BRAF V600E Mutation in Malignant Melanoma-A Romanian Research Experience.

Background and Objectives: The most common mutation in malignant melanoma (MM) is the single-point mutation of v-raf murine sarcoma viral oncogene homolog B1 (BRAF) oncogene. Our study aims to evaluate BRAF V600E mutation, highlighting its frequency differences in primary versus metastatic MM. Materials and Methods: The study group comprised 133 patients diagnosed with MM in several county hospitals of the north-eastern region of Romania who have been assigned for investigation into BRAF V600E mutation in the private medical system. The material consisted of archived formalin-fixed paraffin-embedded (FFPE) blocks. BRAF V600E mutation was identified using the fully automated IdyllaTM BRAF mutation test system. Results: Out of the total of 133 cases, 78 cases were primary tumors, while 55 cases were metastatic MMs. Genetic analysis revealed the presence of BRAF V600E mutation in 66 cases (49.62%) and the wild-type genotype in 67 cases (50.37%). We found a statistically significant difference of the mutation frequency according to age (p = 0.0072). The mutated genotype was found in 45 cases out of 78 primary MMs (57.69%) and in 21 cases out of 55 secondary MMs (38.18%), with a statistically significant difference in favor of primary tumors (p = 0.0413). The correlations between the histopathological types, Clark's level, Breslow index, ulceration, and lymphovascular invasion, respectively, and the mutated genotype were not statistically significant. BRAF V600E mutation was identified in 15 out of 40 secondary tumors with lymph node location (37.5%) and in 6 out of 15 secondary tumors with another location (40%) without statistically significant differences between the mutation frequency and the location of the secondary tumors. Conclusions: Our results support MM high genetic heterogeneity, pointing out the relationship between BRAF V600E mutation and several clinicopathological characteristics, in primary and metastatic MMs, stressing the importance of BRAF testing implementation in Romania.

Significance of the Galectin-8 Immunohistochemical Profile in Ovarian Cancer.

Ovarian cancer (OC) still registers a high prevalence in female gynecological pathology. Given the aggressiveness of the tumor and the lack of response to conventional therapies, a current research interest is the identification of new prognostic markers. Gal-8, a member of the galectin family of molecules, involved in tumorigenesis, disease progression, and metastasis, has been assigned as a valuable tumor prognostic factor, and its inhibition may open new perspectives in cancer therapeutic management. Few studies have been carried out so far to evaluate OCs' galectin profiles. Our study aimed to characterize the Gal-8 profile in different types of ovarian neoplasia and to demonstrate its prognostic value. Our study group comprised 46 cases of OCs that were histologically and immunohistochemically investigated, introduced to Gal-8 immunoreactivity, qualitatively and semi-quantitatively evaluated, and correlated with clinicopathological characteristics. Gal-8 immunoexpression was identified in tumor epithelial cells, showing a dominant nuclear labeling, followed by cytoplasmic and mixed, nuclear, and cytoplasmic labeling. Significant differences between tumor histotypes were found in the statistical analysis between low and high Gal-8 immunoscore levels and clinicopathological features: HGSC (eng.= high-grade serous carcinoma) vs. LGSC (eng. = low-grade serous carcinoma), pathogenic types (type I vs. type II), and tumor grades. Our results reflect Gal-8 expression variability depending on the histological type and subtype, the progression stages, and the degree of differentiation of ovarian tumors, supporting its value as a prognostic factor. Our findings open perspectives for larger studies to validate our results, along with a potential Gal-8 transformation into a future therapeutic target.

Novel titanium-apatite hybrid scaffolds with spongy bone-like micro architecture intended for spinal application: In vitro and in vivo study.

