Unveiling The Neuropathology Tumour Landscape: 10-Year Statistical Analysis With Global Comparison - Single Centre Experience.

Introduction: Central nervous system (CNS) tumours represent a significant public health issue worldwide, and their incidence and distribution vary across different populations. Although studies on CNS tumours have been conducted in various countries, there is a lack of information regarding their patterns in Macedonia. Therefore, this study is aimed at investigating the distribution, histopathological types and subtypes and demographic features of CNS tumours in our country. Materials and Methods: A cross sectional study was conducted using the electronic database of the Institute of Pathology - Medical Faculty, University "Ss. Cyril and Methodius" in Skopje which contains data from 3286 received and analysed surgical specimens, mainly from the University Clinic of Neurosurgery in Skopje, and a smaller number of surgical specimens from the University Surgical Centre "St. Naum Ohridski" in Skopje between 2012 and 2022. The collected and analysed data includes patient age, sex and histopathological types and subtypes of the tumours. Results: The majority of CNS tumours were diagnosed in adults aged between 50-70, with a male to female ratio of 1.5:1. The most common location of the tumours was the cerebrum, followed by the pituitary gland and cerebellum. The most frequent histological groups were gliomas, with glioblastoma as the most common diagnosis, followed by meningiomas. Conclusion: Following a detailed and thorough review of the CNS tumours in our study, we can conclude that the R. of Macedonia follows global statistics and trends regarding brain tumours.

Glomerulopathies with Fibrillary Deposits.

The glomerulopathies associated with the deposition of extracellular fibrils in the glomeruli are subdivided into Congo red positive (amyloidosis) and Congo red negative (non-amyloidotic glomerulopathies) based on Congo red staining. The non-amyloidotic glomerulopathies are divided into immunoglobulin-derived and non-immunoglobulin-derived glomerulopathies. The immunoglobulin-derived glomerulopathies: fibrillary glomerulopathy (FGn) and immunotactoid glomerulopathy (ITG) are rare glomerulopathies. The diagnosis of fibrillary-immunotactoid glomerulopathy depends on electron microscopy, which shows the presence of microfibrils in the glomeruli. The microfibrils in FGn are randomly arranged with diameters less than 30 nm. The microfibrils in ITG are larger than 30 nm with a visible lumen (microtubules), focally arranged in parallel bundles. Patients with fibrillary-immunotactoid glomerulopathy present with proteinuria (usually in the nephrotic range), microscopic hematuria, arterial hypertension, and chronic kidney disease that progresses to kidney failure over months to years. Currently, there are no guidelines for the treatment of fibrillary-immunotactoid glomerulopathy, although immunotactoid glomerulopathy could be associated with underlying hematologic disorders with the need for clone-directed therapy.

Immunotactoid Glomerulopathy: A Rare Glomerular Disease Case Study.

Immunotactoid glomerulopathy (ITG) is a rare glomerular disease with variable responsiveness to the immunosuppressive therapy and with uncertain prognosis. ITG was diagnosed in two patients with type 2 diabetes mellitus with nephrotic syndrome and chronic kidney disease. The absence of diabetic retinopathy in the first case and the recent onset of diabetes in the second case accompanied with sudden increase in the 24-hour proteinuria and rapid decline in kidney function, prompted us to perform kidney biopsy. The electron microscopy set the diagnosis of ITG in both cases. There is no consensus for the treatment of ITG. The first patient was treated with combination of steroids and mycophenolate mofetil with reduction of the 24-hour proteinuria, but with persistence of the chronic kidney disease. The second patient received high doses of steroids with continuous deterioration of kidney function with the need of hemodialysis treatment.

Anti-inflammatory and immunomodulatory effects of rosuvastatin in patients with low-to-moderate cardiovascular risk.

Statins have shown anti-inflammatory pleiotropic effects in subjects with/at risk of cardiovascular disease. The aim of this study was to evaluate the inflammatory/immunomodulatory properties of rosuvastatin in subjects at low-to-moderate cardiovascular risk. Data was collected from patients' records, physical examination and blood sampling. Subjects were assigned to rosuvastatin 20 mg per day. Rosuvastatin significantly decreased C-reactive protein (p = 0.045), and increased vascular endothelial growth factor (p = 0.004) and epidermal growth factor (p = 0.009). A multivariate analysis identified total cholesterol (p = 0.027) and vascular endothelial growth factor (p = 0.011) to be independently associated with rosuvastatin treatment. Given beneficial/harmful role of growth factors, vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF), in cardiovascular disease, one would suggest the need for routine monitoring of growth factor levels, especially in patients on long-term statin therapy.

