MOLECULAR CHARACTERISTICS OF THE HETEROGENEITY OF NON-INVASIVE PAPILLARY UROTHELIAL CARCINOMAS AND THE MARKERS OF THEIR RECURRENCE.

Urothelial carcinoma represents the most common type of bladder cancer (>90%) and is the most frequent malignancy of the urinary tract. Most of the urothelial carcinomas are non-invasive at the time of diagnosis, however they are characterised with the risk of recurrence after surgical treatment. The aim of our study was to investigate the characteristics of tumor heterogeneity and markers of its progression in urothelial papillary carcinomas. Study included following groups: normal urothelial epithelium, urothelial papilloma, urothelial neoplasms with low malignant potential (PUNLM), non-invasive low grade papillary urothelial carcinomas (LGPUC) and non-invasive high grade papillary urothelial carcinomas (HGPUC). In addition, study included relapsed cases of non-invasive LGPUC and HGPUC. Nuclear features and mitotic counts was assessed using digital pathology software QuPath in standard H&E stained specimens. In addition, the presence of mitosis was detected as PHH3 labelled cells by immunohistochemistry. Proliferation was measured as Ki67 labelling index by immunohistochemistry. Tumor heterogeneity was investigated by the differential expression pattern of CK5, CK7 and CK20 by immunohistochemistry. Study results showed, that Nuclear features, as well as the number of mitosis, proliferation index and intratumoral heterogeneity significantly correlate with the presence of higher grade non-invasive urothelial lesions. In addition, it is possible to distinguish two major groups of non-invasive LGPUC and HGPUC, based on nuclear and phenotypic heterogeneity and mitotic and proliferative activity, I group which is characterised with higher intratumoral heterogeneity, higher mitotic count and higher proliferative activity, represents the high risk group of non-invasive LGPUC and HGPUC recurrence.

DISTRIBUTION OF STEM CELLS IN DIFFERENT THYROID LESIONS IN PATIENTS OF REPRODUCTIVE, MENOPAUSAL AND POST-MENOPAUSAL AGE.

Different studies indicate that cancer stem cells (CSCs) play an important role in the progression and therapy resistance in different cancer types. The aim of our study was to analyse the distribution of CSCs in different thyroid lesions, in reproductive, menopausal and post-menopausal women. Study included altogether 200 formalin-fixed and paraffin-embedded tissue material, with the diagnosis of NIFTP, Hashimoto's thyroiditis, papillary thyroid carcinoma (PTC) and PTC and Hashimoto's thyroiditis co-occurred cases. Normal thyroid gland was used as a control tissue. Stem cell marker - CD44, as well as other markers including Ki67, BCL2, CK19, CD56, ER were investigated with standard immunohistochemical procedure. The results of our study indicated that CD44 stem cell marker, as well as proliferation marker Ki67 is significantly upregulated in PTC cases in all age groups. However, the expression of CD44 and Ki67 is significantly higher in reproductive age patients, compared to patients in menopause and post-menopause. In addition, the expression of CD44 and Ki67 is significantly higher in PTC and Hashimoto's thyroiditis co-occurred cases, compared to cases with PTC only.

HISTOPATHOLOGICAL, PROLIFERATIVE, APOPTOTIC AND HORMONAL CHARACTERISTICS OF VARIOUS TYPES OF LEIOMYOMAS.

