 Sprague-Dawley rats was incised in six locations. Before the incisions were made, skin was injected either with lidocaine and epinephrine (one side) or with these agents supplemented with 0.015%, 0.03%, or 0.045% NAC (contralaterally). Photographic documentation of the wound healing process was made at 11 time points. Rats were sacrificed 3, 7, 14, or 60 days after incision to excise scars for histological analysis. They included: Abramov scale scoring, histomorphometry analysis, and collagen fiber arrangement assessment. Skin pretreated with 0.03% NAC produced the shortest scars at all analyzed time points, though this result was statistically insignificant. At this NAC concentration the scars had smaller areas on the third day and were narrower on the day 4 compared with all the other groups (p < 0.05). On day 7, at the same concentration of NAC, the scars had a higher superficial concentration index (p = 0.03) and larger dermal proliferation area (p = 0.04). NAC addition to pre-incisional anesthetic solution decreased wound size and width at an early stage of scar formation at all concentrations; however, with optimal results at 0.03% concentration.

Higher Mutation Burden in High Proliferation Compartments of Heterogeneous Melanoma Tumors.

Melanoma tumors are the most heterogeneous of all tumor types. Tumor heterogeneity results in difficulties in diagnosis and is a frequent cause of failure in treatment. Novel techniques enable accurate examination of the tumor cells, considering their heterogeneity. The study aimed to determine the somatic variations among high and low proliferating compartments of melanoma tumors. In this study, 12 archival formalin-fixed paraffin-embedded samples of previously untreated primary cutaneous melanoma were stained with Ki-67 antibody. High and low proliferating compartments from four melanoma tumors were dissected using laser-capture microdissection. DNA was isolated and analyzed quantitatively and qualitatively. Libraries for amplicon-based next-generation sequencing (NGS) were prepared using NEBNext Direct Cancer HotSpot Panel. NGS detected 206 variants in 42 genes in melanoma samples. Most of them were located within exons (135, 66%) and were predominantly non-synonymous single nucleotide variants (99, 73.3%). The analysis showed significant differences in mutational profiles between high and low proliferation compartments of melanoma tumors. Moreover, a significantly higher percentage of variants were detected only in high proliferation compartments (39%) compared to low proliferation regions (16%, p < 0.05). Our results suggest a significant functional role of genetic heterogeneity in melanoma.

Colorectal Adenocarcinomas Harboring ALK Fusion Genes: A Clinicopathologic and Molecular Genetic Study of 12 Cases and Review of the Literature.

This study determined the frequency and the clinicopathologic and genetic features of colorectal carcinomas driven by oncogenic fusions of the anaplastic lymphoma kinase gene (ALK). Of the 8150 screened tumors, 12 (0.15%) were immunohistochemically ALK-positive with D5F3 antibody. These cancers harbored CAD-ALK (n=1), DIAPH2-ALK (n=2), EML4-ALK (n=2), LOC101929227-ALK (n=1), SLMAP-ALK (n=1), SPTBN1-ALK (n=4), and STRN-ALK (n=1) fusions, as detected by an RNA-based next-generation sequencing assay. ALK fusion carcinomas were diagnosed mostly in older patients with a 9:3 female predominance (median age: 72 y). All tumors, except a rectal one, occurred in the right colon. Most tumors were stage T3 (n=7) or T4 (n=3). Local lymph node and distant metastases were seen at presentation in 9 and 2 patients. These tumors showed moderate (n=6) or poor (n=3) glandular differentiation, solid medullary growth pattern (n=2), and pure mucinous morphology (n=1). DNA mismatch repair-deficient phenotype was identified in 10 cases. Tumor-infiltrating lymphocytes were prominent in 9 carcinomas. In 4 carcinomas, tumor cells showed strong, focal (n=3), or diffuse programmed death-ligand 1 immunoreactivity. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 tumors. Four patients died of disease within 3 years, and 7 were alive with follow-up ranging from 1 to 8 years. No mutations in BRAF, RAS, and in genes encoding components of PI3K-AKT/MTOR pathway were identified. However, 1 tumor had a loss-of-function PTEN mutation. Aberration of p53 signaling, TP53 mutations, and/or nuclear accumulation of p53 protein was seen in 9 cases. ALK fusion colorectal carcinomas are a distinct and rare subtype of colorectal cancers displaying some features of mismatch repair-deficient tumors.

miR-410-3p is induced by vemurafenib via ER stress and contributes to resistance to BRAF inhibitor in melanoma.