Calcification of the cavernosal bodies may be responsible for development of erectile dysfunction in uremic apolipoprotein E deficient (apoE-/-) mice / Calcificación de cuerpos cavernosos y desarrollo de disfunción eréctil en la falta de apolipoproteína urémica E (ratones ApoE-/-) en un modelo de ratón

INTRODUCTION AND OBJECTIVE: Erectile dysfunction's physiopathology in uremia is complex and multifactorial, involving a combination of classical risk factors and specific uremia-related risk factors such as increased oxidative stress, endothelial dysfunction and inflammation. The aim of the study is to investigate the effect of chronic kidney disease (CKD) on vascular calcification and endothelial function of cavernosal bodies in apolipoprotein E deficient (apoE−/−) mice, a well known model of erectile dysfunction. MATERIALS AND METHODS: Eight-week-old male apoE−/− mice were randomly assigned to the following 3 groups: (I) subtotally nephrectomised (SNX apoE−/−, 12 mice), (II) uninephrectomised (UNX apoE−/−, 11 mice) or (III) sham operated (sham-op apoE−/−, 15 mice). At 16 weeks after surgery, aortas and penile erectile tissues were harvested for histological studies to assess atherosclerosis, vascular calcification, nitrotyrosine staining, total collagen content and macrophage staining. RESULTS: At sacrifice, SNX and UNX mice had significantly higher serum urea, total cholesterol, and triglyceride concentrations than sham-op controls. Atherosclerotic lesions in thoracic aorta were significantly larger in uremic apoE−/− mice than in controls. There were no atheromatous lesions in cavernosal bodies or penile artery observed in any group. However, SNX and UNX animals showed a significant increase in calcification score, collagen content and nitrotyrosine staining in cavernosal bodies when compared with controls. The degree of macrophage infiltration was comparable between the 3 groups. CONCLUSION: In conclusion, even mild renal dysfunction, i.e., after uninephrectomy increases calcification score and aggravates endothelial function of cavernosal bodies in apoE−/− mice and this effect might be linked to increased oxidative stress in penile endothelium

INTRODUCCIÓN Y OBJETIVOS: La fisiopatología de la disfunción eréctil en la uremia es compleja y multifactorial, e incluye una combinación de factores de riesgo clásicos y factores específicos asociados a la uremia, como el aumento del estrés oxidativo, la disfunción endotelial y la inflamación. El objetivo de este trabajo es examinar el efecto de la enfermedad renal crónica (ERC) sobre la calcificación vascular y la función endotelial de los cuerpos cavernosos en caso de deficiencia de apolipoproteína E en ratones (ratones ApoE−/−), un modelo bien conocido de disfunción eréctil. MATERIALES Y MÉTODOS: Los ratones machos de 8 semanas de edad con «ApoE−/−mice» se distribuyen aleatoriamente en 3 grupos: 1) con heminefrectomía (SNX ApoE−/−), 12 ratones; 2) con nefrectomía única (UNX ApoE−/−), 11 ratones, y 3) operación de placebo (sham-op ApoE−/−), 15 ratones. Dieciséis semanas después de la cirugía, se retiraron los tejidos eréctiles de la aorta y el pene para realizar estudios histológicos con el fin de evaluar la aterosclerosis, la calcificación vascular, las sombras de nitrotirosina, el contenido de colágeno total y las sombras de macrófagos. RESULTADOS: Durante el sacrificio, los ratones con SNX y UNX reflejaron valores de urea sérica, colesterol total y concentración de triglicéridos significativamente más elevados, en comparación con los casos controlados con placebo. Las lesiones ateroscleróticas en la aorta torácica fueron mucho mayores en los ratones urémicos «ApoE−/−» en comparación con los controles. No hubo lesiones ateromatosas en los cuerpos cavernosos ni en la arteria del pene en ninguno de los grupos. Sin embargo, los animales con nefrectomía seminal y única mostraron un aumento significativo en la calcificación, presencia de colágeno y manchas de nitrotirosina en cuerpos cavernosos en comparación con los controles. El grado de infiltración de macrófagos fue comparable entre los 3 grupos. CONCLUSIÓN: Se ha concluido que incluso una disfunción renal menor, es decir, tras una nefrectomía única, aumenta la calcificación y exacerba la función endotelial de los cuerpos cavernosos en ratones «ApoE−/−», y este efecto puede estar asociado a un aumento del estrés oxidativo en el endotelio del pene