Uterine leiomyomas decrease the quality of life by causing significant morbidity among women of reproductive age. Histologically various types of leiomyoma's can be differentiated. We have analysed the histopathological, proliferation, apoptotic and hormonal profile in different types of leiomyomas. Study included altogether 140 cases distributed into following groups: group I - normal myometrium (20 cases), group II-classic leiomyoma (69 cases), group III-cellular leiomyoma (15 cases), group IV - bizarre cell/atypical leiomyoma (22 cases), group V - smooth muscle tumors of uncertain malignancy potential (STUMP) (8 cases) and group VI - leiomyosarcoma (6 cases). Together with classic histopathological features such as nuclear atypia, cellularity, presence of mitoses, vasculature and necrosis, immunohistochemical phenotype using antibodies against Ki67, Cas3, ER and PR were analysed. The results of our study showed that leiomyomas are characterised with variable histopathological and immunohistochemical phenotype. Especially, two entities, bizzare/atypical leiomyoma and STUMP, which can be divided into two subgroups according to the presence of the degree of atypia and the expression of proliferation, apoptosis and hormonal markers, which might be the explanation of their different prognosis. Presented histopathological and immunohistochemical features should be considered in the diagnosis of myometrial smooth muscle tumors.

LC3A positive "stone like structures" are differentially associated with survival outcomes and CD68 macrophage infiltration in patients with lung adenocarcinoma and squamous cell carcinoma.

AIMS: The aim of the study was to analyse the prognostic and predictive value of LC3A positive' 'Stone Like Structures'' (SLSs) in a large cohort of patients with non-small cell lung carcinoma (NSCLC) and to check its relationship with tumor infiltrating lymphocytes (TILs) and PD-L1 expression. METHODS: Tissue microarrays from 1015 patients diagnosed at the Institute of Pathology and Molecular Pathology, University Hospital Zurich, Switzerland, were stained for LC3A, PD-L1, CD3 and CD68 using automated tissue stainer Ventana Benchmark Ultra (Roche). TILs were assessed in matched haematoxylin and eosin stained slides. RESULTS: LC3A positive SLSs, were significantly associated with worse overall (OS) and disease-free survival (DFS) outcomes in patients with lung adenocarcinoma (LADC) (HR = 2.4, 95 %CI(.994-1.008, p = 0.029) and HR = 3.9, 95 %CI (1.002-1.014), p = 0.002 respectively), whilst it was associated with better OS and DFS in patients with lung squamous cell carcinoma (LUSC), with marginal significance (HR = .99, 95 %CI(.975-1.011),p = 0.042 and HR = .99, 95 %CI (.975-1.008), p = 0.026). Multivariate analysis showed that LC3A SLSs are independent poor prognostic factor only in patients with LADC. In addition, LC3A SLSs, were negatively associated with CD68 count in LADC, whilst there was a positive correlation in LSCC. CONCLUSIONS: LC3A SLSs are differentially associated with the survival outcomes and CD68 count in LADC and LSCC. Further studies are justified for the understanding the underlying biological mechanisms of this phenomenon.

PHENOTYPIC CHARACTERISTICS OF RELAPSED LEIOMYOMA AND SMOOTH MUSCLE TUMORS OF UNCERTAIN MALIGNANCY POTENTIAL IN REPRODUCTIVE WOMEN.

Uterine leiomyoma represents the most common pelvic tumor in females, including numerous histological subtypes, from which smooth muscle tumors of uncertain malignancy potential (STUMP) represents the diagnostic challenge. On the other hand, the study of the relapse risk markers after laparoscopic myomectomy is of high interest. We investigated the molecular phenotype of different types of leiomyoma after hysterectomy or laparoscopic surgery in reproductive and menopausal women. Standard immunohistochemistry was used to detect proliferation markers Ki67 and cyclin D1, apoptotic markers Bcl2 and Cas3, and ER and PR. The results of our study indicated that ER expression is significantly higher in relapsed leiomyoma, compared to control group. In addition, relapsed leiomyomas are characterised with high proliferation and apoptotic index. With regard to STUMP despite histological homogeneity, this entity is characterised with the presence of three distinct molecular subtypes, based on proliferation and apoptotic marker expression, which should be used as diagnostic aid in these tumors.

DISTRIBUTION OF SEX HORMONES AND LYMPHOCYTES IN REPRODUCTIVE WOMAN WITH THYROID PAPILLARY CARCINOMA AND HASHIMOTO'S THYROIDITIS.

