MOLECULAR MARKERS OF THE PROGRESSION OF CONJUNCTIVAL NEOPLASTIC EPITHELIAL LESIONS.

Conjunctival epithelial lesions vary from benign to borderline malignancy to malignant, therefore it is extremely important to detect the molecular markers of the malignant progression of conjunctival intraepithelial lesions. The aim of our study was to analyse the molecular markers of the progression of conjunctival intraepithelial lesions. We have analysed Ki67, PHH3, Bcl2, P53, P63 and CK7 using standard immunohistochemistry. In addition, we have calculated the squamous-glandular index based on the evaluation of H&E stained specimens and as the ratio of P63/CK7. The results of our study indicated that the presence of squamous epithelium is significantly increased during the progression of conjunctival intraepithelial lesions, and therefore the squamous-glandular index is also increased. In addition, it is possible to divide conjunctival intraepithelial lesions as low grade and high grade lesions based on the distribution of proliferation and apoptosis markers.

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.

MICROENVIRONMENT ALTERATIONS IN CONJUNCTIVAL NEOPLASTIC LEOSIONS WITH DIFFERENT PROLIFERATION-APOPTOTIC CHARACTERISTICS.

Different studies indicate that tumor infiltrating lymphocytes (TILs) and tumor associated neutrophils (TANs) play an important role during the progression of malignant tumors. We have analysed the distribution of tumor associated neutrophils (TANs) and tumor infiltrating lymphocytes (TILs) in different conjunctival lesions, with different proliferation and apoptotic characteristics. The distribution of TILs and TANs were evaluated in standard haematoxylin and eosin (H&E) stained sections using the digital pathology software QuPathin normal conjunctiva, actinic keratosis, pterigea, conjunctival intraepithelial neoplasias (CoIN1-3) and conjunctival squamous cell carcinoma (CSCC). In addition, the expression of following markers were investigated using standard immunohistochemistry: Ki67, Bcl2, p53, CD3, CD8 and Foxp3. The study results indicated that the number of TILs and TANs are significantly increased during the progression of conjunctival intraepithelial lesions. Also, the number of TILs and TANs significantly correlate with higher proliferation index, lower apoptotic index and the p53 mutation status.

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.

LOSS OF CAS3 AND INCREASE OF BAX EXPRESSION ASSOCIATED WITH PROGRESSION OF CERVICAL INTRAEPITHELIAL NEOPLASIA.

Apoptosis plays one of the major roles in the progression of human cancers including cervical carcinoma. The aim of our study was to analyse the expression of Cas3, Bax and their correlation with the proliferation index and ER expression status during the progression of cervical intraepithelial neoplasia (CIN). Study included altogether 140 specimens, divided into two major groups, such as: cervical lesions without co-infections and with co-infections. Standard immunohistochemistry was used to detect antigens: Ki67, Cas3, Bax and ER. The study results showed that the expression of Cas3 is significantly decreased whilst the expression of Bax is significantly increased during the progression of CIN in both groups with and without co-infections. The expression of Bax negatively correlates with the expression of Cas3 (r=-42.4, p<0.05) and ER (r=-33.4, p<0.05) and positively correlates with the expression of proliferation marker Ki67 (r=56.3, p<0.05). The results indicate that the deregulated apoptosis measured as increased expression of Bax and Cas3 loss, as well as the increase in proliferation index measured as Ki67 expression is significantly related to the progression of CIN into cervical carcinoma. Therefore, the measuring of mentioned protein expression could be used as the markers of the CIN progression.

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.

STEM CELL INDEX IN THE PROGRESSION OF CERVICAL INTRAEPITHELIAL NEOPLASIA.