Calcification of the cavernosal bodies may be responsible for development of erectile dysfunction in uremic apolipoprotein E deficient (apoE-/-) mice.

INTRODUCTION AND OBJECTIVE: Erectile dysfunction's physiopathology in uremia is complex and multifactorial, involving a combination of classical risk factors and specific uremia-related risk factors such as increased oxidative stress, endothelial dysfunction and inflammation. The aim of the study is to investigate the effect of chronic kidney disease (CKD) on vascular calcification and endothelial function of cavernosal bodies in apolipoprotein E deficient (apoE-/-) mice, a well known model of erectile dysfunction. MATERIALS AND METHODS: Eight-week-old male apoE-/- mice were randomly assigned to the following 3 groups: (i) subtotally nephrectomised (SNX apoE-/-, 12 mice), (ii) uninephrectomised (UNX apoE-/-, 11 mice) or (iii) sham operated (sham-op apoE-/-, 15 mice). At 16 weeks after surgery, aortas and penile erectile tissues were harvested for histological studies to assess atherosclerosis, vascular calcification, nitrotyrosine staining, total collagen content and macrophage staining. RESULTS: At sacrifice, SNX and UNX mice had significantly higher serum urea, total cholesterol, and triglyceride concentrations than sham-op controls. Atherosclerotic lesions in thoracic aorta were significantly larger in uremic apoE-/- mice than in controls. There were no atheromatous lesions in cavernosal bodies or penile artery observed in any group. However, SNX and UNX animals showed a significant increase in calcification score, collagen content and nitrotyrosine staining in cavernosal bodies when compared with controls. The degree of macrophage infiltration was comparable between the 3 groups. CONCLUSION: In conclusion, even mild renal dysfunction, i.e., after uninephrectomy increases calcification score and aggravates endothelial function of cavernosal bodies in apoE-/- mice and this effect might be linked to increased oxidative stress in penile endothelium.

Measurement-oriented deep-learning workflow for improved segmentation of myelin and axons in high-resolution images of human cerebral white matter.

BACKGROUND: Standard segmentation of high-contrast electron micrographs (EM) identifies myelin accurately but does not translate easily into measurements of individual axons and their myelin, even in cross-sections of parallel fibers. We describe automated segmentation and measurement of each myelinated axon and its sheath in EMs of arbitrarily oriented human white matter from autopsies. NEW METHODS: Preliminary segmentation of myelin, axons and background by machine learning, using selected filters, precedes automated correction of systematic errors. Final segmentation is done by a deep neural network (DNN). Automated measurement of each putative fiber rejects measures encountering pre-defined artifacts and excludes fibers failing to satisfy pre-defined conditions. RESULTS: Improved segmentation of three sets of 30 annotated images each (two sets from human prefrontal white matter and one from human optic nerve) is achieved with a DNN trained only with a subset of the first set from prefrontal white matter. Total number of myelinated axons identified by the DNN differed from expert segmentation by 0.2%, 2.9%, and -5.1%, respectively. G-ratios differed by 2.96%, 0.74% and 2.83%. Intraclass correlation coefficients between DNN and annotated segmentation were mostly >0.9, indicating nearly interchangeable performance. COMPARISON WITH EXISTING METHOD(S): Measurement-oriented studies of arbitrarily oriented fibers from central white matter are rare. Published methods are typically applied to cross-sections of fascicles and measure aggregated areas of myelin sheaths and axons, allowing estimation only of average g-ratio. CONCLUSIONS: Automated segmentation and measurement of axons and myelin is complex. We report a feasible approach that has so far proven comparable to manual segmentation.