The incidence of papillary thyroid carcinoma is characterised with increasing tendency, with unknown reasons. Frequently the co-occurrence of papillary thyroid carcinoma and Hashimoto's thyroiditis has been observed. The aim of our study was to analyse the expression of hormone receptors, lymphocytic infiltration and thyreocyte/lymphocyte proliferation index in thyroid papillary carcinoma and in Hashimoto's thyroiditis. Study included 115 formalin-fixed and paraffin-embedded tissue material from the teaching, research and diagnostic laboratory of Tbilisi State Medical University. Study material was divided into following groups: normal thyroid gland (n=15), Non-invasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features (NIFTP) (n=15), classic papillary carcinoma (CPC)(n=20), follicular variant of papillary carcinoma (FPC) (n=17), cylindric-cell variant of papillary carcinoma (CCPC)(n=9), Hashimoto's thyroiditis (HT) (n=25) and the co-occurrence of Hashimoto's thyroiditis and papillary carcinoma (HTPC) (n=14). Standard immunohistochemistry was used to detect ER, PR, Ki67, CK19, CD56. In addition, lymphocytic infiltration was evaluated in H&E stained specimens. Study results showed that ER and PR expression is higher in FPC, CCPC and HTPC compared to CPC (p<0.001), whilst lymphocytic infiltrate is lower in FPC and CCPC compared to CPC (p<0.05). In addition, ER and PR expression is higher in HTPC compared to HT only (p<0.001). The thyreocyte/lymphocyte proliferation index is increased in FPC and CCPC compared to CPC and it is also higher in HTPC compared to only HT and CPC (p<0.05). The expression of sex steroid hormones plays an important role in the pathogenesis of papillary thyroid carcinoma. The expression level of ER and PR is even higher in cases where Hashimoto's thyroiditis and papillary carcinoma co-occur. Therefore, we can conclude that Hashimoto's thyroiditis may play an important role in the development of papillary thyroid carcinoma.

DISTRIBUTION OF INTRAEPITHELIAL LYMPHOCYTES AND MACROPHAGES IN CERVICAL MICROENVIRONMENT DURING THE PROGRESSION OF CERVICAL INTRAEPITHELIAL NEOPLASIA.

Microenvironment plays central role in the development of cervical precancerous and cancerous lesions. Cervical intraepithelial neoplasia (CIN) represents a group of precancerous lesions, divided into three degrees. We investigated the distribution of intraepithelial lymphocytes and macrophages in different grades of CIN. We analysed lymphocyte marker CD103, macrophage marker CD68 and proliferation marker Ki67 using standard immunohistochemistry. In addition, we investigated the distribution of lymphocytes using standard haematoxylin and eosin method. The results of our study indicated thatgrade I CIN which subsequently progressed into grade II CIN was characterised with low lymphocytic infiltration, low lympho-epithelial index and low lymphocyte proliferation index. Similar results were seen in cases of CINII which were later progressed into CINIII or in carcinoma. Therefore, we would like to recommend the analysis of microenvironment alterations in CIN lesions, in order to assess their progression potential.

EPIGENETIC CHANGES - HISTONE 3 PHOSPHORYLATION - EPITHELIAL OVARIAN TUMORS.

Histone modifications represent one of the types of epigenetic changes. Histones, undergo different types of epigenetic modifications, including the phosphorylation of serine residues. pHH3 antibodies specifically detect histon-3 protein, when phosphorylated at 10th and 28th serine residues. Traditionally pHH3 antibodies are used as proliferation marker, as it detects cells in late G2 and M phase. We studied the distribution of phosphor-histon-3 in epithelial tumors of the ovary and its relationship with ER, PR, Ki67, p53 and BCL2. Altogether, we investigated postoperative material from 160 patients. Standard immunohistochemistry was used to detect, phosphohistone-H3 (pHH3), ER, PR, Ki67, p53 and BCL2. The results of our study showed that phosphohistone-H3 expression is negatively associated with the expression of ER and PR expression, as well as with BCL2 expression, on the other hand it positively correlates with Ki67 and mutant p53 (p<0.05). In addition, the expression of phosphohistone-H3 is detected in Ki67 negative cases and its expression is increased along with the increase of malignancy grade. Our study results indicate that PHH3 might be used as an additional marker for the assessment of proliferation and malignancy potential of epithelial tumors of the ovary.

