ROMANIAN JOURNAL of MORPHOLOGY and EMBRYOLOGY

Vol. 50 No. 3, 2009

1. Gastrointestinal stromal tumors: review on morphology, diagnosis and management

Emoke Fulop, Simona Marcu, Doina Milutin, Angela Borda

Gastrointestinal stromal tumors (GISTs) are the most frequent mesenchymal tumors of the gastrointestinal tract. Major advances in their definition and classification and the understanding of their molecular mechanisms have recently been made. These advances have become a model of targeted therapy in oncology. The diagnosis of GISTs relies on histological arguments - proliferation of spindle-shaped cells in 70% of cases, of epithelioid cells in 20%, histological variants are rare -, and on immunohistochemical arguments - expression of CD117 in 95%, usually associated with CD34 expression in 70% of cases. Most GISTs are associated with molecular abnormalities in low target genes: KIT and PDGFRA. The differential diagnosis of GISTs includes the other mesenchymal tumors of the gastrointestinal tract, such as leiomyomas, leiomyosarcomas, schwannomas and intra-abdominal fibromatosis. The evaluation of the prognosis is essential and is based on a simple algorithm using two histoprognostic parameters, tumor size and mitotic index. The treatment of localized GISTs is surgical resection and that of advanced or unresecable GISTs is based on the use of targeted therapy, imatinib, which is a pharmacological antagonist of the c-kit protein. Proper understanding and utilization of the diagnostic criteria and classification of GISTs by pathologists are essential for good patient management.

Corresponding author: Emoke Fulop, MD, PhD candidate, e-mail: emifulop@yahoo.com

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2. Histopathological changes in acute ischemic stroke

Otilia Margaritescu, L. Mogoanta, Ionica Pirici, D. Pirici, Daniela Cernea, Cl. Margaritescu

We study here the histopathological changes in twenty-two cases of acute ischemic stroke. The average age of the patients was 62-year-old, and the interval from the onset of the disease to the death varied from 6 hours to 15 years. The brain lesions after acute stroke were observed in all regions. Their evolution allowed us to classify them in fourth stages. Phase one changes (1-2 days after onset) (n=2 patients) included red hypoxic and "ghost" neurons and other acute neuronal injury and spongiosis. The second phase (n=14 patients) was subdivided into: (a) a phase of acute inflammation (3-37 days after onset) (n=5 patients), where we observed especially features of acute inflammation together with coagulative necrosis, and (b) phase of chronic inflammation (10 days-53 years after the onset) (n=9 patients), in which prevail mononuclear and macrophage infiltrate, astrogliosis, spongiosis and neo-vascularization. In the third phase (26 days-23 years after the onset), we included six cases characterized by the absence of an inflammatory reaction, presence of cavitation, astrogliosis and macrophages. Our study describes the heterogeneity of brain injury after acute ischemic stroke with the participation of all brain components, and the chronology in which these lesions develop and evolve.

Corresponding author: Claudiu Margaritescu, MD, PhD, e-mail: c_margaritescu2000@yahoo.com

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3. Analysis of the immunohistochemical expression of mammaglobin A in primary breast carcinoma and lymph node metastasis

M. Raica, Anca Maria Cimpean, Adriana Meche, Aurora Alexa, C. Suciu, Anca Muresan

Mammaglobin A is a specific marker of the normal and neoplastic mammary tissue that usually is detected by RT-PCR. Few data are available about the immunohistochemical expression of this marker in mammary carcinoma and about the significance of the positive reaction. Our purpose was to investigate the sensitivity of the mammaglobin expression in breast cancer and to determine its correlations with conventional prognostic parameters. There were investigated 47 patients with breast carcinoma, and slides from paraffin blocks were stained with an antibody against mammaglobin. The immunohistochemical reaction was scored based on the percentage of positive tumor cells in both primary tumors and lymph node metastasis. Positive reaction for mammaglobin was found in the normal mammary tissue adjacent to the tumor in all cases, in 78.72% primary breast carcinoma, and in 58.06% of cases with lymph node metastases. A significant correlation was found between the mammaglobin expression in the primary tumor, grade, and lymph node status, but not with the age of the patient, pathologic subtype of carcinoma and stage of the tumor. The ductal in situ carcinoma associated to the invasive tumor did not influence significantly the prognostic value of mammaglobin expression. Out results suggest that mammaglobin is a sensitive marker of breast carcinoma, it defines a subgroup of patients with better prognosis and is a useful method to detect breast cancer metastases.

Corresponding author: Marius Raica, MD, PhD, e-mail: raica@umft.ro

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4. Effects of 60Co gamma-rays on human osteoprogenitor cells

C. I. Tomuleasa, V. Foris, Olga Soritau, Emoke Pall, Eva Fischer-Fodor, V. Lung-Illes, Ioana Brie, Piroska Virag, Maria Perde-Schrepler, I. D. Postescu, Gabriela Chereches, Otilia Barbos, Corina Tatomir

Background: Radiation therapy is one of the most efficient treatments of neoplastic diseases used worldwide. However, patients who undergo radiotherapy may develop side effects that can be life threatening because tissue complications caused by radiation-induced stem cell depletion may result in structural and functional alterations of the surrounding matrix. This treatment also damages the osteogenic activity of human bone marrow by suppressing osteoblasts, leading to post-irradiation sequelae. Even if widely used in oncology, there is still little information on the fate and potential therapeutic efficacy of electromagnetic rays. Material and Methods: We addressed this question using both human mesenchymal stem cells and osteoblasts. Monoclonal antibody characterization identified specific surface markers for stem cells (SSEA-4, CD29, CD105, Oct 3, Nanog and SOX2) and osteoblasts (Osteopontin and Osteonectin). The technique of anti-alkaline phosphatase FITC-staining demonstrated the presence of this specific ectoenzyme. Cells were cultured in complex osteogenic medium (DMEM, 15% fetal calf serum, non-essential amino acids, L-glutamine, dexametazone, ascorbic acid, insulin, TGF-beta, BMP-2 and beta-glycero-phosphate) after being irradiated at 0.5 Gy, 1 Gy, 2 Gy and 4 Gy using a Theratron 1000 60Co source. The viability of irradiated cells was assessed using Trypan Blue staining. The comparison between cell lineages after culture in osteogenic media regarding phenotypical characterization and the intensity of the mineralization process included histology stainings (Alizarin Red S, Alcian Blue and von Kossa), and the MTT-based proliferation assay. Results: After irradiation, the proliferation and differentiation of osteoprogenitor cells is dose-dependent. Conclusions: This study is one among the first papers investigating the biophysics of low-dose gamma-irradiation on stem cell culture, focusing on the potential applications in radiation oncology and various palliative treatments.