Stem cells represent the small subpopulation of healthy and cancerous tissues, which are characterised with increased proliferation and self-renewal properties. From the many different markers of stem cells, we have investigated the stem cell index during the progression of cervical intraepithelial neoplasia (CIN), based on the immunohistochemical expression of CD44 in total of 140 tissue samples from uterine cervix. In addition, we have performed the profound correlation analysis of CD44 with different epithelial-mesenchymal, proliferation, apoptosis and hormonal markers at both protein and mRNA level. The results of our study indicated that, stem cell index based on the CD44 detection is significantly increased with the progression of cervical intraepithelial neoplasia. In addition, CD44 expression significantly correlates with the epithelial-mesenchymal transition, proliferation-apoptotic features and ER status in both protein and mRNA level. Two, groups of cervical intraepithelial lesions as well as carcinoma can be identified based on the expression of CD44, Ki67, Cas3 and ER. To the best of our knowledge we are first to demonstrate such findings in CIN and cervical carcinoma and identified characteristics could be used for the early assessment of CIN patient prognosis and for the relevant clinical management.

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.

EVALUATION OF THE RISK OF CERVICAL INTRAEPITHELIAL NEOPLASIA PROGRESSION BASED ON CELL PROLIFERATION INDEX, EPITHELIAL-MESENCHYMAL TRANSITION AND CO-INFECTIONS.

Human papilloma virus (HPV) infection, especially with high risk types, represent the major etiological factor for the development of cervical precancerous and cancerous lesions. However, other factors including cell proliferation index, epithelial-mesenchymal transition and the presence of co-infections might also influence the progression of cervical intraepithelial neoplasia (CIN). The aim of our study was to analyse, the expression of cell proliferation markers and epithelial-mesenchymal transition markers during the progression of cervical intraepithelial neoplasia, in cases with and without co-infections. Standard immunohistochemistry was used to detect, Ki67, cyclin D1, phosphohiston-H3, p63, E-cadherin, ß-catenin and vimentin. The results of our study indicated that the expression of Ki67, phosphohiston-H3 and p63 is significantly increased during the progression of CIN disease, whilst the expression of E-cadherin and ß-catenin are progressively lost. The expression of mesenchymal marker vimentin is also increased in CINIII and in invasive carcinoma. Proliferation index based on Ki67 labelling is significantly higher in cases with co-infections and the expression on E-cadherin is significantly lower in cases with co-infections compared to cases without co-infections. In conclusion, the measurement of proliferation index, based on Ki67 labelling, as well as mitotic index based on phosphohiston-H3 detection can reliably indicate high and low risk groups of the progression of CIN. Similarly, higher p63 expression, loss of E-cadherin and ß-catenin and higher vimentin expression can indicate the progression risk of CIN. The presence of co-infections is associated with the increased expression of proliferation marker Ki67 and the loss of E-cadherin and therefore it can be considered as an additional marker of CIN progression.

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.

ASSESSMENT OF THE VALUE OF METHYLATION FEATURES IN DIFFERENT TISSUES FOR PREOPERATIVE IDENTIFICATION OF HIGH-RISK BREAST TUMORS.

Epigenetic changes in breast cancer patients have long been believed to be a promising marker for clinical management - early detection, prognostication, etc. Various approaches are available to test global DNA or target gene methylation status. We used some of them in different tissues of patients with breast cancer to analyze possible links between epigenetic parameters and their value for predicting clinicopathological characteristics (most importantly stage and lymph node status) of tumors. Patients with ductal and lobular invasive carcinoma were included in the study along with age-matched controls. Blood, tumor tissue and normal breast ductal epithelial cells were investigated using MS-HRM technique (for BRCA1 gene promoter methylation assessment), COBRA-PCR (for Alu element methylation quantification) and ELISA-based method (for global DNA methylation estimation). These parameters were analyzed in comparison to clinical, histologic and phenotypic characteristics of the tumors. BRCA1 promoter methylation was detected exclusively in tumor tissues and in only two cases, both of which had aggressive phenotype. Alu and global DNA methylation showed markedly low levels in all tissues of cancer patients compared to healthy controls, with extreme hypomethylation in tumor tissue. Changes in these two parameters were not always concordant. Methylation levels detected in blood showed rather weak relationship with clinicopathological characteristics of tumors. We could conclude that tested parameters are not useful for preoperative determination of tumor stage or lymph node status, neither any other clinicopathological characteristics of tumors. Epigenetics signatures of different tissues of the same patient are not homogenous. Given the uniform picture of DNA methylation in blood in some of investigated groups, additional data and very careful approach are required when considering using it as a diagnostic or predictive tool.