PHENOTYPIC CHARACTERISTICS OF TROPHOBLASTIC HYPERPLASIA AND MICROENVIRONMENT ALTERATIONS IN CHORIONIC VILLI IN SPONTANEOUS ABORTIONS.

Hydatidiform mole represents the major cause of the molar pregnancy, which is a special cause of spontaneous abortions. We analysed phenotypic characteristics of epithelial hyperplasia and tumor microenvironment alterations in different types of hydatidiform moles. Standard immunohistochemistry was used for the detection of Ki67, Cyclin D1, p53, BCL2, E-cadherin, p63, Vimentin, CD34, CD3, CD4, CD8 and CD68. In addition, epithelial hyperplasia has been assessed in standard diagnostic haematoxylin and eosin stained tissue specimens. The results of our study indicated that both cytotrophoblast and sincitiptrophoblast layers are characterised with marked epithelial hyperplasia and high proliferation index in partial and complete moles, whilst apoptotic index is minimal. Early complete mole resembles the partial mole, rather than complete mole. Lymphocyte infiltration, marked by CD3, CD4 and CD8 is also higher in complete and partial moles, whilst macrophage infiltration is relatively lower. Macrophage infiltration marked by CD68 correlates with microvessel density marked by CD34. The evaluation of proliferation and apoptotic markers, as well as microenvironment, might serve as additional diagnostic markers in patients with hydatidiform moles and hydropic abortions.

MORPHOPHENOTYPIC CHARACTERISTICS OF OVARIAN SEROUS BORDERLINE TUMORS.

Borderline ovarian tumors (BOTs) represent particular challenge for diagnosis and clinical management as they are characterized with the features of both benign cystadenomas and malignant carcinomas. The aim of our study was to investigate histomorphological and immunohistochemical characteristics of ovarian serous-papillary borderline tumors, compared to serous cystadenomas and low- and high-grade serous carcinomas. Altogether, 80 formalin fixed and paraffin embedded tissue specimens, distributed in four groups, including serous cystadenoma (group I), serous BOTs (group II), Low (group III) and high (group IV) grade serous carcinomas, were investigated by standard immunohistochemistry, using antibodies against CK7 CK20, WT1, Vimentin, CDX2, CEA, ER, cyclin D1, BCL2, E-cadherin, calretinin, СA125, Ki67, P53. Study results showed, that ovarian serous BOTs are characterized with slightly increase proliferative potential compared to benign cystadenomas, whilst apoptotic potential is retained with the difference from malignant serous carcinomas. p53 mutation is not present, as well as the expression of Vimentin. Overall, ovarian serous BOTs are characterized with highly variable immunohistochemical phenotype and the use of multiple immunohistochemical markers are recommended for the differential diagnosis from low grade serous carcinomas and benign cystadenomas.

PHENOTYPIC CHARACTERISTICS OF CHORIONIC VILLI DURING GESTATIONAL TROPHOBLASTIC DISEASES.

Differential diagnosis of gestational trophoblastic diseases (GTP) using standard micromorphological examination is complicated and less reliable. Therefore, the aim of our study was to investigate the immunohistochemical phenotype of chorionic villi during GTP, as well as in physiological cases. Study included five groups: I group - normal chorionic villi, II group - chorionic villi with hydropic changes, III group - Partial mole, IV group - Complete mole, V group - early Complete mole. Following markers were examined using standard immunohistochemistry: CK7, CK20, P63, PLAP, P57, CK5, CK8/18, CEA, CD34 Ki67, P53, E-cadherin, vimentin, ß-catenin and inhibin. Study results showed that chorionic villi are characterized with marked phenotypic heterogeneity in normal tissue, as well as in cases of GTP, which can be used as an additional criterion for the differential diagnosis of GTP.