Corresponding author: Olga Soritau, MD, e-mail: osoritau@hotmail.com

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5. P53, p63 and Ki-67 assessment in HPV-induced cervical neoplasia

Florina Vasilescu, M. Ceausu, Cristina Tanase, Ruxandra Stanculescu, Teodora Vladescu, Zenaida Ceausu

Carcinoma of the uterine cervix is the most frequent malignancy in women, with an incidence of approximately 456.000 cases per year, leading to 200.000 deaths per year. Twenty-six archived formalin-fixed paraffin-embedded samples of squamous cell carcinoma, selected from 30 Papanicolaou-positive smears, have been analyzed using standard HE stain and the IHC indirect tristadial ABC peroxidase method for four antibodies: p53, p63, Ki-67, HPV. Statistical analysis has been done using the Student t-test, one-group two tails, "paired two samples for mean" variant. Two thirds of the cases were medium and poor differentiated carcinomas. The expression pattern of the proliferation and prognostic factors was biologically correlated with the histopathological type and HPV-infection. Two statistically significant correlations were found between p63 and Ki-67 and between p63 and p53 (p<0.001). The significant increase of the expression of the analyzed immunomarkers was observed in most of the cases with late stage of cervical neoplasm. P63, followed by Ki-67, showed better correlation with cancer progression than p53. This observation could be used in clinical practice with the purpose of identifying those patients requiring more aggressive treatment.

Corresponding author: Mihai Ceausu, MD, PhD, e-mail: ceausu_mihai@yahoo.com

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6. Immunohistochemical and transmission electron microscopy study regarding myofibroblasts in fibroinflammatory epulis and giant cell peripheral granuloma

Ana Maria Filioreanu, Eugenia Popescu, C. Cotrutz, Carmen Elena Cotrutz

Fibroblasts represent the main cellular population in the connective tissue; they have a central role in extracellular matrix (ECM) synthesis, degradation and remodeling. These cells may express a substantial heterogeneity regarding their morphology and functions in pathological conditions and during tissue remodeling. Myofibroblasts are a good example for heterogeneity and phenotypical changes. These cells can be morphologically and immunologically defined by the expression of specific cytoskeleton proteins. Myofibroblasts show cytoplasmic actin microfilaments organized as stress fibers and interconnected by gap or adherens junctions. These cells come also in contact with extracellular matrix by focal contacts. Myofibroblasts play fundamental roles in pathologic conditions, even by activation and proliferation or by deletion. Moreover, these cells seem to be involved in formation and repair of the ECM compounds, proliferation and differentiation of the epithelial, vascular or neurogenic elements. The purpose of the present study is to emphasize the presence and distribution of myofibroblasts in the reactive stromal tissue of granulation tumors in the oral area, fibroinflammatory epulis and giant cells peripheral granuloma, by means of immunocytochemical and transmission electron microscopy studies. Both tumor types shown a common characteristic of the presence of reactive inflammatory stromal tissue and myofibroblasts are a common issue.

Corresponding author: Ana Maria Filioreanu, MD, PhD, e-mail: afilioreanu@yahoo.com

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7. Gastric cancer: correlation between clinicopathological factors and survival of patients (III)

Daniela Lazar, Sorina Taban, I. Sporea, Alis Dema, Marioara Cornianu, Elena Lazar, A. Goldis, C. Vernic

Purpose: The purpose of this study was to identify the clinicopathological factors that influence the prognosis of patients having undergone surgery for gastric cancer. The analysis of the potential prognosis factors has included in this study parameters concerning the patient (sex, age), as well as parameters related with the tumor (histological type according to the WHO classification; degree of tumor differentiation; tumor location; stage of disease; pT and pM parameters according to the TNM classification of AJCC/UICC). Material and Methods: From the total number of 265 patients (186 men and 79 women) diagnosed with gastric cancer in the period 1998-2002, 61 operated patients were selected. On this group, we performed a prospective study regarding the evolution and aggressiveness of gastric cancer, on a duration of 5 years. Survival time was calculated from the month of the surgical intervention until the month of death or confirmation of survival, and survival rate was represented by the percentage of survivals at the end of the observed interval (in years and months). Results: The studied group consisted of 61 patients (43 men and 18 women), with ages between 30 and 80 (average age = 59.34 years). According to the maximum level of tumor invasion we identified: pTis - one case (1.6%); pT1 - four cases (6.6%); pT2 - nine cases (14.7%); pT3 - 17 cases (27.9%); most gastric cancers were included in the pT4 category (49.2%). We identified 43 gastric carcinomas with metastases in regional lymph nodes (70.5% of the cases). According to the number of lymph nodes involved: pN0 - 18 cases (29.5%); pN1 - 16 cases (26.2%); pN2 - 23 cases (37.7%); pN3 - four cases (6.6%). Distance synchronous metastases were present in 14 gastric carcinomas (pM1 - 23% of the cases), nine cases with hepatic metastases and five cases with distance peritoneal disseminations. Classification of gastric carcinomas: we identified one single case listed in the 0 stage, three cases in the IA stage, five cases in the IB stage, seven cases in the II stage, 11 cases in the IIIA stage, eight cases in the IIIB stage, and 26 cases in the IV stage. From the total of carcinomas, stage IV consisted of the greatest number of tumors, representing 42.6%. We generally remarked the increase of the number of cases in advanced stages. Conclusions: In the studied group, we noted a great number of gastric carcinomas diagnosed in stages T3 and T4 (77.1% of cases). Cardial tumor locations and gastric stump locations, as well as "signet-ring" cell and undifferentiated carcinomas prove to be aggressive, being diagnosed in advanced stages. 70.5% of gastric neoplasms presented lymph node metastases, most cases representing pN2 tumors (37.7%). 23% of neoplasms studied presented distance metastases at the time of diagnosis. We noted a significant correlation between the degree of tumor differentiation and the level of invasion, as well as the presence of distance and lymph node metastases. Survival at 5 years of patients included in the study was correlated significantly with the level of tumor invasion, the presence of lymph node and distance metastases, and the TNM stage.