Despite significant development of melanoma therapies, death rates remain high. MicroRNAs, controlling posttranscriptionally gene expression, play role in development of resistance to BRAF inhibitors. The aim of the study was to assess the role of miR-410-3p in response to vemurafenib-BRAF inhibitor. FFPE tissue samples of 12 primary nodular melanomas were analyzed. With the use of Laser Capture Microdissection, parts of tumor, transient tissue, and adjacent healthy tissue were separated. In vitro experiments were conducted on human melanoma cell lines A375, G361, and SK-MEL1. IC50s of vemurafenib were determined using MTT method. Cells were transfected with miR-410-3p mimic, anti-miR-410-3p and their non-targeting controls. ER stress was induced by thapsigargin. Expression of isolated RNA was determined using qRT-PCR. We have found miR-410-3p is downregulated in melanoma tissues. Its expression is induced by vemurafenib in melanoma cells. Upregulation of miR-410-3p level increased melanoma cells resistance to vemurafenib, while its inhibition led to the decrease of resistance. Induction of ER stress increased the level of miR-410-3p. miR-410-3p upregulated the expression of AXL in vitro and correlated with markers of invasive phenotype in starBase. The study shows a novel mechanism of melanoma resistance. miR-410-3p is induced by vemurafenib in melanoma cells via ER stress. It drives switching to the invasive phenotype that leads to the response and resistance to BRAF inhibition.

Colonic Adenocarcinomas Harboring NTRK Fusion Genes: A Clinicopathologic and Molecular Genetic Study of 16 Cases and Review of the Literature.

This study was undertaken to determine the frequency, and the clinicopathologic and genetic features, of colon cancers driven by neurotrophic receptor tyrosine kinase (NTRK) gene fusions. Of the 7008 tumors screened for NTRK expression using a pan-Trk antibody, 16 (0.23%) had Trk immunoreactivity. ArcherDx assay detected TPM3-NTRK1 (n=9), LMNA-NTRK1 (n=3), TPR-NTRK1 (n=2) and EML4-NTRK3 (n=1) fusion transcripts in 15 cases with sufficient RNA quality. Patients were predominantly women (median age: 63 y). The tumors involved the right (n=12) and left colon unequally and were either stage T3 (n=12) or T4. Local lymph node and distant metastases were seen at presentation in 6 and 1 patients, respectively. Lymphovascular invasion was present in all cases. Histologically, tumors showed moderate to poor (n=11) differentiation with a partly or entirely solid pattern (n=5) and mucinous component (n=10), including 1 case with sheets of signet ring cells. DNA mismatch repair-deficient phenotype was seen in 13 cases. Tumor-infiltrating CD4/CD8 lymphocytes were prominent in 9 cases. Programmed death-ligand 1 positive tumor-infiltrating immune cells and focal tumor cell positivity were seen in the majority of cases. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 cases. No mutations in BRAF, RAS, and PIK3CA were identified. However, other genes of the PI3K-AKT/MTOR pathway were mutated. In several cases, components of Wnt/ß-catenin (APC, AMER1, CTNNB1), p53, and TGFß (ACVR2A, TGFBR2) pathways were mutated. However, no SMAD4 mutations were found. Two tumors harbored FBXW7 tumor suppressor gene mutations. NTRK fusion tumors constitute a distinct but rare subgroup of colorectal carcinomas.

Primary malignant melanoma of esophagus: clinicopathologic characterization of 20 cases including molecular genetic profiling of 15 tumors.