TLR9 EXPRESSION, LANGERHANS CELL DENSITY AND LYMPHOCYTIC INFILTRATION IN PROGRESSING CERVICAL INTRAEPITHELIAL NEOPLASIA.

Toll like receptors (TLRs), NK cells, Langerhans cells and T cells play an important role in the protection of host organism from human papilloma virus (HPV) infection. The aim of our study was to analyse TLR9 expression, Langerhans cell density and NK cell and Lymphocytic infiltration in the progression of cervical intraepithelial neoplasia (CIN). By using standard immunohistochemistry, we have investigated TLR9, CD1a Langerhans cell marker, CD56 NK cell marker, CD3 general T cell marker, CD4 T helper cell marker and CD8 cytotoxic T cell marker. We have introduced NK cell epithelial index and Langerhans cell epithelial index. In addition, we have introduced proliferation apoptotic index of lymphocytes using Ki67 and BCL2 markers. The results of our study showed that TLR9 expression, T cell infiltration and NK epithelial index is significantly increased during the progression of CIN disease. Whilst Langerhans cell epithelial index is significantly decreased. Proliferation apoptotic index of lymphocytes is also significantly decreased during the progression of CIN. Based on our study results we recommend the assessment of TLR9 and proliferation apoptotic index as an additional markers for defining CIN progression potential.

Overexpression of filamin-A protein is associated with aggressive phenotype and poor survival outcomes in NSCLC patients treated with platinum-based combination chemotherapy.

An actin-binding protein filamin A connects the actin filament network to cell membrane receptors, and acts as a scaffold for various signaling pathways related to cancer growth and progression. Recently, it has been reported that filamin A is required for efficient regulation of early stages of DNA repair process. Moreover, some in vitro studies showed that the overexpression of filamin A determines resistance to various cytotoxic drugs, including cisplatin. We aimed to analyse the expression of filamin A protein in resected NSCLC (Non Small Cell Lung Cancer) specimens, to investigate the association of the level of filamin A protein expression and other clinicopathological features, and possible relationship between the expression of filamin A and survival outcome in NSCLC patients, treated with platinum-based combination chemotherapy. We performed filamin A protein immunohistochemistry on formalin-fixed and paraffin-embedded (FFPE) tissue sections from 135 NSCLC patients, using EP2405Y antibody against C-terminus of filamin A. Cytoplasmic, membranous and nuclear positivity of filamin A was evaluated semi-quantitatively and correlated with available clinicopathological data. Patients were divided into two groups for survival analysis (I group - patients treated with adjuvant platinum-based chemotherapy, II group - patients with surgical treatment only). We found significant positive correlation between filamin A protein expression and NSCLC stage (r=0.249; p<0,05), presence of lymph node (N)(r=0.205; p<0,05) and distant metastases (M) (r=0.332; P<0.01). Increased filamin A protein expression was significantly related with poor survival outcomes in patients with adjuvant platinum-based chemotherapy: OS (HR=1.005, 95%CI[1.000;1.010], p=0.037), DFS (HR=1.004, 95%CI [1.001:1.008], p=0,017). Multivariate Cox proportional hazards regression analysis also showed that overexpression of filamin A represents an independent risk factor for disease relapse, in addition to tumor size, stage, and metastases status (HR=1.723, 95%CI [1.021:2.909], p<0.05). Thus, filamin A expression might be a new prognostic marker in patients with NSCLC.

Cytologic, colposcopic and histopathologic correlations of LSIL and HSIL in reproductive and menopausal patients with hyperkeratosis.