Corresponding author: Daniela Lazar, MD, PhD, e-mail: lazar_daniela@yahoo.com

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8. Fractal analysis of astrocytes in stroke and dementia

D. Pirici, L. Mogoanta, Otilia Margaritescu, Ionica Pirici, Valerica Tudorica, Marieta Coconu

The varied morphological forms in which astrocytes occur in brain of ischemic/hemorrhagic stroke and Alzheimer's disease (AD) patients are complex and the mechanisms that drive their formation are not yet properly understood. Subjective differences can be described between these pathologies in what it concerns astrocyte implication, but these have not been yet subjected to a morphometrical quantification. Here we apply a fractal dimension (FD) analysis algorithm to differentiate both between fibrous, protoplasmatic and activated astroglia; but also between the three pathological conditions studied. Analyzing more than 1000 astroglia, we show here first that FD can clearly differentiate between the three morphological subtypes. Second, we describe resemblances of the FD values for ischemic and hemorrhagic lesions, and significant differences when these are compared to AD patients. These results are further discussed and integrated in what it regards the preferential regions proved to be affected in these conditions, and which parallels our results. This work illustrates that fractal dimension analysis of astroglia is a useful method for quantitatively describing gliosis in different pathologies, and that it may offer more insight into the pathogenesis of brain diseases.

Corresponding author: Daniel Pirici, MD, PhD, e-mail: daniel.pirici@yahoo.com

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9. Alveolar bone dehiscences and fenestrations: an anatomical study and review

Vanda Roxana Nimigean, V. Nimigean, Maria Angelica Bencze, Nicoleta Dimcevici-Poesina, R. Cergan, Simona Moraru

Aim: To determine the prevalence and distribution of fenestrations and dehiscences of the jaw bones among the Caucasian population, to find if any correlations can be established between their occurrence and certain teeth characteristics and to discuss the clinical implications the defects of alveolar process could have. Material and Methods: 138 skulls of specimens ranging from 21 to 54 years of age, having either complete dentition or reduced number of missing teeth were studied. Teeth found to have one of the two defects were examined for signs of faceting (attrition) that was considered an indicative for excessive occlusal forces and were submitted (except for the case of the third molars) to an analysis concerning their bucco-lingual inclination in the jaw. Results: High-prevalence rates for both osseous entities were found. Fenestrations were present in 69.565% of the skulls and dehiscences were present in 53.623% of the skulls. More fenestrations were found in the maxilla: 74.679% and more dehiscences were found in the mandible: 71.613%. No correlations could be established between the presence of dehiscences and fenestrations and the development of high-occlusal forces, whereas all teeth affected either by dehiscences or by fenestrations were found to have modifications of the normal bucco-lingual inclination angle's values (p<0.05). Discussion: The interest regarding the correlation between the alveolar processes morphology and the teeth dates back to 1963. The published studies are somewhat consistent with regard to prevalence and distribution of dehiscences and fenestrations, while opinions concerning their etiology are heterogenous. According to our study and the data provided by the specialized literature references, dehiscences and fenestrations are common findings related to the presence of the teeth. Conclusions: The potential of developing fenestrations and dehiscences must be carefully evaluated through oral surgery procedures. With regard to implant placement, this study aims to help the clinician design and manage treatment, in order to clinically correct the conditions and identify the principles of bone augmentation, so that endo-osseous implants can be properly placed.

Corresponding author: Vanda Roxana Nimigean, DMD, DDS, PhD, e-mail: vandanimigean@yahoo.com

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10. Diagnostic and differential diagnostic criteria of lymphoid neoplasms in bone marrow trephine biopsies: a study of 87 cases

Emoke Horvath, T. Mezei, Z. Pavai, M. Turcu, Smaranda Demian, Erika Toth, Liliana Chira, I. Jung

The aim of this study is to present the diagnostic and differential diagnostic criteria of the bone marrow specimen involved by lymphomas based on the histomorphological immunophenotype features and clonality of the tumor cells, patterns of lymphoproliferation and diagnostic pitfalls. BMB material obtained from the right posterior iliac crest was represented from 87 untreated and treated patients with BM involving malignant lymphoma, stained with Hematoxylin-Eosin, Giemsa, Periodic Acid Schiff and Gomori's Silver. In order to perform immunohistochemistry examination we used a large antibody panel. B-cell clonality was determined in six cases. We found eight reactive lymphoproliferative responses and 79 lymphoid neoplasms of which 45 were diagnosed as de novo lymphoma, the rest of 34 samples being examined for staging. The predominant lymphoma was CLL (30 cases), over followed by DLBCL (18 cases). The most frequent patterns of involvement were the interstitial (29%) and mixed (15%) ones. In eight cases, we found reactive lymphoid aggregates. The B-cell clonality test showed four monoclonal, one oligoclonal and one polyclonal diseases form. Diagnosis of lymphoma versus reactive aggregate has been based on the combination of a lot of antibodies and involvement pattern. Although investigation of gene rearrangement was necessary for the establishment of the correct diagnosis in only 6.9% of cases, it should be emphasized that it is of great importance in disease monitoring.