Primary malignant melanoma of esophagus is very rare, and its clinicopathologic and genetic features have not been extensively investigated. In this study, 20 tumors from 14 male and 6 female patients (40-79 years old) were evaluated. Dysphagia, chest pain, and weight loss were frequent symptoms. Thirteen melanomas, including two with multiple lesions, involved the distal third of esophagus. The median tumor diameter was 6 cm. Epithelioid morphology, moderate atypia, and pigmentation were typical findings. None of the patients had melanoma elsewhere, and all tumors exhibited a junctional peri-epithelial component consistent with a primary lesion. The median mitotic activity was 11 per 10 high-power fields (range, 0-31). Nine patients died of tumor within 4-22 months, however, two showed long-term (96 and 104 months) survival. In 15 cases, tissue for further immunohistochemical and molecular studies were available. BRAF, KIT, and NRAS mutation status was assessed by Sanger sequencing in all 15 tumors. The next-generation sequencing of 50 or 409 genes was performed in five and three cases, respectively. IGF1R expression indicating activation of the IGF axis was seen in 82% (9/11) of tumors. However, no BRAF mutations were identified. In 33% (5/15) of tumors, NRAS mutations were detected. KIT expression was seen in 50% (7/14) of melanomas including single KIT mutant. Two of three tumors evaluated with 409 genes panel revealed multiple driver mutations indicating sub-clonal expansion, whereas a single mutation (TSC1 p.H371Q) was the sole change in the third case. SF3B1 p.K666T and p.R625C mutations were detected in two cases. However, no co-occurrence of SF3B1 and GNAQ or GNA11 mutations, seen in uveal melanoma, was detected. FBXW7 p.R465C and p.R479G mutations, linked to cancer progression, were found in two of eight tumors. In summary, esophageal melanoma mutation profile indicates complexity of molecular mechanisms underlying its pathogenesis.

Anti-inflammatory Action of Metformin with Respect to CX3CL1/CX3CR1 Signaling in Human Placental Circulation in Normal-Glucose Versus High-Glucose Environments.

Upregulation of chemokine CX3CL1 and its receptor CX3CR1 occurs in the diabetic human placenta. Metformin, an insulin-sensitizing biguanide, is used in the therapy of diabetic pregnancy. By preventing the activation of NF-κB, metformin exhibits anti-inflammatory properties. We examined the influence of hyperglycemia (25 mmol/L glucose; HG group; N = 36) on metformin-mediated effects on CX3CL1 and TNF-α production by placental lobules perfused extracorporeally. Additionally, CX3CR1 expression and contents of CX3CR1, TNF-α receptor 1 (TNFR1), and NF-κB proteins in the placental tissue were evaluated. Placentae perfused under normoglycemia (5 mmol/L glucose; NG group; N = 36) served as the control. Metformin (2.5 and 5.0 mg/L; subgroups B and C) lowered the production of CX3CL1 and TNF-α in a dose-dependent and time-dependent manner. Hyperglycemia did not weaken the strength of these metformin effects. Moreover, CX3CL1 levels after perfusion with 5.0 mg/L metformin were reduced by 33.28 and 33.83% (at 120 and 150 min, respectively) in the HG-C subgroup versus 24.98 and 23.66% in the NG-C subgroup, which indicated an augmentation of the metformin action over time in hyperglycemia. CX3CR1 expression was significantly higher in the HG-B and HG-C subgroups compared to that in the NG-B and NG-C subgroups. Increased CX3CR1 protein content in the placental lysates was observed in subgroups B and C. The two higher metformin concentrations significantly decreased the levels of NF-κBp65 protein content in both groups. However, the decrease was significantly stronger in hyperglycemia. TNFR1 upregulation in the HG group was not affected by metformin. Further studies on metformin therapy during pregnancy are needed, including safety issues.

High Glucose Level Disturbs the Resveratrol-Evoked Curtailment of CX3CL1/CX3CR1 Signaling in Human Placental Circulation.