The significance of hyperkeratotic cells in Pap smear is not yet fully understood. Hyperkeratosis represents benign structural change of cervical squamous epithelium, which may mask displastic lesions and make an accurate colpo-cytological examination difficult. We investigated the cytologic, colposcopic and histopathologic correlations of low grade squamous intraepithelial lesion (LSIL) and high grade squamous intraepithelial lesion (HSIL) in hyperkeratotic patients, in order to estimate the influence of hyperkeratosis on the accuracy of cyto-colposcopic examination. We examined conventional Pap smears of 766 reproductive and 384 menopausal women. Patients with hyperkeratotic cells in Pap smear were subsequently investigated by colposcopy and in case of colposcopically proved hyperkeratosis, direct biopsy with subsequent histopathological examination of H&E stained sections were performed. LSIL was found in 2,74% and 1,08% of Pap smears, whilst CIN1 was detected in 6,46% and 4,85% of biopsy specimens inreproductive and menopausal women respectively. ASC-H/HSIL were found in 2,74% and 2,71% of Pap smears and CIN2/3 in 4,18% and 7,76% of biopsy specimens in reproductive and menopausal women respectively. The reliability of Pap test significantly decreases under the presence of hyperkeratosis and it must be an indication for colposcopy. In cases of colposcopically proved hyperkeratosis direct biopsy with subsequent histopathological examination must be performed.

Cytologic, colposcopic and histopathologic correlations of hyperkeratosis in reproductive women.

The significance of leucoplakia/hyperkeratosis in colposposcopy or hyperkeratotic cells in Pap smear, is not fully understood. It is considered that hyperkeratosis itself is a benign change, but it may mask other pathologies, including invasive carcinoma. We studied 123 woman with colposcopically identified hyperkeratosis and its' possible relationship with dysplastic lesions of uterine cervix, based on correlation with cytopathological and histopathological data. The results of our study showed that 1) cervical dysplastic lesions are identified 2 times less frequently in conventional Pap smears from patients with thick leukoplakia, than in patients with thin leukoplakia and 2) histopathologically identified cervical dysplasias are 1,5 times more common in patients with thick leukoplakia. Pathologists and gynecologists in practice should be aware of this difference.

[Lynch syndrome (case report)].

The authors have analyzed the ethology, pathogenesis, treatment of Lynch Syndrome and described a case history of a 65-year-old woman with right breast cancer who was admitted into a hospital with intestines impassability and operation was performed. During the operation tumour, which caused the acute intestine impassability was identified. Apart from this, after the inner organ examination metastasis on stomach and tumour in upper and middle rectum were found. Histology and colonoscopy revealed the rectum adenocarcinoma (morphologic code 8140/3) with mild differentiation. Examination revealed adenocarcinoma with high and middle area differentiation (morphologic code 8480/3). Post-operational period was satisfactory. On the 10-th day the stitches were removed, chemical therapy was carried out according to the scheme set. Patient was examined after a year and two months. General health was satisfactory; she conducts an active life.

[Value of thrombolytic therapy in acute myocardial ischemia after administration of kinin system inhibitors]. / Znachenie tromboliticheskoi terapii pri ostroi ishemii miokarda na fone predvaritel'nogo vozdeistviia ingibitorov kininovoi sistemy.

An experimental study in dogs showed that thrombolytic revascularization after 4 to 6 hours of coronary arterial occlusion resulted in further deterioration of ischemia and an impairment of cardiac contractility and hemodynamics. Micromorphologically, multiple extravasations were demonstrated in the ischemized region in combination with insignificant glycogen content. The administration of thrombolytics was accompanied by enhanced fibrinolytic activity, yet the activation of the first three phases of blood coagulation showed no decline. In a series with thrombolytic therapy preceded by contrykal and heparin infusions, micromorphology revealed no intramyocardial hemorrhages in the ischemized region. Active functional cells showed micro- and macrogranular glycogen that accumulated into clusters in myocardial fibres. The significant postrevascularization decline in ischemia was accompanied by improved myocardial contractility and cardiac hemodynamics in the presence of hypocoagulation and increased fibrinolytic activity.