A Rare Case of Soft Tissue Erdheim Chester Disease: Diagnostic Dilemma and Management.

BACKGROUND: Erdheim Chester disease (ECD) is a rare form of non-Langerhans histiocytosis that still presents a diagnostic and clinical dilemma. CASE PRESENTATION: We present a rare case of ECD, young 31 male with atypical localisation and soft tissue presentation and no bone involvement. He started clinical investigations due to subcutaneous tumour mass in the lumbar spine that caused severe back pain. Skin biopsy revealed ECD with Immunohistochemistry CD68+, CD10+, CD11c+, vimentin+, S100A4+. Activating BRAFV600E mutation was positive from the tumour tissue. The patient was referred to the haematology department. PET CT was performed for initial disease staging. Treatment was started with corticosteroids (methylprednisolone 0.5 mg/kg per day), and after 7 days, a significant clinical improvement was noticed in terms of pain disappearance with no need for pain killers. After two weeks, treatment with interferon Alfa (IFN-α) was started in a dose of 3 million units 3 times per week. After 4 months of interim treatment PET, CT revealed a significant reduction of the tumour mass. Therapy with IFN-α was continued, and the patient is still clinically in good condition. CONCLUSION: It can be concluded that shortening the time of diagnosis of ECD is essential in treatment outcome of this disease. Still, large studies have to confirm the best treatment of this rare condition.

Hepatocellular carcinoma - clinicopathological characteristics, survival, and expression of various histologic molecular markers.

The aim of this study was to make a clinical characterisation of patients with hepatocellular carcinoma and to investigate the expression of a set of molecular markers in patients from the Republic of North Macedonia. We analysed 60 patients for clinicopathologic factors, and we investigated tumour tissue and surrounding liver tissue for immunoexpression of E-cadherin, ß-catenin, cyclin D1, and p53. Infection with hepatitis virus B and C (p < 0.001), tumour dimension (p < 0.001), vascular invasion (p < 0.002), and tumour differentiation (p < 0.021) significantly influenced the survival of the patients. E-cadherin and ß-catenin expression reduction and cyclin D1 and p53 overexpression were significantly higher in the tumour than in the non-tumour tissue (p < 0.001; p < 0.001; p = 0.001; p < 0.001, respectively). No significant correlation was found between clinicopathological characteristics and the analysed molecules nor between the molecules themselves. The immunoexpression of E-cadherin, ß-catenin, cyclin D1, and p53 was not related to the tumour aggressiveness and prognosis. However, their significantly higher expression in HCC tissue compared to that in non-tumour tissue indicate their important role in hepatocarcinogenesis. The clinicopathological characteristics of the neoplasm remain highly predictive factors for the survival of the patients.

EBV Positive Gastric Carcinomas and Their Clinicopathological Characteristics.

BACKGROUND: The understanding of the etiopathogenesis of gastric carcinoma (GC) can be a base for development of new therapeutic methods to reduce mortality and to increase survival in patients with GC. The percentage of Epstein - Barr virus (EBV) positive gastric carcinomas is uncertain, and the etiologic importance of EBV in the pathogenesis of GC has still not been elucidated. AIM: This study aimed to determine the percentage of EBV associated GC as well as to determine their clinicopathological characteristics. MATERIAL AND METHODS: The study included 80 patients with GC who were analysed for ethnicity, local growth of a tumour (T status), the presence of nodal metastases (N), the presence of distant metastases (M), stage of the disease and degree of carcinoma differentiation. For detection of EBV, immunostainings were performed on tumour tissue and the peripheral non-tumour gastric mucosa. RESULTS: Positive immunostaining with an antibody against EBV was found in 19 (23.75%) of the 80 patients with gastric carcinomas. EBV immunostainings were significantly different in patients with or without metastasis and between patients of Macedonian and Albanian ethnicity (p < 0.0001, p < 0.009, respectively). EBV immunoexpression was significantly associated with the presence of distant metastases and with patients of Albanian ethnicity. CONCLUSION: Association of EBV immunostainings with distant metastasis in patients with GC suggests the influence of EBV infection on the progression of gastric carcinoma. Due to scarce and doubtful literature data on EBV associated GC, further studies are necessary to determine the role of EBV regarding aetiology, treatment and prognosis in patients with EBV associated gastric carcinoma.