Titanium alloy scaffolds with novel interconnected and non-periodic porous bone-like micro architecture were 3D-printed and filled with hydroxyapatite bioactive matrix. These novel metallic-ceramic hybrid scaffolds were tested in vitro by direct-contact osteoblast cell cultures for cell adhesion, proliferation, morphology and gene expression of several key osteogenic markers. The scaffolds were also evaluated in vivo by implanting them on transverse and spinous processes of sheep's vertebras and subsequent histology study. The in vitro results showed that: (a) cell adhesion, proliferation and viability were not negatively affected with time by compositional factors (quantitative MTT-assay); (b) the osteoblastic cells were able to adhere and to attain normal morphology (fluorescence microscopy); (c) the studied samples had the ability to promote and sustain the osteogenic differentiation, matrix maturation and mineralization in vitro (real-time quantitative PCR and mineralized matrix production staining). Additionally, the in vivo results showed that the hybrid scaffolds had greater infiltration, with fully mineralized bone after 6 months, than the titanium scaffolds without bioactive matrix. In conclusion, these novel hybrid scaffolds could be an alternative to the actual spinal fusion devices, due to their proved osteogenic performance (i.e. osteoinductive and osteoconductive behaviour), if further dimensional and biomechanical optimization is performed.

Insights into molecular pathways of endometriosis and endometriosis-related ovarian carcinoma.

BACKGROUND: Endometriosis is a benign estrogen-dependent gynecological disease involving components of the female genital tract (uterus, Fallopian tubes, ovaries, large, round, and utero-sacral ligaments) and intra- and extraperitoneal regions. Since the moment of its etiopathogeny has been identified, the intrinsic capacity of endometriosis malignant transformation has been hypothesized. PATIENTS, MATERIALS AND METHODS: Our study included a total number of 50 patients diagnosed with endometriosis (31 cases) and endometriosis-related ovarian carcinoma (EOC) (19 cases). A clinicopathological and immunohistochemical study directed towards the detection of atypical transition lesions and the similitudes in epithelial-mesenchymal transition (EMT) phenomenon [E-cadherin∕ß-catenin∕cytokeratin 18 (CK18)], apoptosis [B-cell lymphoma 2 (Bcl-2)∕Bcl-2-associated X (Bax)], and hormonal dynamics mirrored by the immunoexpression of estrogen receptor (ER) and progesterone receptor (PR) in endometriosis and EOC glands and stroma has been performed. RESULTS: Our study showed a higher immunoexpression of CK18 and E-cadherin in endometriosis than in neoplastic counterparts, while ß-catenin had a stronger immunoexpression in tumors compared with endometriotic areas, with statistically significant differences between the studied groups. Bcl-2∕Bax higher rate in endometriosis had a statistically significant association to a more aggressive tumor behavior (p=0.020). ER immunoexpression was stronger in endometriosis, with less negative scores compared to EOC, while PR immunoexpression was stronger in endometriosis, with a lower percent of negative scores compared to EOC. PR immunostaining was correlated to ovarian location of endometriosis (p=0.004) and tumor grade of EOC (p=0.027). Stromal ER and PR immunoexpression has been significantly lower in endometriosis in comparison to tumor stroma (p=0.001) and PR stromal immunoexpression had been higher in more differentiated tumors compared to less differentiated types (p=0.005). CONCLUSIONS: Our study supports that endometriosis is a precursor of EOC by the identification and the coexistence of both lesions in the investigated cases, the identification of intermediate lesions, as well as the expression of EMT immunomarkers, along with apoptosis and steroid receptors immunoexpression.

The endometrial regeneration frontiers: from mechanisms to applications in regenerative medicine.

The endometrium is a unique and remarkable tissue characterized by a constant regeneration activity and this has been speculative for scientists, regarding its mechanism, regulatory factors, and their significance for fertility and endometrial pathology. Relatively recent scientific progresses due to genomics, proteomics, and transcriptomics have changed the knowledge in respect with endometrial regeneration and uterine-derived diseases. Our review is designed to highlight the recent progresses in understanding the endometrial physiology and its alterations involvement in uterine-derived diseases, addressing the current paradigm regarding endometrial regeneration, based on endometrial regenerative cells. In an attempt to explain the complex process of endometrial regeneration, different mechanisms have been proposed during time, from proliferation of basal glandular cells, to mesenchymal-epithelial transition, and lately to differentiation of stromal cells, based on endometrial regenerative cells or stem cells. Their unlimited potential of reconstruction of any type of tissue has been demonstrated and is currently in different trial stages in cell-based therapies of regenerative medicine, opening promising perspectives in severe or lethal diseases, by exploitation of stem cells. Currently, beside uterine acquired diseases and infertility, endometrial stem cells have been tested in a large spectrum of clinical applications. The great potential of endometrial cells for cell therapies arise from their accessibility, completely xeno-free derivation, allogenic use, possibility of large-scale therapeutic doses production, safety, reproducibility, and chance to overcome the drawbacks associated with autologous therapies. In order to overcome hostile environment of an injured tissue, the association of endometrial stem cells with other cells or added medium opens the perspective of specific combination available as standardized therapeutic means in the next future.