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.

PROLIFERATIVE/STEM CELL INDEX AND PHENOTYPIC CHARACTERISTICS OF PROLIFERATIVE PROCESSES IN ENDOMETRIUM.

The aim of the study was to explore and identify the relationship between endometrial proliferative/stem cell index and phenotypic characteristics under proliferative processes of endometrium using statistical correlation analysis. The study represents a retrospective research. The coded and depersonalized material data from Acad. N. Kipshidze Central University Clinic was used in the study. 5 study groups (83 cases) were selected from routine histopathology tissue specimens of uterus. Hematoxilyn-eosin technology and immunohistochemistry with markers ki67, CD146, PTEN was performed. The proliferative/stem cell index (PR/ST index) was calculated by the ratio of Ki67-positive cell percentage value divided by CD146-positive cell percentage value. PTEN expression score was evaluated by next scheme - PTEN positive cell numbers <10% - conditional negative, PTEN positive cell numbers 10-50 % - heterogenic, PTEN positive cell numbers >50% - conditional positive. The study showed that, PR/ST index in 1st study group Endometrial Hyperplasia ranges within the interval 15-23, PTEN is diffusely positive with focal heterogeneity 11.1%. 2nd study group Endometrial Dysplasia, the PR/ST index ranges within the interval 16.5-23.3. PTEN heterogeneity is 18.3%, negativity - 6.3%. 3rd study group Endometrioid Carcinoma Grade 1, the PR/ST index ranges between 21.7 and 25.5. PTEN is heterogenic in 35.7% of cases, negative in 14.3% of cases. 4th study group Endometrioid Carcinoma Grade 2, the PR/ST index ranges within the interval 23.2-27.8. PTEN is heterogenic in 43.5% of cases, negative in 30.4% of cases. 5th study group Endometrioid Carcinoma Grade 3, the PR/ST index ranges within the interval 25.8-29.4. PTEN is heterogenic in 33.3% of cases, negative in 41.7% of cases. It was found that, in endometrial hyperplasia and dysplasia cases the PR/ST index do not correlate with phenotypic characteristics, while in the cases of endometrial carcinoma different degrees of malignancy the PR/ST Index and phenotypic characteristics intensely and directly correlates. The high attention should be given to the fact that heterogeneity peak of PTEN expression takes place in the cases of endometrial carcinoma G2, but the negativity of PTEN protein is increasing in parallel with the malignancy increasing process and reaches peak performance in cases of endometrial carcinoma G3.

LINE-1 METHYLATION IN BLOOD AND TISSUES OF PATIENTS WITH BREAST CANCER.

Methylation is an epigenetic alteration proved to be involved in many disease processes including cancer. This change affects mainly gene promoters and repetitive sequences in genome. Long Interspersed Nuclear Element-1 (LINE-1) is a family of retrotransposons - repetitive elements that modify gene activity and can themselves be targeted by epigenetic mechanisms. LINE-1 methylation level is a surrogate marker for global methylation. In many conditions this parameter is found to be altered not only in affected cell groups, but also throughout other tissues. The aim of our study was to compare LINE-1 methylation pattern in DNA extracted from blood of the patients with benign and malignant breast tissue. In addition, we investigated correlation of LINE-1 methylation in blood and tissues of same patients and relationship of all variables with histopathologic and phenotypic characteristics of tumors. Patients with biopsy-proved ductal invasive carcinoma of breast and no preoperative chemo/radiotherapy were chosen for the study group. Another pool of patients with various benign breast lesions represented controls. Blood samples from both group members were collected preoperatively. Tumor tissue sections were processed for pathology report and part of remaining tissue was used for methylation study. LINE-1 methylation level was quantified using ELISA-based assay. It was analyzed in combination with histologic and phenotypic tumor parameters and compared between different tissues and different study groups. LINE-1 was found to be significantly hypomethylated in breast cancer tissue compared to blood. Blood samples of patients with malignant tumors showed slightly lower methylation level, than samples obtained from control group members. Lymphovascular invasion was the only aggressiveness-determining factor that was found to be at least weakly correlated with LINE-1 hypomethylation in blood. We can conclude, that global hypomethylation measured by LINE-1 methylation level is significant in tumor tissue. But there is no significant difference between LINE-1 methylation levels in blood of patients with benign and malignant breast tumors; therefor LINE-1 hypomethylation in blood cannot be used as a marker for early tumor detection. Neither is it valid for determination of tumor behavior.