Thyroid cancer detection rate and associated risk factors in patients with thyroid nodules classified as Bethesda category III.

Background Ultrasound guided fine-needle aspiration (FNA) is a standard procedure for thyroid nodules management and selecting patients for surgical treatment. Atypia of undetermined significance (AUS) or follicular lesion of undetermined significance (FLUS), as stated by The Bethesda System for Reporting Thyroid Cytopathology, is a diagnostic category with an implied malignancy risk of 5-15%. The aim of our study was to review cytology and histopathology reports, as well as clinical and ultrasound data, for thyroid nodules reported as AUS/FLUS, in order to evaluate the malignancy rate and to assess factors associated with malignant outcome. Patients and methods A total of 112 AUS/FLUS thyroid nodules in 105 patients were evaluated, of which 85 (75.9%) were referred to surgery, 21 (18.8%) were followed-up by repeat FNA and 6 nodules (5.3%) were clinically observed. Each was categorized in two final diagnostic groups - benign or malignant, which were further compared to clinical data of patients and ultrasonographic features of the nodules. Results Final diagnosis of malignancy was reached in 35 cases (31.2%) and 77 (68.8%) had benign lesions. The most frequent type of cancer was papillary thyroid carcinoma (PTC) - 58.1% PTC and 25.8% had follicular variant of PTC. Patients' younger age, smaller nodule size, hypoechoic nodule and presence of calcifications were shown to be statistically significant risk factors for malignancy. Conclusions The rate of malignancy for the AUS/FLUS diagnostic category in our study was higher than estimated by the Bethesda System. Clinical and ultrasound factors should be considered when decision for patient treatment is being made.

HER2 Positive Gastric Carcinomas and Their Clinico-Pathological Characteristics.

BACKGROUND: HER2 protein expression in gastric carcinoma, in correlation with existing, acknowledged prognostic factors which include the parameters that determine the TNM stage of the disease, could become the basis for ongoing research in the field of molecular targeted and personalised therapy. AIM: To determine the expression of the HER2 protein in gastric carcinoma and to correlate the expression of a HER2 protein with clinicopathological characteristics of the disease. MATERIAL AND METHODS: The data of HER2 protein expression and the parameters of the TNM classification were obtained from the histopathological reports of the Institute of Pathology in Skopje, and for the clinical stage we used patient's files from the University Clinic for Abdominal Surgery in Skopje. RESULTS: The analysis of the correlation of HER2 protein expression and TNM classification parameters pointed out a significant correlation between HER2 protein expression and intragastric localisation of gastric carcinoma (P = 0.005), and the tumour grade of differentiation (P = 0.034). There was also a positive correlation between HER2 protein expression pattern and positive lymph nodes in patients with gastric carcinoma (P = 0.03). The expression pattern of HER2 +++ was significantly more common registered in patients with positive lymph nodes (P = 0.03). CONCLUSION: The expression of HER2 protein could represent a biological marker with prognostic and predictive value in patients with gastric carcinoma. Considering the high mortality rate in patients with gastric carcinoma and lack of international standardised therapeutic approach, research of the role and significance of HER2 overexpression and Trastuzumab therapy may prove useful in the development of new therapeutic strategies.

Presentation of the Molecular Subtypes of Breast Cancer Detected By Immunohistochemistry in Surgically Treated Patients.