Adipocytes spectrum - From homeostasia to obesity and its associated pathology.

Firstly identified by anatomists, the fat tissue is nowadays an area of intense research due to increased global prevalence of obesity and its associated diseases. Histologically, there are four types of fat tissue cells which are currently recognized (white, brown, beige, and perivascular adipocytes). Therefore, in this study we are reviewing the most recent data regarding the origin, structure, and molecular mechanisms involved in the development of adipocytes. White adipocytes can store triglycerides as a consequence of lipogenesis, under the regulation of growth hormone or leptin and adiponectin, and release fatty acids resulted from lipolysis, under the regulation of the sympathetic nervous system, glucocorticoids, TNF-α, insulin, and natriuretic peptides. Brown adipocytes possess a mitochondrial transmembrane protein thermogenin or UCP1 which allows heat generation. Recently, thermogenic, UCP positive adipocytes have been identified in the subcutaneous white adipose tissue and have been named beige adipocytes. The nature of these cells is still controversial, as current theories are suggesting their origin either by transdifferentiation of white adipocytes, or by differentiation from an own precursor cell. Perivascular adipocytes surround most of the arteries, exhibiting a supportive role and being involved in the maintenance of intravascular temperature. Thoracic perivascular adipocytes resemble brown adipocytes, while abdominal ones are more similar to white adipocytes and, consequently, are involved in obesity-induced inflammatory reactions. The factors involved in the regulation of adipose stem cells differentiation may represent potential pathways to inhibit or to divert adipogenesis. Several molecules, such as pro-adipogenic factors (FGF21, BMP7, BMP8b, and Cox-2), cell surface proteins or receptors (Asc-1, PAT2, P2RX5), and hypothalamic receptors (MC4R) have been identified as the most promising targets for the development of future therapies. Further investigations are necessary to complete the knowledge about adipose tissue and the development of a new generation of therapeutic tools based on molecular targets.

Clinicopathological significance and prognostic value of myoinvasive patterns in endometrial endometrioid carcinoma.

Endometrioid endometrial carcinoma has an overall good prognosis. However, variable five-year survival rates (92%-42%) have been reported in FIGO stage I, suggesting the involvement of other factors related to tumor biological behavior. These may be related to the role played by epithelial-mesenchymal transition (EMT) and cancer stem cells in endometrial carcinogenesis. In this context, our review highlights the prognostic significance of several types of myoinvasion in low grade, low stage endometrioid endometrial carcinoma, as a reflection of these molecular changes at the invasive front. According to recently introduced myoinvasive patterns, the diffusely infiltrating and microcystic, elongated, and fragmented (MELF) patterns show loss of hormone receptors, along with EMT and high expression of cancer stem cell markers, being associated with a poor prognosis. Additionally, MELF pattern exhibits a high incidence of lymphovascular invasion and lymph node metastases. Conversely, the broad front pattern has a good prognosis and a low expression of EMT and stem cells markers. Similarly, the adenomyosis (AM)-like and adenoma malignum patterns of invasion are associated to a favorable prognosis, but nevertheless, they raise diagnostic challenges. AM-like pattern must be differentiated from carcinoma invasion of AM foci, while adenoma malignum pattern creates difficulties in appreciating the depth of myoinvasion and requires differential diagnosis with other conditions. Another pattern expecting its validation and prognostic significance value is the nodular fasciitis-like stroma and large cystic growth pattern. In practice, the knowledge of these patterns of myoinvasion may be valuable for the correct assessment of stage, may improve prognosis evaluation and may help identify molecules for future targeted therapies.

Single nucleotide polymorphisms of XRCC3 gene in hepatocellular carcinoma - relationship with clinicopathological features.