Hyperglycemia-induced hyperactivity of chemokine CX3CL1 (fractalkine) occurs in the human placenta. Anti-inflammatory/antioxidant activities of resveratrol (3,5,4'-trihydroxy-trans-stilbene) are related to the modulation of chemokine CX3CL1 and its receptor, CX3CR1, signaling pathways. We examined the influence of high glucose (25 mmol/L glucose; HG group; N = 36) on resveratrol-mediated effects on CX3CL1 and TNF-α production by the placental lobule, CX3CR1 expression and contents of CX3CR1, TNF-α receptor 1 (TNFR1), and NF-κB proteins in placental tissue. The placental lobules perfused under normoglycemic conditions formed the control NG group (N = 36). Resveratrol (50 and 100 µM; subgroups B and C) administered into the perfusion fluid lowered the production of both CX3CL1 and TNF-α. The reductions in CX3CL1 levels were more evident in the NG group. CX3CR1 expression was significantly higher in the NG subgroups B and C compared to the HG subgroups B and C (385.2 and 426.5% versus 199.3 and 282.4%, resp.). An increase in CX3CR1 protein content in placental lysates was observed in the NG subgroups B and C. Also, resveratrol significantly decreased NF-κBp65 protein content only in the NG group, not affecting hyperglycemia-elicited TNFR1 upregulation. In conclusion, euglycemia assures optimal effects of resveratrol pertaining to CX3CL1/CX3CR1 signaling in the placenta. Future studies on resveratrol are needed, especially those including maternal-fetal risk assessments.

Human beta-defensin 1, 2 and 3 production by amniotic epithelial cells with respect to human papillomavirus (HPV) infection, HPV oncogenic potential and the mode of delivery.

BACKGROUND: Human beta-defensins (HBD) produced by human amniotic epithelial cells (HAEC) co-create an innate antiviral immune response in the materno-placento-fetal unit. Oncogenic potential of HPV may reflect its ability to avoid immune recognition. In this study we assessed the risk of HAEC infection with human papillomavirus (HPV) in relation to the type of labor and the impact of the oncogenic potential of HPV on HBD production in HAEC. METHODS: A comparative analysis [HPV(+) vs. HPV(-)HAEC] of the production of HBD were performed. HAEC were isolated from placentas of 116 HPV(+) and 36 HPV(-) parturients (groups I and II, respectively) using trypsin-based method. The cases of premature rupture of membranes (PROM), natural labors (NL) and cesarean sections (CS) were analysed in respective subgroups. High-risk (HR-HPV) and low-risk (LR-HPV) genotypes of HPV in cervical smears and HAEC were identified using the Roche Linear Array(®) HPV Genotyping Test. HBD-1,-2,-3 concentrations in the HAEC culture supernatant were assessed using ELISA. RESULTS: The highest percentage (42.1%) of HPV transmission to HAEC occurred in PROM, an intermediate value was observed after NL (38.5%), and the lowest (25.6%) after CS. The mean concentrations of HBD-2 and HBD-3 in group I were up to 3.1- and 2.8-fold higher (p < 0.05), respectively. The mean concentration of HBD-2 was higher (p < 0.05) in LR-HPV infection compared with HR-HPV. CONCLUSIONS: The course of labor and the mode of delivery influence the risk of HPV transmission to the HAEC. HPV infection upregulates HBD-2 and HBD-3 production in HAEC. Smaller increases in HBD-2 level after HR-HPV infection as compared to LR-HPV may affect cancerogenesis. Therapeutic potential of HBD-2 for HR-HPV infection should be assessed in future studies.

Perianal infection of highly oncogenic human papilloma virus: how to prevent from cancer? Case report.

The presence of oncogenic types of human papilloma virus (HPV) in location of the anus is related to anal carcinoma. However, there is little knowledge about the natural history of such infections in patients outside risk groups, its relation to cervical cancer, the risk of anal cancer development as well as any way to prevent it. There are no standard procedures in the case of finding of HPV-associated anal intraepithelial neoplasia in the perianal area. Case report describes an incidental finding of a highly oncogenic type of HPV discovered in a histopathological assessment of a 48-year old woman after a haemorrhoidectomy. This paper presents the approach taken for this patient in terms of diagnosis, treatment and methods of prevention of anal and cervical cancer development.

PEComa of the mesentery coexisting with colon cancer: a case report.

Perivascular epithelioid cell tumor (PEComa) is a rare entity originating from mesenchymal tissue, which stains for both melanocytic and smooth muscle markers. We would like to present an unusual case of the PEComa of the mesentery which was unexpected discovery in a female patient with colonic adenocarcinoma. The tumour was revealed on the computer tomography and then resected during surgery, with subsequent chemotherapy for the colon adenocarcinoma. Furthermore we would like to discuss PEComa biology, emphasizing histological criteria of malignancy, possible treatment options and differential diagnosis which is mostly based on immunohistochemistry. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1809062291157051 .