INTRODUCTION: The detection of estrogen, progesterone and HER-2 neu receptors on the surface of the tumour cell is a significant prognostic factor, alone or in combination. The presence or absence of receptors on the surface of the tumour cell is associated with the conditional gene expression in the tumour cell itself. Based on these genetically determined expressions of the tumour cell, five molecular subtypes of breast cancer have been classified on the St. Gallen International Expert Consensus in 2011 that can be immunohistochemically detected, with each subtype manifesting certain prognosis and aggression. AIM: Analyzing the presentation of molecular subtypes of breast cancer that are immunohistochemically detected in surgically treated patients at the Clinic for Thoracic and Vascular Surgery. MATERIAL AND METHODS: We used the international classification on molecular subtypes of breast cancer which divides them into: Luminal A (ER+ and/or PR+, HER-2 negative, Ki-67 < 14%), Luminal B with HER-2 negative (ER+ and/or PR+, HER-2 negative, Ki-67 ≥ 14%), Luminal B with HER-2 positive (ER+ and/or PR+, HER-2+, any Ki-67), HER-2 enriched (ER-, PR-, HER-2+), and basal-like (triple negative) (ER-, PR-, HER-2 negative, CK5/6+ and/or EGFR+). A total of 290 patients, surgically treated for breast cancer, were analysed during 2014. RESULTS: In our analysis, we found that Luminal A was present in 77 (26.55%) patients, Luminal B HER-2 negative was present in 91 (31.38%) patients, Luminal B HER-2 positive was present in 70 (24.14%) patients, HER-2 enriched was present in 25 (8.62%) patients and basal-like (or triple negative) was present in 27 (9.31%) patients. CONCLUSION: Detecting the subtype of breast cancer is important for evaluating the prognosis of the disease, but also for determining and providing an adequate therapy. Therefore, determining the subtype of breast cancer is necessary for the routine histopathological assay.

Tumour Lymphocytic Infiltration, Its Structure and Influence in Colorectal Cancer Progression.

BACKGROUND: The role of the immune system in the control of tumour progression has been stressed, recently. Many studies indicate the fact that the immune system can prevent tumour progression in several types of human malignant neoplasms including colorectal cancer. According to some authors, a higher density of "tumour-associated lymphocytes" (TAL), in malignant neoplasms, correlate with prolonged survival of patients. AIM: This study aims to determine the structure and the influence of the immune cells, TAL, in the progression of colorectal cancer (CRC). PATIENTS AND METHODS: The study included 103 patients with CRC operated at the University Clinic of Digestive Surgery in Skopje, whose operative material was analysed at the Institute of Pathology, Medical Faculty in Skopje. The structure of tumor-associated cells and their density were determined and were correlated with neoplasm's grade, local growth (T), positive lymph nodes, lymphatic invasion and stage of the disease. RESULTS: CD4+, CD8+ and CD20+ lymphocytes (Ly) were found in TAL. The density of TAL was significantly different in neoplasms with different T status, lymphatic invasion, patients with and without nodal metastasis and patients with a different stage of the disease. The density of CD4+, CD8+, and CD20+ cells were significantly different in neoplasms with different T. The density of CD8+ and CD20+ lymphocytes was lower in patients with nodal metastasis and higher stage. CONCLUSION: The density of tumor-associated lymphocytes can anticipate the disease progression in patients with colorectal cancer, and the density of TAL influences the control of tumour progression.

Correlation of Immunohistochemistry and Fluorescence in Situ Hybridization for HER-2 Assessment in Breast Cancer Patients: Single Centre Experience.

BACKGROUND: Accurate assessment of HER-2 is imperative in selecting patients for targeted therapy. Most commonly used test methods for HER-2 are immunohistochemistry (IHC) and fluorescence in situ hybridisation (FISH). We evaluated the concordance between FISH and IHC for HER-2 in breast cancer samples using Food and Drug Administration approved tests. MATERIAL AND METHODS: Archived paraffin tissue blocks from 73 breast cancer patients were used. HER-2 immunostaining was performed using Ventana anti-HER-2 monoclonal antibody. The FISH assay was performed using PathVysion™ HER-2 DNA Probe Kit. RESULTS: Of the 73 cases 68.5% were IHC 0/1+, 15.07% were IHC 2+ and 16.44% were IHC 3+. Successful hybridisation was achieved in 72 cases. HER-2 FISH amplification was determined in 16.67% cases. Ten IHC 3+ and two IHC 2+ cases were FISH positive. Two of the IHC 3+ cases were FISH negative. Concordance rate was 100%, 18.18% and 83.33% for IHC 0/1+, 2+ and 3+ group, respectively. Total concordance was 84.72%, kappa 0.598 (p < 0.0001). The sensitivity of IHC in detecting IHC 2+ and IHC 3+ cases was 16.7% and 83.3%, and the specificity was 85% and 96.67%, respectively. CONCLUSION: The consistency between the methods was highest for IHC negative and lowest for IHC equivocal cases. The immunohistochemistry showed high sensitivity for IHC 2+/3+ cases and high specificity for IHC 3+ cases. Our results support the view that false-positive rather than false-negative IHC results are a problem with HER-2/IHC testing, and that IHC should be used as an initial screening test, but IHC 2+/ 3+ results should be confirmed by FISH.