Recent studies support the involvement of XRCC3 gene polymorphisms in carcinogenesis. Our study focuses on the identification of polymorphic variants of XRCC3 in hepatocellular carcinoma (HCC) and an analysis of the relationship between these polymorphic variants and clinicopathological (including the genotype specific risk) and survival characteristics. Fifty cases of HCC were genotyped using molecular biology techniques for Thr241Met, rs861539 (c.722C>T) and 5'-UTR, rs1799796 (c.562-14A>G) polymorphisms. Statistical analysis was based on 2, Fisher's, logistic regression (odd ratio - OR), and log-rank tests. Statistically significant differences were shown only for rs1799796 A>G and tumour grade, between wild type (AA) and heterozygote (AG) genotypes, and wild type (AA) and heterozygote & homozygote (AG & GG) genotypes. The logistic regression analysis found an OR of rs1799796 polymorphism occurrence in HCC related to tumour grade. The statistical analysis revealed, for the rs861539 C>T polymorphism, a better survival only for the homozygote genotype (TT) compared to the heterozygote (CT), and for rs1799796 A>G polymorphism, a longer survival for the wild type (AA) compared to heterozygote (AG) and to heterozygote & homozygote (AG & GG) genotypes, respectively. Our results suggest that XRCC3 gene SNPs could influence the tumour aggressiveness expressed by tumour grade.

Heterogeneous Periostin Expression in Different Histological Variants of Papillary Thyroid Carcinoma.

BACKGROUND: Periostin (PN) epithelial and stromal overexpression in tumor pathology has been studied according to tumor growth, angiogenesis, invasiveness, and metastasis, but a limited number of studies address PN in thyroid tumors. AIM: Our study aimed to analyze PN expression in different histological variants of PTC and to correlate its expression with the clinicopathological prognostic factors. MATERIAL AND METHODS: PN expression has been immunohistochemically assessed in 50 cases of PTC (conventional, follicular, oncocytic, macrofollicular, and tall cell variants), in tumor epithelial cells and intratumoral stroma. The association between PN expression and clinicopathological characteristics has been evaluated. RESULTS: Our results show that PTC presented different patterns of PN immunoreaction, stromal PN being significantly associated with advanced tumor stage and extrathyroidal extension. No correlations were found between PN overexpression in tumor epithelial cells and clinicopathological features, except for specific histological variants, the highest risk of poor outcome being registered for the conventional subtype in comparison to the oncocytic type. CONCLUSIONS: Our study demonstrates differences in PN expression in histological subtypes of PTC. Our results plead in favor of a dominant protumorigenic role of stromal PN, while the action of epithelial PN is less noticeable.

Pointing at Ito cell, from structure to function (⃛ or Cinderella story in liver histology).

For more than 130 years, the hepatic stellate cells (also known as Ito cells) have been the object of numerous studies that aimed at their characterization both in normal and postlesional status, where they play a key role in fibrosis progression specific for chronic hepatic pathology. Converged studies on their histophysiology have revealed other functions, namely the involvement in liver embryological development and regeneration, metabolisms regulation, modulation of local inflammatory and immune reactions. Ito cells plasticity is surprising, as they are able to provide the connection between the complex sinusoidal and parenchymal microenvironment, influencing by autocrine and paracrine mechanisms the extracellular matrix content in tight correlation to growth and repair necessities. Last but not least, Ito cells take an active part in systemic homeostasis maintenance by their capacity to store and mobilize vitamin A, respectively. The evolving interest in their research will be undoubtedly followed by a better knowledge of the physiopathological sequences responsible for liver diseases, as new targets for the development of new therapeutic approaches directed toward improvement of prognosis and patients' quality of life.

Prognostic significance of MMP-9 and TIMP-1 in liver metastases.