Corresponding author: Emoke Horvath, MD, PhD, e-mail: horvath_emoke@yahoo.com

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11. Role of oxidative stress in the pathogenesis of chronic hepatitis C (CHC)

Carmen Fierbinteanu-Braticevici, Maria Mohora, D. Cretoiu, Sanda Cretoiu, Ana Petrisor, R. Usvat, Daniela Adriana Ion

Oxidative stress is an important pathophysiological mechanism in chronic hepatitis with C-virus infection (CHC). Steatosis is frequently observed in CHC and seems to have a significant impact on the natural history of the disease with respect to development of fibrosis. The aim of this study was to investigate the relationship between systemic parameters of oxidative stress, insulin resistance, steatosis degree, and fibrosis in CHC. Fifty patients with chronic hepatitis C (29 men and 21 women with the average age 45 years), with or without steatosis, were tested for: oxidative stress and antioxidant status by measuring serum malondialdehyde (MDA), total blood non-proteic thiols concentration (GSH), gamma glutamyl transpeptidase (GGT) activity, lipid parameters, and liver function tests. Our results show that the prevalence of insulin resistance (IR) in chronic hepatitis with C-virus genotype 1 was 32% and the association with hepatic steatosis was in a proportion of 48%, IR is mediated by both metabolic factors as well as viral factors. Hepatic steatosis was associated with an increase of MDA, correlated with its severity, and secondary with a decrease of GSH. The activity of serum GGT was net superior, in patients with steatosis, proportional with its degree. Conclusions: In patients infected by HCV genotype 1, oxidative stress and insulin resistance contribute to steatosis, which in turn exacerbates both insulin resistance and oxidative stress and accelerates the progression of fibrosis. The induction of GGT is an adaptive response against oxidative damage elicited by lipid peroxidation and it may be critical in the progression of the disease.

Corresponding author: Carmen Fierbinteanu-Braticevici, MD, PhD, e-mail: cfierbinteanu@yahoo.com

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12. Tumor biomarkers in cervical cancer: focus on Ki-67 proliferation factor and E-cadherin expression

E. Ancuta, Codrina Ancuta, Laurette Graziella Cozma, Cristina Iordache, Ivona Anghelache-Lupascu, E. Anton, E. Carasevici, Rodica Chirieac

Despite recent advances in the immune mechanisms of cervical cancer (CC) and complex management opportunities, relapse remains still an actual issue. While predictive factors are required, current research is directed towards proliferation and tumor aggressiveness biomarkers as potential negative factors in CC. The main objectives were to assess tumor proliferation and invasiveness biomarkers (Ki-67, E-cadherin) and to identify potential correlation between biomarkers and classic prognostic factors in CC. Radical hysterectomy specimens from 61 consecutive CC were immunohistochemically investigated for Ki-67 and E-cadherin. Nuclear immunostaining for Ki-67 proliferation index was assigned scores 1 to 3, "+" meaning low (10-30%), "++" moderate (30-50%), "+++" high-proliferation rate (>50%); cell membrane E-cadherin staining was either negative or positive. Statistical analysis was performed in SPSS-13 software, p<0.05. Results: no significant correlation between Ki-67 and classical prognostic factors (p>0.05) was reported; however, in relapsed CC, Ki-67 correlates with tumor grading (r=0.386, p<0.05). Significant correlation between E-cadherin and tumor size (r=-0.280, p=0.029), relapse (r=-0.386, p=0.002) and disease free survival (r=0.374, p=0.003) were demonstrated. Indirect statistically significant moderate correlation between Ki-67 and E-cadherin (r=-0.461, p<0.00001) was shown, mainly in invasive squamous CC (r=-0.549, p=0.0001), stage IB (r=0.578, p=0.009), IIB (r=-0.585, p=0.003), relapsed CC (r=-0.525, p<0.01), HPV-infection (r=-0.504, p=0.033). Conclusions: CC aggressiveness, particularly in invasive squamous carcinoma, either 16 or 18 HPV-positive cases, FIGO stage IB and IIB, and cases with relapse, depends on two pivotal factors, tumor proliferation rate (Ki-67) and tumor invasiveness (E-cadherin).

Corresponding author: Eugen Ancuta, MD, e-mail: eugen01ro@yahoo.com

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13. BRCA1 expression in invasive breast carcinomas and clinicopathological correlations

Maria Comanescu, Carmen Florina Popescu

BRCA1-positive tumors characterize a heterogeneous group of breast cancers with a specific range of histopathological and immuno-histochemical features. We evaluated the relationship between morphological characteristics and immunohistochemical profile of 14 BRCA1-positive breast tumors from an initial study group of 40 breast tumors. Morphological parameters of all cases were studied on Hematoxylin-Eosin-stained sections and their immunophenotypic profile was assessed using antibodies against estrogen receptors (ER), progesteron receptors (PR), c-erbB2 oncoprotein (Her2/neu), BRCA1-protein, and the proliferative rate of the tumoral cells was assessed by Ki-67-proliferative index. All patients were females with an average age of 43.71-year-old. Fourteen out of the 40 tumors were BRCA1-positive. All breast carcinomas in this study were invasive. The most common histological type in our study was invasive ductal carcinoma. The majority of the tumors were high-grade G3. The examination of the lymph node found metastasis in eight cases. We found 11 cases of triple negative (ER, PR and Her2/neu), while the rest showed positivity for all these three markers. We observed a high-celullar proliferation index in all cases. The immunohistochemical study of BRCA1-protein is important to the study of the development and progression of the disease.

Corresponding author: Maria Comanescu, MD, PhD, e-mail: mariacomanescu@yahoo.com

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14. Fahr's syndrome: diagnosis issues in patients with unknown family history of disease

M. Lazar, Daniela Adriana Ion, A. Streinu-Cercel, Anca Ioana Badarau

Fahr's disease (FD) is a rare clinical neurodegenerative entity, occurring in fourth or fifth decade or elderly patients, consisting in symmetric polytopic calcifications, in one ore more of the following areas: basal ganglia, cerebral white matter, thalami, internal capsulae, cerebellum, which can lead to pyramidal, extrapyramidal, cerebellar symptoms, alteration of sensitive perception and psychiatric manifestations. The purpose of this paper is to present the FD-diagnosis with unknown family history of disease, based on calcification pattern, symptomatology and lab tests. A three years retrospective study was effectuated on 1942 patients, aged between 20 and 96-year-old, presenting neurological and psychiatric symptoms, which required differential diagnosis with FD. All the patients were evaluated by CT-scans and levels of serum calcium and alkaline phosphatase were measured in cases with cerebral calcification, in order to exclude abnormal calcium-phosphorus metabolism. Cerebral and cerebellar calcification were found in 176 cases, seven cases presenting a calcification pattern suggestive for FD and in six from the seven cases a positive diagnosis of FD was established.