Nested variant of urothelial carcinoma of renal pelvis - a case report.

We report a case of an 80-year-old woman with a very rare subtype of urothelial carcinoma - nested variant of urothelial carcinoma mimicking physiological von Brunn's nests. Optimal treatment of NVUC has not been determined due to the small number of cases, as well as the lack of randomized and follow-up studies. In our case the right retroperitoneal nephroureterectomy was chosen.

Thrombospondin and VEGF-R: is there a correlation in inflammatory bowel disease?

Up to date several authors discussed interactions between cells forming inflammatory infiltrates in the course of inflammatory bowel disease (IBD), mainly dealing with endoscopic biopsy specimens. These usually contain only mucosa. We have evaluated full bowel wall sections, which seems to be especially important in patients with Crohn's disease (CD). The purpose of our study was to evaluate the relationship between vascular density and expression of thrombospondin-1 (TSP-1) and vascular endothelial growth factor receptor 1 (VEGFR-1) in full-thickness tissue fragments of intestinal wall taken from patients after colectomy, comparing those with IBD to non-IBD control group. Histological sections were immunostained with antibodies against CD-31, TSP-1, and VEGFR-1 and analyzed by pathologists with the use of computer-assisted morphometrics. Our research showed significantly higher vascular density and vascular area percentage in all layers of bowel wall in patients with CD when compared to control. We have also demonstrated differences in vascular density distribution between ulcerative colitis (CU) and CD and between CU and control. However we have not found statistically significant correlation between those findings and VEGFR-1 or TSP-1 expression. Our results might suggest existence of different, TSP-1 independent pathways of antiangiogenesis in IBD.

[Epithelioma calcificans malherbe]. / Przypadek rodzinnego wystepowania nabloniaka wapniejacego malherbe'a (pilomatrixoma).

Authors described the case of familial occurrence of epithelioma calcificans Malherbe (pilomatricoma) in 6-year-old boy and his 68-year-old grandfather. Tumors were clinically diagnosed as atheromas of nucha and shoulder.

Pathological analysis of lesions within intestines resected due to ulcerative colitis.

INTRODUCTION: Ulcerative colitis (UC) and Lesniowski-Crohn's disease together constitute a type of intestinal pathology known as Inflammatory Bowel Disease. The etiology of UC still remains unknown, however some epidemiological data suggest the role of bacteria and viruses and also some habitual as well as environmental factors like smoking, diet, drugs, geographical and social status, as well as stress. The genetic predisposition is also suggested. UC affects young people in 2nd - 4th decades of life. Exacerbations of the disease may result in the necessity of surgical treatment, typically in the form of total proctocolectomy accompanied by the subsequent formation of ileo-pouch-anal anastomosis. The aim of our study was to analyze morphological pictures of resected specimens. MATERIAL AND METHODS: We analyzed 67 cases (40 women and 27 men) of UC with the special interest being focused at macro- as well as microscopic features of the intestines resected. We reviewed macroscopic characteristics of intestines (i.e. the length of resected fragments, localization, shape and diameter of the ulcers, polyps, number of resected lymph nodes), as well as microscopic descriptions concerning, among others the character and localization of inflammatory infiltrate, the architecture of glands, the presence of crypt abscesses and Paneth's metaplasia. Special attention was paid to the morphology of intestinal wall vasculature. RESULTS: In 42% of the cases macroscopically the inflammation covered the whole length of the resected colon. In 58% macroscopically detected inflammatory changes were segmental in distribution. In four cases the disease had clinically the fulminant course and the inflammation was transmural. There were 3 cases, in which histological assessment revealed the presence of malignancy (2 cases of mucus producing adenocarcinoma and one case of carcinoma in situ situated in the anal canal). Generally, microscopic findings were typical for the active phase of UC. We found intensive vascularization and hyperemia of the intestinal wall to be the common features accompanying the inflammation. CONCLUSIONS: Young people in the 3rd and 4th decades of life constitute the group being relatively commonly affected by the UC, and undergo the surgical proctocolectomy. Some of the cases present with the fulminant course of the disease. A rich vascular network is a common finding in the inflamed intestinal wall. We hypothesize, that intensive vascularization may play a significant role in the pathogenesis of UC.