Magdalena Uvirova

The changes of angiogenesis and immune cell infiltration in the intra- and peri-tumoral melanoma microenvironment.

Malignant melanoma (MM) urgently needs identification of new markers with better predictive value than currently-used clinical and histological parameters. Cancer cells stimulate the formation of a specialized tumor microenvironment, which reciprocally affects uncontrolled proliferation and migration. However, this microenvironment is heterogeneous with different sub-compartments defined by their access to oxygen and nutrients. This study evaluated microvascular density (MVD), CD3+ lymphocytes (TILs) and FOXP3+ T-regulatory lymphocytes (Tregs) on formalin-fixed paraffin-embedded tissue sections using light microscopy. We analyzed 82 malignant melanomas, divided according to the AJCC TNM classification into four groups—pT1 (35), pT2 (17), pT3 (18) and pT4 (12)—and 25 benign pigmented nevi. All parameters were measured in both the central areas of tumors (C) and at their periphery (P). A marked increase in all parameters was found in melanomas compared to nevi (p = 0.0001). There was a positive correlation between MVD, TILs, FOXP3+ Tregs and the vertical growth phase. The results show that MVD, TILs and FOXP3+ Tregs substantially influence cutaneous melanoma microenvironment. We found significant topographic differences of the parameters between central areas of tumors and their boundaries.

Immunoglobulin G4, autoimmune pancreatitis and pancreatic cancer.

Background: Immunoglobulin G4 (IgG4)-related diseases are a group of diseases characterized by enlargement of the affected organs, elevation of serum IgG4, massive infiltration of affected organs with lymphocytes and plasma cells with IgG4 positivity and tissue fibrosis. Type I autoimmune pancreatitis is one form of IgG4-related disease. For IgG4-related diseases, various localizations are described for up to 10% of malignancies. The aim of our study was to examine IgG4 serum levels and pancreatic tissue with respect to the simultaneous presence of autoimmune pancreatitis in patients with pancreatic cancer. Methods: IgG4 serum levels were examined In 106 patients with histologically confirmed pancreatic cancer. The level of 135 mg/dl was considered as the normal value. Pancreatic tissue was histologically examined with respect to the presence of markers of autoimmune pancreatitis. Results: A higher IgG4 level than the cut-off value of 135 mg/dl was proven in 11 patients with pancreatic cancer. Of these 11 patients, 7 had levels twice the normal limit (65.6%). Autoimmune pancreatitis was diagnosed in these individuals. In the case of 1 patient, it was basically an unexpected finding; another patient was initially diagnosed with autoimmune pancreatitis. Repeated biopsy of the pancreas at the time of diagnosis did not confirm the presence of tumour structures, therefore steroid therapy was started. At a check-up 6 months after starting steroid therapy, the condition of the patient improved subjectively and IgG4 levels decreased. However, endosonographically, malignancy was suspected, which was subsequently confirmed histologically. This patient also demonstrated an IgG4 level twice the normal limit. Conclusion: IgG4-related diseases can be accompanied by the simultaneous occurrence of malignancies, which also applies to autoimmune pancreatitis. Chronic pancreatitis is considered a risk factor for pancreatic cancer. It cannot be reliably confirmed whether this also applies to autoimmune pancreatitis. In accordance with other works, however, it is evident that, despite the described high sensitivity and specificity for IgG4 elevation in the case of autoimmune pancreatitis, even levels twice the normal limit are demonstrable in some individuals with pancreatic cancer, without the presence of autoimmune pancreatitis. We believe that patients with IgG4-related disease, including autoimmune pancreatitis, must be systematically monitored with respect to the potential presence of malignancy.

Detection of nano- and micro-sized particles in routine biopsy material - pilot study

BACKGROUND Nanotechnology is receiving enormous funding. Very little however is known about the health dangers of this technology so far. Chronic tonsillitis is one of a number of diseases called idiopathic. Among other factors, the tonsils are exposed to suspended particles in inhaled air including nano particles. The objective of this study was to detect and evaluate metallic particles in human tonsil tissue diagnosed with chronic tonsillitis and in amniotic fluid as a comparison. METHODS . Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX) was used for identification of solid particles in a total of 64 samples of routinely analyzed biopsy and cytologic material. RESULTS Almost all samples were found to contain solid particles of various metals. The most frequent, regardless of diagnosis, were iron, chromium, nickel and aluminium. The size, determined using SEM, varied from around 500 nm to 25 µm. The majority formed aggregates of several micrometers in size but there were a significant number of smaller (sub-micrometer or nano-sized) particles present. The incidence of metallic particles was similar in child and adult tissues. The difference was in composition: the presence of several metals in adults was due to occupational exposure. CONCLUSIONS The presence of metallic particles in pathologically altered tissues may signal an alternative causation of some diseases. The ethiopathogenic explanation of these diseases associated with the presence of nano-sized particles in the organism has emerged into a new field of pathology, nanopathology.