Corresponding author: Mihai Lazar, MD, e-mail: mihai.i.lazar@gmail.com

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15. The genotoxicity study of resveratrol in primary gastric adenocarcinoma cell cultures

P. Mitrut, Fl. Burada, Aurelia Enescu, R. Scorei, Daniela Badea, Amelia Genunche-Dumitrescu, I. Rogoz, Madalina Manea

Gastric cancer is the second most common cause of cancer-related death in the world. Some studies indicate that polyphenolic compounds and antioxidants exert a protective action against gastric cancer. Among the polyphenolic compounds tested and proven effective against gastric cancer is resveratrol, a natural polyphenol present in red wines and various human food items. Resveratrol has been shown to suppress proliferation of a wide variety of tumor cells. We tested the genotoxic activity of resveratrol in primary cell cultures from gastric adenocarcinoma, obtained by mucosal biopsy at upper digestive endoscopy. The adenocarcinoma cells were analyzed for the presence of micronuclei at different concentrations of resveratrol at 48 hours and at 72 hours. The results showed that resveratrol induced micronuclei dose-dependently. The frequency of micronuclei increased progressively with the dose of resveratrol used, the high frequency is in the primary culture initiated from gastric adenocarcinoma: signet ring cell type. The high frequency of micronuclei is at 72 hours at the 20 micro-g/mL resveratrol and is decreased at low concentrations (5 micro-g/mL, 10 micro-g/mL resveratrol). This results show the genotoxic activity of resveratrol in adenocarcinoma gastric cell and the anticancer property of this substance.

Corresponding author: Paul Mitrut, MD, PhD, e-mail: centrul_renasterea@rdscv.ro

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16. The use of scanning electron microscopy in evaluating the effect of a bleaching agent on the enamel surface

Diana Dudea, A. Florea, Carmen Mihu, R. Campeanu, Codruta Nicola, Gh. Benga

In this paper we present the results of an experiment with a commercial gel containing carbamide peroxide (CP) 15% (Opalescence 15% PF), aimed to assess the effects of this bleaching agent on the enamel surface of extracted human teeth, by using scanning electron microscopy (SEM). Opalescence 15% PF was applied on two quadrants of twelve extracted permanent, decay-free, human teeth, for 3 hours or 8 hours; for four teeth the application was performed once, while for eight teeth the treatment was repeated daily for 14 days (in order to simulate the usual clinical protocol of at-home bleaching). One quadrant of each tooth was used for control and the remaining quadrant was etched for 40 seconds with a gel containing 37% phosphoric acid. No differences concerning the micromorphology of tooth surface were observed by SEM, on the samples treated with Opalescence 15% PF once, for 3 or 8 hours, when compared to the control. Various, mainly minor changes occurred in samples treated with Opalescence 15% PF for 3 hours or 8 hours daily, for 14 days. On the other hand, the acid-etched samples had an irregular surface, which suggests important alterations of the prismatic structure of the enamel. These are the first studies of this type performed in Romania.

Corresponding author: Diana Dudea, DMD, PhD, e-mail: ddudea@umfcluj.ro

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17. Clinical, histological and therapeutic study regarding the variations of the edentulous ridge's mucosa

Monica Mihaela Craitoiu, Mihaela Pauna, Veronica Mercut, Stefania Craitoiu, Elena Annabel Nitulescu

The oral mucosa of the edentulous ridges presents many morphological macroscopic and microscopic variations, from one area of the edentulous ridge to another, as well as from one patient to another. Because the mobile prosthetic treatment realizes a direct contact between the dental prosthesis and the mucosa of the denture supporting structures and of the peripheral structures, the prosthetic success depends on establishing a correct and long lasting relation between them. The present article's purpose is to present the pro-prosthetic and prosthetic treatment possibilities for partial or complete edentulous patients who have variations of the mucosa of the residual ridges, as well as to present the histopathological importance of these variations. The mucosal characteristics essentially influence the attitude in the different treatment phases of a mobile dental prosthesis, the prosthetic success depending on the establishment of a correct and long lasting relation between the dental prosthesis and the oral mucosa.

Corresponding author: Monica Mihaela Craitoiu, MD, PhD candidate, e-mail: mcraitoiu@yahoo.com

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18. Modular adaptive bone plate for humerus bone osteosynthesis

Daniela Tarnita, D. N. Tarnita, N. Bizdoaca, Corina Tarnita, C. Berceanu, C. Boborelu

The present paper describes a bionics application of shape memory alloy in construction of orthopedic implant. The main idea of this paper is related to design modular adaptive implants for fractured bones. In order to target the efficiency of medical treatment, the implant has to protect the fractured bone, for the healing period, undertaking much as is possible from the daily usual load of the healthy bones. The adaptability of this design is related to medical possibility of the doctor to made the implant to correspond to patient specifically anatomy. Using a CT-realistic numerical humerus bone model, the mechanical simulation of the osteosyntesis process for humerus bone using staples made out of Nitinol. The stress and displacements diagrams for bone, for plate modules and for staples, are presented.