The Spectrum of Histopathological Changes in the Renal Allograft - a 12 Months Protocol Biopsy Study.

INTRODUCTION: Renal transplantation became a routine and successful medical treatment for Chronic Kidney Disease in the last 30 years all over the world. Introduction of Luminex based Single Antigen Beads (SAB) and recent BANFF consensus of histopathological phenotypes of different forms of rejection enables more precise diagnosis and changes the therapeutic approach. The graft biopsies, protocol or cause, indicated, remain a golden diagnostic tool for clinical follow up of kidney transplant recipients (KTR). AIM: The study aimed to analyse the histopathological changes in renal grafts 12 months after the surgery in KTR with satisfactory kidney function. MATERIAL AND METHODS: A 12-month protocol biopsy study was performed in a cohort of 50 Kidney transplant recipients (42 from living and 8 from deceased donors). Usual work-up for suitable donors and recipients, standard surgical procedure, basic principles of peri and postoperative care and follow up were done in all KTR. Sequential quadruple immunosuppression including induction with Anti-thymocyte globulin (ATG) or Interleukin-2R antagonist (IL-2R), and triple drug maintenance therapy with Calcineurin Inhibitors (CNI), Mycophenolate Mofetil (MMF) and Steroids were prescribed to all pts. Different forms of Glomerulonephritis (16), Hypertension (10), End Stage Renal Disease (13), Hereditary Nephropathies (6), Diabetes (3) and Vesicoureteral Reflux (2) were the underlying diseases. All biopsies were performed under ultrasound guidance. The 16 gauge needles with automated "gun" were used to take 2 cores of tissue. The samples were stained with HE, PAS, Trichrome Masson and Silver and reviewed by the same pathologist. A revised and uploaded BANFF 2013 classification in 6 categories (Cat) was used. RESULTS: Out of 48 biopsies, 15 (31%) were considered as normal, 4 (8%), Borderline (BL-Cat 3), 5 (10%) as Interstitial Fibrosis/Tubular Atrophy (IF/TA-Cat 5), 5 (10%) were classified as non-immunological (Cat 6), 2 as a pure antibody-mediated rejection (ABMR-Cat 2) and T-cell Mediated Rejection (TCMR-Cat 4). The remaining 17 samples were classified as a "mixed" rejection: 7 (41%) ABMR + IF/TA, 5 (29%) ABMR + BL + IF/TA, 2 (11%) BL + IF/TA, 1 (5%) ABMR + BL, 1 (5%) ABMR + TCMR and 1 (5%) TCMR + IF/TA. The mean serum creatinine at the time of the biopsy was 126.7 ± 23.4 µmol/L, while GFR-MDRD 63.4 ± 20.7 ml/min, which means that the majority of the findings were subclinical. Among the non-immunological histological findings (Cat 6), 3 cases belonged to CNI toxicity, 1 to BK nephropathy and 1 to recurrence of the primary disease. CONCLUSION: Our 12-month protocol biopsy study revealed the presence of different forms of mixed subclinical rejection. Use of recent BANFF classification and scoring system enables more precise diagnosis and subsequently different approach to the further treatment of the KTR. More correlative long-term studies including Anti HLA antibodies and Endothelial Cell Activation- Associated Transcripts (ENDAT) are needed.

Free Gingival Graft versus Mucograft: Histological Evaluation.