Využití fluorescenční in situ hybridizace v diagnostice melanocytárních lézí

Melanocytarni leze vykazuji znacnou morfologickou heterogenitu. Jejich diagnostika je narocna i přes soucasne využiti modernich histopatologických a imunohistochemických metod. Významným přinosem v diagnostice melanocytarnich lezi je zavedeni molekularně geneticke metody fluorescencni in situ hybridizace s použitim ctyřbarevne sondy znacici geny CCND1, RREB1, MYB a centromeru chromozomu 6. V letech 2008–2012 bylo v nasi laboratoři uspěsně metodou fluorescencni in situ hybridizace vysetřeno 160 vzorků různých typů melanocytarnich lezi, 44 nevů a 116 melanomů. Změny asociovane s diagnozou melanomu byly prokazany u 116 vzorků (72,5 %), normalni nalez u 41 vzorků (25,6 %), ve dvou připadech bylo rozhodnuti na hranici hodnotitelnosti metody (1,2 %) a v jednom připadě se jednalo o vysetřeni recidivy melanomu v jizvě, který byl svým nalezem atypický (monozomie chromozomu 6) (0,6 %). Na zakladě dosažených výsledků byla stanovena senzitivita metody 96,6 % a specifita metody 91%. Tyto výsledky ukazuji, že fluorescencni in situ hybridizace je vhodnou metodou pro zajistěni přesne a rychle diagnostiky nejednoznacných melanocytarnich lezi. Pravě rychlost a spravnost diagnozy je u vysoce malignich melanomů zasadni a ma neoddiskutovatelný vliv na prognozu a uspěsnost lecby pacienta.

IDH Mutation Analysis in Glioma Patients by CADMA Compared with SNaPshot Assay and two Immunohistochemical Methods

Mutations in IDH1/2 genes are a marker of good prognosis for glioma patients, associated with low grade gliomas and secondary glioblastomas. Immunohistochemistry and Sanger sequencing are current standards for IDH1/2 genotyping while many other methods exist. The aim of this study was to validate Competitive amplification of differentially melting amplicons (CADMA) PCR for IDH genotyping by comparison with SNaPshot assay and two immunohistochemical methods. In our study, 87 glioma patients (46 from Olomouc and 41 from Ostrava) were analyzed. IDH1/2 mutations in native bioptical samples were analyzed at DNA level by CADMA and SNaPshot while IDH1 mutations in FFPE samples were analyzed at protein level by two IHC methods. CADMA PCR sensitivity for IDH1 was 96.4% and specificity 100% for 86 concluded samples. SNaPshot assay sensitivity was 92.9% and specificity of 100% for 85 concluded samples. IHC in the laboratory no. 2 reached sensitivity 85.7% and specificity 100% for 86 concluded samples. IHC in the laboratory no. 4 reached sensitivity of 96.4% and specificity of 79.7% in 74 concluded samples. Only one IDH2 mutation was found by SNaPshot while CADMA yielded false negative result. In conclusion, CADMA is a valid method for IDH1 p.(R132H) testing with higher sensitivity than SNaPshot assay. Also, molecular genetic methods of IDH1 testing from native samples were more robust than IHC from FFPE.

Crohn's disease - genetic factors and progress of the disease

BACKGROUND AND OBJECTIVES Crohns disease is a multifactorial inflammatory disease affecting mainly the gastrointestinal tract. The genetic factors that are involved in the disease include mainly three mutations of the gene NOD2/CARD15 (R702W, G908R, 3020insC). The aim of this study was to determine the relationship between the presence of these variants and disease phenotype. MATERIAL AND METHODS 70 patients with Crohns disease were examined for the presence of the above-mentioned mutations. The researchers used the medical records to retrospectively obtain clinical data and together with the information obtained prospectively according to the protocol they analysed the connection between gene mutations and disease phenotype. RESULTS At least one mutation was found in 22 patients with Crohns disease (32%), four patients were found to have two different mutations (composed heterozygotes - 6%) and six patients (9%) were homozygotes for the 3020insC gene. No significant differences were found between the groups with wild-type form and the mutated form of the NOD2 / CARD15 gene with respect to age at the time of diagnosis, form of the disease or localization according to the Montreal classification. CONCLUSION Mutations of the NOD2 / CARD15 gene did not significantly affect the frequency of reoperations, homozygotes with 3020insC gene mutations, however, represented a high risk group. The phenotype was not related significantly to the presence of the examined mutations.