SPECIFICITIES OF ENDOMETRIAL PROLIFERATION/STEM CELL INDEX DISTRIBUTION IN ENDOMETRIOID CARCINOMA OF DIFFERENT GRADE OF MALIGNANCY.

Endometrial neoplasia is the most common malignant tumor of female genital system in developed countries. The incidence of endometrial cancer has increased in the last years and despite advances in diagnosis and treatment, the death rates have steadily been increasing over the past 20 years. Therefore aspects of endometrial cancer development, pathogenesis and effective treatment is especially urgent to this day, as much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Endometrial stem cells take the special place among somatic stem cells of female reproductive system-the detection of them and identification of their location in the complex cellular hierarchy still remains challenging. Further study of endometrial stem cells will clarify their role in gynecologic pathologies associated with hyper-proliferative states of endometrium. The aim of our study was to explore the specificities of endometrial proliferative/stem cell index distribution under endometrioid carcinoma of different grade of malignancy. The study represents a retrospective research. The coded and depersonalized material data from Acad. N. Kipshidze Central University Clinic was used in the study. 3 study groups - 1st study group "Endometrioid Carcinoma Grade 1" (14 cases), 2nd study group "Endometrioid Carcinoma Grade 2" (23 cases) and 3rd study group "Endometrioid Carcinoma Grade 3" were selected from routine histopathology tissue specimens of uterus. Hematoxilyn-eosin technology and immunohistochemistry with proliferation marker ki67 and stem cell marker CD146 was performed. The proliferative/stem cell index was calculated by the ratio of Ki67-positive cell percentage value divided by CD146-positive cell percentage value. The study showed that in the 1st study group labeled as "Endometrioid Carcinoma Grade 1", the proliferative/stem cell index ranges between 21.7 and 25.5. Its mean average value in the age distribution subgroups accounts for: 1.1) reproductive age - 22.4; 1.2) menopause - 23.5; 1.3) post-menopause - 24.8. Proliferative/stem cell index reaches its maximum in the samples retrieved from post-menopause age, and decreases significantly in reproductive age individuals. In the 2nd study group labeled as "Endometrioid Carcinoma Grade 2", the proliferative/stem cell index increases and ranges within the interval 23.2-27.8. Its mean average value in the age distribution subgroups accounts for: 2.1) reproductive age -23.7; 2.2) menopause - 24.2; 2.3) post-menopause - 25.8. In the 3rd study group labeled as "Endometrioid Carcinoma Grade 3", the proliferative/stem cell index markedly increases and ranges within the interval 25.8-29.4. Its mean average value in the age distribution subgroups accounts for: 3.1) reproductive age - 28.4; 3.2) menopause - 28.5; 3.3) post-menopause - 28.5. It was found that average value of proliferative/stem cell index in the 1st and 2nd study groups (EC Grade 1/2) keeps the same tendencies of increase in age subgroups as well as at endometrial hyperplasia conditions - in particular in both study groups increase in value of the proliferative/stem cell index in age subgroups makes about 1% (1st study group-0,97%, 2nd study group-0,96%). What about 3rd study group (EC Grade 3) average value of proliferative/stem cell index in age subgroups is almost the same. It was found that average value of proliferative/stem cell index in endometrioid carcinoma most markedly differs from the norm in post-menopause period. The study showed that average value of proliferative/stem cell index in endometrioid carcinoma cases (EC Grade 1/2) tends to increase with age like endometrial hyperplasia conditions, in contrast with the norm, where it is observed to progressively decrease with aging. The attention should be given to the fact that the mean average value of proliferative/stem cell index in endometrioid carcinoma Grade 3 is almost constant.