Prevalence and Prognostic Value of HPV among Tunisian Patients with Laryngeal Cancer and Relationship between DNA HPV and p16, IGF-1R, Survivin, p53 Expressions.

OBJECTIVES: Tobacco and alcohol are the main etiological factors common to laryngeal cancers. However, the Human Papilloma Virus (HPV) constitutes an alternative risk factor according to several studies. In Tunisia, despite the annual increasing incidence of laryngeal squamous cell carcinoma (LSCC), the prevalence and prognostic significance of HPV have never been explored.In this study, we sought to highlight HPV DNA in 70 biopsies of laryngeal cancer, and to analyze the status of HPV infection in association with p53, p16, survivin, and IGF-1R expressions. METHODS: HPV high risk (HPV HR) DNA was detected in tumors by in situ hybridization. However, the expression of p53, p16, survivin and IGF-1R were stained by immunohistochemistry test. The correlations of HPV status with clinicopathological parameters, overall survival, disease-free survival and proteins expressions were statistically evaluated. RESULTS: HPV HR DNA was detected in 39 out of 70 (55.71%) laryngeal tumors. HPV+ patients have a better overall survival (P = .081) and long disease-free-survival (P = .016) with a low rate of recurrence (P = .006) than HPV- patients. No significant correlations were found between HPV HR status and clinicopathological parameters (all P > .005). Moreover, HPV+ tumors were not associated with expression of p53, p16 and survivin. However, HPV HR status correlates with weak to moderate IGF-1R expression (P = .043). CONCLUSION: The substantial detection of HPV HR in LSCC tumors suggest that this virus plays an important part in laryngeal cancer in Tunisia. It is a good prognostic factor. In addition, HPV infection could act to block the pathway of IGF-1R expression.

Effect of Hypericum humifusum aqueous and methanolic leaf extracts on biochemical and histological parameters in adult rats.

Hypericum genus is traditionally known for its medicinal use and its therapeutic and antioxidant effects. However, the toxic effect of this plant has not been much explored. Our study aimed at investigating the effect of Hypericum humifusum (Hh) leaf extracts on oxidative stress parameters in male rats. For it, we first focused on the phytochemical analysis of the aqueous and methanolic extracts of Hh leaves. Hence, Wistar rats were treated per gavage for 30 days and divided into Control (1 mL/rat, distilled water), A200 group (200 mg/kg body weight (bw) aqueous extract), A400 group (400 mg/kg bw aqueous extract), M10 group (10 mg/kg bw methanolic extract), M20 group (20 mg/kg bw methanolic extract). The phytochemical analysis revealed the presence of tannins, flavonoids, steroids, carbohydrates, and phenolic compounds. Biochemical and histological investigations were performed in plasma and liver tissue. Liver tissue homogenates were used for the measurement of malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) levels. At the same time, alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) were assayed in plasma samples. Histological study was also conducted in liver. We showed that Hh extracts reduced relative liver weight and increased ALT, AST, LDH activities in treated groups compared to control group. These results were associated with an increase of MDA levels and a decrease of antioxidant enzyme activities (CAT and SOD) in liver tissues of treated rats. Histology of liver demonstrated several alterations showing necrosis, altered hepatocytes and lymphocyte migration mainly in A200 group and dilated sinusoids, foamy appearance of hepatocytes and lymphocyte accumulation in the other treated groups. This original work indicated that chronic consumption of Hh leaf extracts has no antioxidant effect but instead it induces oxidative stress and enhances markers of cell damage which was confirmed by histological study of liver rats.

Chronic consumption of Hypericum humifusum leaf extracts impairs epididymis spermatozoa characters in association with oxidative stress in adult male Wistar rats.

Recently, there has been increasing interest in Hypericum (Hypericaceae) genus. The first part of the present study focused on the phytochemical analysis of the methanolic and aqueous extracts of Hypericum humifusum leaves. The second part of the study investigated the effect of Hypericum humifusum leaf extracts on male reproductive parameters. 30 male rats were grouped into control (1mL/rat, distilled water), treated by 200mg/kg body weight (bw) aqueous extract (A200), 400mg/kg bw aqueous extract (A400), 10mg/kg bw methanolic extract (M10) and 20mg/kg bw methanolic extract (M20) groups. The phytochemical analysis revealed the presence of tannins, flavonoids, steroids, carbohydrates, and phenolic compounds. After thirty-day treatment, body and reproductive organs were weighed. Testes in all rat groups were processed for biochemical assays and histopathological examinations. Epididymis sperm analyses were also performed. Testicular tissue homogenate samples were used for Malondialdehyde (MDA), catalase and superoxide dismutase (SOD) measurements. We showed that Hh extracts induced a severe seminiferous tubular damage with an increase in the percentage of empty seminiferous tubules. Epididymis sperm analysis revealed a significant reduction in density and viability of sperm with alteration of spermatozoa morphology. Also, we found that Hh leaf extracts decreased plasma total cholesterol, HDL-cholesterol and triglycerides levels. These results were associated with an increase of MDA levels and a decrease of catalase and SOD activities in testis tissues. Our finding revealed that chronic consumption of Hh extracts induces disruption of normal spermatogenesis by alteration of sperm density, viability, and morphology. This action may be due to an inhibition of the antioxidant-defense system.

Expression profile of biomarkers altered in papillary and anaplastic thyroid carcinoma: Contribution of Tunisian patients.