Corresponding author: Daniela Tarnita, Eng, PhD, e-mail: dtarnita@yahoo.com

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19. Clinical and histopathological aspects in otomastoiditis

Cristina Popescu, Elena Ionita, Carmen Aurelia Mogoanta, Cristiana Simionescu, Emilia Patru

We aimed to establish correlations between the clinical manifestations and histopathological changes occurring in otomastoiditis. To this effect, we have made a retrospective clinical and statistical study on a number of 146 patients diagnosed with otomastoiditis, admitted in the Otorhinolaryngology Clinic of the Emergency County Hospital, Craiova, during 2003-2007. Of the 146 patients, 128 (87.67%) were suggested to follow a radical surgical treatment (total petromastoidian evidation), in 10 cases (6.85%) a partial petromastoidian evidation was performed, and in eight cases (5.48%) there were made reappraisals of the petromastoidian evidation cavities. When the surgical procedures were performed, there were collected fragments of mucous from the middle ear or from the mastoid cavities, which were sent for examination to the Laboratory of Anatomical Pathology, where there were performed classical histological (Hematoxylin-Eosin and trichromic Van Gieson) and immunohistochemistry staining. The clinical and statistical study emphasized the fact that the onset of the disease was chronic in 143 patients (97.95%) and of acute type in three patients (2.05%). Taking into account the medical history of the patients, repeated episodes and incorrectly treated of acute infections of the middle ear (otitis) were found in most cases, amounting to a percentage of 97.95%. The distribution of the patients on the five years was relatively uniform, in 2003 - 24 cases (16%), in 2004 - 36 cases (25%), in 2005 - 28 cases (19%), in 2006 - 25 cases (17%), in 2007 - 33 cases (23%), and the most affected age groups were 21-30-year-old (19.86%) and 51-60-year-old (21.92%). Analyzing the cases included in the studied group emphasized the fact that for a relatively large percentage of patients (87.67%) it was required as an immediate necessity the surgical procedure of total petromastoidian evidation, due to the extensive damages, the negligence of the patients that did not contact in proper time a medical specialist. The most frequent histopathological diagnose was of cholesteatoma in 60 cases (41.10%), fact which also grounded the surgical procedure. Immunohistochemical and microscopic study highlighted in the epithelium areas of necrosis, associated with areas of reactive hypertrophy and hyperplasia, and in the chorion, the existence of a chronic inflammatory infiltrate made up of lymphocytes, plasmocytes and macrophages. The immunohistochemistry studies stressed the preponderant presence of T-lymphocytes and macrophages in the chorion of the mucous of the middle ear, confirming the presence of an immune reaction, predominantly of cellular type.

Corresponding author: Cristina Popescu, MD, e-mail: cristina.popescu25@yahoo.com

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20. E-cadherin and p63 immunoexpression in dysplastic lesions and urothelial carcinomas of the bladder

A. Stepan, Cl. Margaritescu, Cristiana Simionescu, Raluca Ciurea

In this study, we investigated 30 cases of dysplastic lesions and bladder carcinomas. The lesions presented different degree of differentiation and depth of invasion. The aim of this study was to appreciate the immunoexpression of some proteins, like E-cadherin and p63, which are implicated in the cellular adhesiveness and the normal development of the urothelium, depending on the degree of tumoral differentiation and the depth of invasion. The immunoreactivity of the markers was qualitative and quantitative evaluated. Both markers presented an immunoexpresion diminishing with depth of invasion, the carcinomas with muscularis propria invasiveness having a minimum intensity reaction (0 or 1), and a medium percentage of marked cells by 10-50% for E-cadherin, and 30% for p63. The immunostaining intensity for E-cadherin decreased with the tumoral degree of differentiation, the poorly differentiated carcinomas having a reaction with minimum intensity (0 or 1). The p63-immunoexpression was correlated with the degree of differentiation in superficially cases of chorion invasiveness carcinoma. The immunostaining intensity in cases with muscularis propria invasiveness was diminished as the number of epithelial layers becomes bigger, but there was no variation depending on tumoral differentiation. In conclusion, E-cadherin and p63 are implicated in tumoral progression and may be used as a prognostic markers.

Corresponding author: Alex Stepan, MD, PhD candidate, e-mail: astepan76@yahoo.com

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21. Videocapsule endoscopy and single balloon enteroscopy: novel diagnostic techniques in small bowel pathology

C. C. Vere, Camelia Foarfa, C. T. Streba, S. Cazacu, D. Parvu, T. Ciurea

Videocapsule endoscopic (VCE) exploration represents a major breakthrough in non-invasive imagistic, especially of the small bowel. Our study group consisted of 29 initial subjects admitted in the 1st Internal Medicine and Gastroenterology Clinic at the Emergency County Hospital of Craiova between June 2008 and March 2009. We have excluded a number of eight subjects from the VCE-group, as their pathology represented an absolute contraindication for capsule ingestion. The remaining 21 patients (11 men - 52.38% vs. 10 women - 47.61%) underwent videocapsule diagnostic investigation followed by single balloon enteroscopy with biopsy or surgical removal, where case. Large resection cups were histologically prepared in the Pathology Department of the Emergency County Hospital of Craiova. We performed a statistical analysis of the data using Fisher's Exact Test, which is applicable to small numbered lots. In five cases (23.80%) VCE investigation did not reveal any abnormalities. Five (23.80%) cases had non-tumoral pathology, all of them being inflammatory lesions. We found tumoral lesions in 11 (52.38%) cases: five (23.80%) benign and six (28.57%) malign. We found that inflammatory lesions predominantly affected subjects in the 15-30 and 30-45 years intervals (p=0.00103), whereas tumoral lesions are positively correlated with old age, predominantly affecting the 45-60 and above 60-year-old groups (p=0.00216). VCE combined with SBE open new frontiers for small bowel exploration. Histology remains the single most accurate test for establishing the nature of a lesion.