INTRODUCTION: The correction of the gingival recession is of esthetical and functional significance, but the tissue regeneration can only be confirmed by a histological examination. AIM: This study aims to make a comparison between the free gingival graft and the autograft. MATERIAL AND METHODS: This study included 24 patients with single and multiple gingival recessions. Twelve patients were treated with a free gingival graft and the other twelve with a micrograft. Six months after the surgical procedure, a micro-punch biopsy of the transplantation area was performed. The tissue was histologically evaluated, graded in 4 categories: immature, mature, fragmented and edematous collagen tissue. The elastic fibres were also examined and graded in three categories: with a normal structure, fragmented rare and fragmented multiplied. RESULTS: Regarding the type of collagen tissue that was present, there was a significant difference between the two groups of patients, with a larger number of patients treated with a micrograft showing a presence of mature tissue, compared to the patients treated with a free gingival graft. A larger number of patients in both of the groups displayed elastic fibres with a rare fragmented structure; 33.3% of the patients showed a normal structure; 50% demonstrated a normal structure. CONCLUSION: The patients treated with a free gingival graft showed a larger presence of fragmented collagen tissue and fragmented elastic fibres, whereas a mature tissue was predominantly present in the surgical area where a Geistlich Mucograft was placed.

Standardization and optimization of fluorescence in situ hybridization (FISH) for HER-2 assessment in breast cancer: A single center experience.

Accurate assessment of human epidermal growth factor receptor 2 (HER-2) is crucial in selecting patients for targeted therapy. Commonly used methods for HER-2 testing are immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Here we presented the implementation, optimization and standardization of two FISH protocols using breast cancer samples and assessed the impact of pre-analytical and analytical factors on HER-2 testing. Formalin fixed paraffin embedded (FFPE) tissue samples from 70 breast cancer patients were tested for HER-2 using PathVysion™ HER-2 DNA Probe Kit and two different paraffin pretreatment kits, Vysis/Abbott Paraffin Pretreatment Reagent Kit (40 samples) and DAKO Histology FISH Accessory Kit (30 samples). The concordance between FISH and IHC results was determined. Pre-analytical and analytical factors (i.e., fixation, baking, digestion, and post-hybridization washing) affected the efficiency and quality of hybridization. The overall hybridization success in our study was 98.6% (69/70); the failure rate was 1.4%. The DAKO pretreatment kit was more time-efficient and resulted in more uniform signals that were easier to interpret, compared to the Vysis/Abbott kit. The overall concordance between IHC and FISH was 84.06%, kappa coefficient 0.5976 (p < 0.0001). The greatest discordance (82%) between IHC and FISH was observed in IHC 2+ group. A standardized FISH protocol for HER-2 assessment, with high hybridization efficiency, is necessary due to variability in tissue processing and individual tissue characteristics. Differences in the pre-analytical and analytical steps can affect the hybridization quality and efficiency. The use of DAKO pretreatment kit is time-saving and cost-effective.

Immunohistochemical Expression of Epidermal Growth Factor Receptor in Hepatocellular Carcinoma.

INTRODUCTION: Epidermal growth factor receptor (EGFR) signaling plays an important role in various cancers, including hepatocellular carcinoma (HCC). We aimed to evaluate immunoexpression of EGFR in HCC and surrounding non-tumor liver tissue and to correlate it to multiple clinicopathologic data. MATERIAL AND METHODS: We analyzed 60 patients with HCC for multiple clinicopathologic characteristics and survival. Presence of the immunosignal and the percentage of positive tumor cells at the whole tumor tissue sample and adjacent cirrhotic liver tissue were semi-quantitatively determined. RESULTS: Nineteen patients (31.67%) were female and 41 (68.33%) were male ranging in age from 31 to 85 years, median 61.88±10.51. Mean survival time for female patients was 8.86±1.76 months, for male 13.03±1.50 months and overall survival was 11.6051±1.19 months. The most patients had: T2 status (41.67%), no enlarged lymph nodes (90%), vascular invasion (63.33%) and well differentiated (43.33%) tumors. EGFR immunoexpression was determined in range from 0% to 100% in both tumor and non-tumor tissue with mean value of 39.58% in tumor and 86.86% in cirrhotic tissue (p<0.00). Higher percent of tumor EGFR positive cells were found in cases with higher T status, higher levels of AFP and poorly differentiated carcinoma, but not significantly. Lower percent of tumor EGFR positive cells were found in patients with vascular invasion and enlarged lymph nodes, but also not significantly. EGFR expression in tumor tissue significantly influenced survival of the patients (p<0.05). CONCLUSION: The study showed that expression of EGFR in lower percentage of tumor cells was associated to favorable prognosis, making it a potential prognostic marker and therapeutic target.