Our research focuses both on the correlations between MMP-9 and TIMP-1 and classical clinicopathological factors and on the prognostic value of MMP-9 and TIMP-1 for survival. The study group included 52 patients diagnosed with hepatic metastases. The tissue specimens have been specifically processed for immunohistochemical exam, by using anti-MMP-9 and anti-TIMP-1 antibodies. For the semi-quantitative assessment, we have used an individualized score, which values allowed the discrimination of two classes of cases (low and high), using two different thresholds: ≤4 and <4. Data have been statistically analyzed by using Fisher 2×2 test and Kaplan-Meier curves. Statistical analysis between MMP-9 and TIMP-1 expression (low versus high, separately for each threshold) and clinicopathological characteristics had not revealed significant differences. In both types of threshold applied in survival analysis, significant differences between MMP-9 and TIMP-1 low and high expression have been demonstrated. For cases with concordant MMP-9-TIMP1 co-expression, low versus high, the survival analysis revealed that threshold value <4 offers a better stratification of cases when compared to threshold value ≤4, based on significant differences registered only for threshold value <4. No significant differences were registered between cases with discordant MMP-9-TIMP-1 co-expression, for both thresholds. Regardless of the used threshold, the survival analysis achieved between the cases with MMP-9-TIMP-1 concordant co-expression and cases with MMP-9-TIMP-1 discordant co-expression had proven significant differences. Our study suggests that the confirmation of MMP-9 and TIMP-1 value as prognostic factors, based on immunohistochemical expression, requires a threshold validation.

Morphological changes in membranoproliferative glomerulonephritis - a quantitative approach.

Our study aimed to investigate the quantitative profile of the renal corpuscle components in membranoproliferative glomerulinephritis (MPGN). We have analyzed digital color images corresponding to relevant microscopic fields from renal biopsies (10 cases type I MPGN and 10 cases type II MPGN). A computerized morphometric algorithm was designed and applied in both red-green-blue (RGB) and hue-lightness-saturation (HLS) color spaces, allowing the automated measurement of areas for the following morphological characteristics of the renal corpuscles (RCs): glomerulus, Bowman space, cells, mesangial matrix and glomerular basement membranes, and capillaries. Student's t-test comparatively applied between the numerical data obtained for the measured morphological characteristics, for each individual color space, showed significant differences between type I MPGN and type II MPGN for Bowman space area (p=0.006) and for mesangial matrix and glomerular basement membranes area - exclusively in RGB color space (p=0.013). We have also demonstrated larger RCs and glomerular size in type II MPGN, comparative to those in type I MPGN. Consequently, we assume that the morphometrical characterization of RCs histological components could be used as an additional criterion not only in the diagnosis of MPGNs, but also in the stratification of evolution and prognosis of patients diagnosed with type I and II MNGN, respectively.

Macrophage profile in primary versus secondary liver tumors.

Macrophages are important components of the tumor-associated infiltrate and are qualified as one of the major players of the cancer-related inflammation. It was shown that tumor cells can either stimulate or mediate apoptosis of tumor-associated macrophages (TAMs). To date, there is no general agreement regarding the influence of TAMs and their numbers on the progression of hepatocellular carcinoma (HCC) and hepatic metastases (HM). To analyze the presence of TAMs and compare their numbers in intratumoral (IT) and peritumoral (PT) areas with the clinical outcome of HCC and HM patients. Biopsies from 35 HCC and 39 HM cases were analyzed. Clinical and follow-up data was enrolled for each case; the colorectal cancer was the origin of 26 HM patients. TAMs were identified by immunohistochemistry using anti-CD68 monoclonal antibody. The quantitative assessment was performed by determining the mean number of CD68-positive cells in IT and PT areas in HCC and HM. Two threshold methods were applied: threshold 1 (T1) was calculated with the use of (-log) Cox method; threshold 2 (T2) was considered as 1/3 TAMs number of group's mean. For statistical analyses Mann-Whitney U-test, Spearman's correlation, Cox proportional hazard and Kaplan-Meier tests were applied. To date, 36.12% HCC and 27.78% HM patients were alive, median survival was 5 and 17 months for HCC and HM, respectively (P = 0.05). We found significant two-fold decrease of TAMs numbers between IT vs. PT territories in both HCC and HM. A positive correlation between numbers of PT and IT TAMs was observed in HM group (rs = 0.48, P < 0.05) but not in HCC. The number of TAMs was not associated with any studied clinical factor. Univariate Cox regression analysis showed that tumor stage ≤ II (P = 0.01) and increased number of PT TAMs (P = 0.06, only when T2 value was applied) were associated with favorable prognosis in HCC (HR = 2.614 and 2.457, respectively). Univariate and multivariate Cox analyses in HM revealed favorable prognosis for histological grade ≤ G2 and one lobe tumors (P = 0.021 and 0.045; HR = 0.395 and 0.438, respectively). Survival analysis retained the impact of increased TAMs numbers in peritumoral areas (P = 0.03), tumor stages in HCC (P = 0.007), lobes' number (P = 0.007) and histological grade (P = 0.005) on HM patients' outcome. In HCC and HM the low number of TAMs in intratumoral areas was related to the tumor cell microenvironment. The increased peritumoral TAMs number in primary liver tumors was associated with better prognosis.