Corresponding author: Vere Cristin Constantin, MD, PhD, e-mail: vere_cristin@yahoo.com

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22. The costoclavicular ligament revisited: a functional and anatomical study

R. Shane Tubbs, N. A. Shah, B. P. Sullivan, N. D. Marchase, A. Comert, H. I. Acar, I. Tekdemir, M. Loukas, M. M. Shoja

Introduction: The sternoclavicular joint is of clinical importance. However, there is scant information in the literature regarding one ligament of this area, the costoclavicular ligament (CCL). Material and Methods: In order to further elucidate this structure, 10 adult formalin-fixed cadavers (17 sides) underwent dissection of the CCL. Once the CCL was identified, measurements were made of its dimensions and observations made of its anatomy. Next, ranges of motion were performed of the upper extremity and the CCL observed for tension or laxity. Results: Of the 17 sternoclavicular regions examined 16 (94%) were found to possess a CCL. The average medial and lateral lengths, width and thickness were 1, 2, 1.2, 0.340 cm, respectively. The width of the CCL was statistically smaller in women that in men. The majority of ligaments were single structures traveling from the inferior surface of the medial clavicle just lateral and sometimes-fused (12.5%) to the lateral edge of the sternoclavicular joint. These fibers then terminated on the medial end of the first rib and first costal cartilage (75%) or exclusively onto the first costal cartilage (25%). Most ligaments were single and not composed of two parts. Arm abduction resulted in tautness of the ligament and increased as the degree of abduction increased. Internal rotation of the arm translated into medial shift of the clavicle, raising the clavicle away from the first rib creating tension on the CCL. Moderate degrees of external rotation were required before the CCL became taut and even began to pull the first rib laterally. Small amounts of protraction and retraction of the scapula both put the CCL under tension. Conclusions: The CCL is a constant structure found just lateral to the sternoclavicular joint. This ligament was a single band in the majority of our specimens and limited most ranges of motion of the proximal upper limb thus stabilizing the sternoclavicular region.

Corresponding author: Richard Shane Tubbs, MD, PhD, e-mail: rstubbs@uab.edu

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23. Malignant fibrous histiocytoma of larynx with giant cell: case report and histological-clinical considerations

Fl. Anghelina, Elena Ionita, Luminita Chiutu, Carmen Aurelia Mogoanta, S. Ciolofan, Cristina Iosif, M. Ceausu

Introduction: Malignant fibrous histiocytoma (MFH) is a tumor which has a mesenchymal origin, and an uncertain histogenesis. MFH with giant cell accounts for 3-15% of all malignant fibrous histiocytomas. Objective: To explore the histopathology, the diagnosis and the treatment of MFH with giant cell of larynx. Patient and Methods: We report a case of a glottic MFH at a 59-year-old male, who has been smoking for 30 years, and was hospitalized in June 2008 at Emergency County Hospital of Craiova. Results: The largely-sized tumor was originated in glottic area, upper a left vocal cord, with no paresis. The tumor determined respiratory failure and dysphonia. Patient underwent surgical excision of the tumor after an emergency tracheotomy. The immunohistochemical techniques proved positive for vimentin, smooth muscle actin, CD68, CD34, bcl2, EGFR, S100, Ki67, and negative for CD117, NFT, chromogranin, c-erbB2, CK34betaE12, MNF116, and p53. Conclusions: Malignant fibrous histiocytomas (MFH) with giant cell of larynx are very rare mesenchymal neoplasm (this case seems the first reported). The diagnosis of MFH of larynx was difficult and the immunohistochemistry could have been helpful.

Corresponding author: Florin Anghelina, MD, PhD candidate, e-mail: dr_anghelina_florin@yahoo.com

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24. The maxillary recess of the sphenoid sinus

Iulia Camelia Ciobanu, A. Motoc, Adelina Maria Jianu, R. Cergan, M. A. Banu, M. C. Rusu

The most studied recess of the sphenoid sinus seems to be the lateral one, which is highly variable. Less attention seems to be paid to the maxillary recess, extending anterior, inferior and lateral towards the maxillary sinus. Twenty patients were referred for CT of the paranasal sinuses (axial CT), during a 3-month period. After examining the morphology of the sphenoid sinuses, the authors found bilateral maxillary recesses in one patient. On either side, the maxillary recess of the sphenoid sinus was directed anterior and lateral, being separated from the maxillary sinus by a distinctive bony wall; the longitudinal size of these recesses was 1.36 cm, on the left, and 1.22 cm, on the right. As it becomes part of the antero-medial wall of the pterygopalatine fossa, the maxillary recess of the sphenoid sinus should be evaluated prior to the endoscopic endonasal approach of the fossa; the detail becomes more relevant when one takes into account the fact that in our case, on the right side, the CT-scan suggested that the sphenopalatine artery could traverse directly through the maxillary recess. Therefore, such a maxillary recess of the sphenoid sinus seems to be anatomically located in the situs of the orbital process of the palatine bone and thus, it assumes the respective topography. The differential diagnosis with a Haller cell is relevant - the surgical procedures that approach a Haller cell could lead to a misdiagnosed maxillary recess of the sphenoid sinus into the middle nasal meatus. This aspect determined us to consider the maxillary recess of the sphenoid sinus as a false Haller cell and to claim the imperative of a CT-evaluation performed prior to interventions.

Corresponding author: Mugurel Constantin Rusu, MD, PhD, e-mail: anatomon@gmail.com

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25. Multiple malignant tumors of the orbit

Bianca Szabo, I. Szabo, Cristina Nicula, Doinita Crisan, Carmen Crivii

he retinoblastoma is the most frequent malign tumor of the children, starting with the maximum frequency between the ages of one to three years. It is part of the categories of rare diseases, whose frequency is estimated at 1:15.000-1:20.000 newborn babies. It is a tumor of the neuroblast-type placed at the retina level. The retinoblastoma can be unifocal, when a single tumor is present, or multifocal, when it is about more tumors at the level of the same eye or with extension to both eyes. The retinoblastoma can have at the basis two mechanisms of appearance, thus distinguishing two types: the hereditary and the sporadic, non-hereditary. In approximately 40% of the cases, the children affected have the hereditary type of the illness; this includes the cases of the children with bilateral retinoblastoma and approximately 5% of those with tumor at a single eye. The hereby paper presents two clinical cases (mother and daughter) with the histopathological diagnosis of retinoblastoma, followed up on a period of four years. The first case, that of a 4-year-old child, with the histopathological diagnosis of bilateral retinoblastoma with left intra-orbital invasion, to which enucleation has been made to the right eye and left orbit exenteration (on three years interval). The second clinical case is that of a 30-year-old woman who shows up at the clinic with a left intra-orbital tumor recidivation; it is significant to reveal the fact that the first surgical intervention (left eye enucleation) took place 29 years ago and the histopathological diagnosis settled post-operation was that of retinoblastoma. The histopathological diagnosis of the tumor recidivation was of squamous carcinoma. The clinical and histopathological aspects of the two clinical cases presented are being debated, the appearance of a tumor formation in the same topographic area after a long period and the importance of the genetic advice in the families diagnosed with retinoblastoma.