Hepatic steatosis background in chronic hepatitis B and C - significance of similarities and differences.

The aim of our study was to investigate comparatively the steatotic background in viral chronic hepatitis B, C and mixed types, in correlation with the severity degree of specific liver lesions. The study group consisted of 1206 liver biopsy specimens, etiologically diagnosed as hepatitis C - 1021 (84.66%) cases, hepatitis B - 100 (8.29%) cases, hepatitis B and C - 39 (3.23%) cases, hepatitis B and D - 39 (3.23%) cases, hepatitis C and toxicity - six (0.49%) cases, hepatitis B, C and D - one case (0.08%). The histopathological assessment focused on the steatotic lesions associated with inflammation and fibrosis. The cases were classified according to necrosis and inflammatory activity (score between 0-12) and fibrosis (score between 0-4). Our data indicates significant association of steatotic lesions in hepatitis C (76.59%) as opposed to other types of viral hepatitis. In mixed hepatitis B and C, steatotic lesions are more frequent (66.66%) than in chronic hepatitis B (47%) and in mixed chronic B and D hepatitis (48.72%). Steatosis was present in all cases with chronic hepatitis C and associated toxicity. These observations confirm the important aggressiveness of hepatitis C virus as opposed to hepatitis B and D virus. The analysis of the pattern of steatosis in correlation with necrosis and inflammatory activity and fibrosis, respectively, lead to the identification of certain specific elements. Thus, for all types of hepatitis, steatosis is associated predominantly with moderate severity (score 6-8) and progressive expansion of fibrosis (score 2-3). The presence of steatosis does not define hepatic lesions with severe inflammation (score 9-12) nor those with extended fibrosis (score 4). The type of steatosis present is mostly macrovesicular, the transformation into lipid cysts being uncommon. Based on the scoring systems applied in the evaluation of the entire investigated study group, we believe that a possible inclusion of a quantifiable criterion for steatosis could be beneficial in order to complete the characterization of the severity of the lesions, from the point of view of the potential for future evolution, reversible or irreversible.

Conventional cytology versus liquid based cytology in cervical pathology: correspondences and inconsistencies in diagnosis, advantages and limits.

Liquid Based Cytology (LBC) has replaced Papanicolaou standard cytology due to its practical advantages. Our study aimed to analyze the diagnosis correlations and differences between the conventional and liquid based cytology. The study group has been composed by 104 patients, diagnosed in the Laboratory of Cytology, Galati County Hospital, Romania by using both methods on the same patients group. Our study revealed a good correlation between the results. Thus, in 78 from the total of 104 (75%) analyzed cases, the diagnoses established by conventional smears have been identical to that obtained in LBC. The diagnoses have been different in 26 (25%) cases, the inherent errors of conventional Pap diagnosis being corrected by LBC. LBC resulted in the diagnosis of some entities missed in conventional cytology, namely: ASC-US (atypical squamous cells of undetermined significance) (one case), ASC-US associated to AGC-NOS (atypical glandular cells not otherwise specified) (one case), and HSIL (high-grade squamous intraepithelial lesion) associated to AIS (adenocarcinoma in situ) (one case). LBC provided the identification of a higher number of cases of associated lesions, as LSIL (low-grade squamous intraepithelial lesion) and AGC-NOS (five versus two cases), and HSIL and AGC-NOS (seven versus five cases). The national experience is mainly based on conventional cytology usage, after Papanicolaou staining method. Extremely rare centers have benefited by the necessary infrastructure for LBC, thus there are no constant reports in the mainstream. The main impediments in large-scale application of this method, in all national screening centers are connected to costs for capital investments and by conditions of exploitation. Our experience and results support the simultaneous use of the two methods.