Corresponding author: Bianca Szabo, MD, PhD, e-mail: bianca.szabo@umfcluj.ro

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26. Unusual high-origin of the pronator teres muscle from a Struthers' ligament coexisting with a variation of the musculocutaneous nerve

L. Jelev, G. P. Georgiev

During routine anatomical dissection of the right upper extremity of a 53-year-old woman cadaver, an unusual high-origin of the pronator teres muscle was discovered. The fibers of the aberrant muscle arose from two bone origins - the medial epicondyle and a small supracondylar process of the humerus, and from a tendinous arch (Struthers' ligament) extending between them. In addition, there was a variation of the musculocutaneous nerve - in the axilary fossa the musculocutaneous was fused to the median nerve and its usual branches arose consecutively from the median nerve stem. The last of these branches - the lateral antebrachial cutaneous arose in the lower part of the arm from the median nerve and companion to it and to the brachial artery passed under the Struthers' ligament. Our findings indicate that in some rare cases of combined muscular-nerve variations, the lateral antebrachial cutaneous nerve can be added to the neurovascular structures possibly entrapped by the Struthers' ligament.

Corresponding author: Georgi P. Georgiev, MD, e-mail: georgievgp@yahoo.com

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27. Subungual exostosis of the big toe

Ligia Stanescu, Carmen Florina Popescu, Carmen Elena Niculescu, Daniela Dumitrescu, S. S. Mogoanta, Iuliana Georgescu

The subungual exostosis is a benign bone tumor on the distal phalanx of a digit, beneath or adjacent to the nail, often bringing in discussion many differential diagnosis. We present a 14-year-old boy with a cutaneous nodular lesion, painful to the easy touch on the latero-internal half of the nail of right big toe with extension in the cutaneous part of this. He suffered many treatments, especially cauterization, but with recurrence. In the present, the radiological findings of the affected finger and the histopathological ones from the fragment excised confirmed the diagnosis of subungual exostosis. The local excision of the entire region with the removal of the cartilaginous cap has been followed by a silent period without recurrences of almost two years when he as revised.

Corresponding author: Ligia Stanescu, MD, PhD, e-mail: ligstanescu@yahoo.com

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28. Unilateral partial ossification of sacrotuberous ligament: anatomico-radiological evaluation and clinical implications

Jyoti Arora, Vandana Mehta, R. K. Suri, Gayatri Rath

The present study describes the topography and morphometry of a unilateral ossified sacrotuberous ligament. It aims to discuss its anatomical, radiological and clinical implications. The pudendal nerve, internal pudendal artery, nerve to obturator internus and coccygeal branch of inferior gluteal artery are all-important structures near sacrotuberous ligament. An ossified sacrotuberous ligament may be an important etiological factor in neurovascular compression syndromes and its anatomical knowledge may help in the development of new treatment for this common clinical problem. The ossified sacrotuberous ligament in the present case was 7.6 cm in length and exhibited a characteristic anterior and posterior segment. The base was at the ischial tuberosity and the apex showed numerous small bony protuberances with deep intervening grooves. The ossified STL may be important in differential diagnosis of soft tissue pain and tenderness after trauma. It may be a challenging puzzle for the present day surgeon and radiologist in interpretation of CT-scans and MRI.

Corresponding author: Jyoti Arora, MBBS, MSc, e-mail: jyotiarora2005@yahoo.co.in

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29. Absence of the peroneus tertius muscle: cadaveric study with clinical considerations

S. Das, Farihah Haji Suhaimi, Azian Abd Latiff, Khin Pa Pa Hlaing, Norzana Abd Ghafar, Faizah Othman

Peroneus tertius (PT) is a muscle of the anterior compartment of the leg. The PT muscle originates from the anterior surface of the fibula and the interosseous membrane and inserts into the medial side of the dorsal region of the fifth metatarsal bone. During routine dissection, we observed the absence of PT on the left lower limb of a cadaver. Usually, the PT is involved in dorsiflexion and eversion of the foot. In many cases, the absence of PT maybe asymptomatic and it may be incidentally detected during cadaveric dissections or autopsies. The existence of PT may help in the swing phase of bipedal walking. The PT may be used for tendon graft surgeries. The pull of the PT may be responsible for causing stress on the fifth metacarpal and account for all stress fractures in any individual. The absence of the PT may puzzle any transplant and foot surgeons performing graft operations. We as anatomists discuss the clinical implications of the absence of PT.

Corresponding author: Faizah Othman, MD, e-mail: faizah@medic.ukm.my

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30. The demonstration of the inferior sternal cleft using three-dimensional reconstruction: a case report

M. Cudi Tuncer, M. Ufuk Aluclu, Ozlen Karabulut, Refik Ulku, E. Savas Hatipoglu, Hasan Nazaroglu

Congenital sternal cleft is a rare disorder in which there is a gap in the midline of the anterior chest wall between the two halves of the sternum. Typically, the contour of the mediastinal structures can be seen beneath the skin. It is rare and the exact incidence is not known. It results from failure of fusion of the two lateral mesodermal sternal bars by 8 weeks of gestation. Most cases are diagnosed shortly after birth and are reported only rarely in adults. We report here one of the congenital major chest wall deformities; inferior sternal cleft is rarely seen, associated with sternal and costal variations in a 22-year young man.

Corresponding author: Mehmet Cudi Tuncer, MD, PhD, e-mail: drcudi@hotmail.com

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