AIMS: The objective of this study was to compare the protein expression profile between well-differentiated (papillary) and undifferentiated (anaplastic) thyroid carcinoma in Tunisian patients. METHODS: This first Tunisian retrospective study concerned data of 38 thyroid cancer cases (19 papillary carcinoma PTC and 19 anaplastic carcinoma ATC) collected at Salah Azaiez Institute of Tunisia. Immunohistochemistry was used to evaluate tumor expression of different molecular markers (p53, Ki67, E-cadherin, cyclin D1, bcl2, S100 and Her-2). The molecular expression was correlated with the clinicopathological characteristics of the tumors. RESULTS: There were 6 differentially expressed markers when comparing anaplastic thyroid carcinoma ATC with papillary thyroid carcinoma PTC. Expression of p53 and Ki67 were significantly increased in 16 and 18 ATC cases respectively, the Ki67 expression was lost in PTC. Cyclin D1, E-cadherin, bcl2 and S100 were overexpressed in PTC tumors; however, they were significantly decreased in ATC. The last marker, Her-2 was expressed in one case of PTC only. CONCLUSION: Our results, similar with findings of other ethnic groups, showed alteration in expression of molecular markers associated with tumor dedifferentiation, indicating loss of cell cycle control with increased proliferative activity in ATC carcinoma. These data support the hypothesis that ATC may derive from dedifferentiation of preexisting PTC tumor.

Triple negative breast cancer: A clinico-epidemiological and histopronostic study of 90 cases.

PURPOSE: The aim of this study was to describe the clinico-epidemiological and histopronostic characteristics of triple negative breast cancer (TNBC) and to evaluate the therapeutic results in tunisian women. METHODS: We reported the results of a retrospective study including 90 patients treated for TNBC between Junuary 2008 and December 2009 in the Salah Azaiz Institute of Tunis. RESULTS: TNBCoccured in 14% of diagnosed breast cancers. The mean age at diagnosis was 53.67 years. Family history of breast cancer was reported in 10% of cases.The majority of tumors were classified as T2 (41%) and associated with invasive ductal carcinoma histological type (99%) and SBR grade-II (54%). Tumor lymph node metastases were detected in 44% of patients.Among operated patients, 46% of patients underwent conservative surgery and 54% radical surgery. Chemotherapy and postoperative radiotherapy were given in97% and 80%of patients, respectively. After a median follow-up of 33.51 months, 61% of patients remained free of disease, 12% hadloco-regional recurrence, 9% had disease progression during chemotherapy and 21% developed systemic disease. CONCLUSION: TNBC diagnosis is often made in the advanced stage and has a tendency to recur after treatment. The variable responseto chemotherapy is due to the molecular tumor heterogeneity. The development of targeted therapies is necessary to improve outcome of chemoresistant TNBC.

Comparative study of two complementary proliferation markers in 200 breast carcinomas: Ki67 and mitotic index.

BACKGROUND: The evaluation of the proliferation in the mammary carcinomas provides useful prognostic and predictive information for subsequent management. The purely morphological evaluation of proliferative activity was represented by the evaluation of mitotic index. New analytical methods were gradually developed and performed. Among these methods, evaluation of Ki67 by immunohistochemistry is particularly interesting. Its expression is significantly increased in the cell cycle. AIM: To correlate the mitotic index as a classic method of assessing cell proliferation and Ki 67 proliferation index detected by immunohistochemistry to identify the most reliable proliferative marker. METHODS: We studied 200 patients with invasive ductal carcinoma breast over a period of 12 months of 2014. We identified in each case the SBR grade, Ki67 proliferation index and the mitotic index. Correlation between the two parameters was identified using the Spearman test. A result is considered significant when p < 0.01. The distribution of these markers by SBR gradewas studied using the ANOVA method. RESULTS: Ki67 is significantly correlated to the mitotic index. Although these two methods are dependent, Ki67 is the most sensitive and bonded to SBR grade. Determination of Ki67 provides interesting information that could replace the mitotic account. It provides reliable and reproducible data that can be incorporated into a prognostic score. CONCLUSION: Ki67 is a more efficient marker mitotic index, reflecting the cell proliferation.

Mucinous breast carcinoma a rare entity to be known: clinico-pathological study of 48 cases.

BACKGROUND: Mucinous carcinoma is a particular type of breast cancer characterized by the presence of extracellular mucin and is linked with a more favorable prognosis than invasive breast carcinoma of no special type. It accounts for 1 to 7% of all breast cancers. We propose in this work to study at first the clinic-pathological characteristics and the evolution of 48 cases of mucinous carcinomas. Secondly, we propose to identify through a review of recent literature, the therapeutic management of these carcinomas. METHODS: This is a retrospective study, conducted in Salah Azaiez carcinological institute, interesting 48 cases of mucinous carcinoma collected over 19 years. Clinical, radiological and pathological information were collected from medical records. RESULTS: The mean age of our patients was 57 years. The tumor was single in 41 cases and in 7 cases bifocal. Mammographic aspects were favor of malignancy in 33 cases (75%). It was mixed subtype in 14 cases and pure in 34 cases. Lymph node involvement was noted in 14 cases. The number of metastatic lymph nodes ranged from 1 to 11 with an average of 3. Hormone receptors were positive in 35 tumors (73%). The HER2 showed overexpression in 5 cases. Surgery consisted of a radical treatment for thirty-two patients (66%). Overall survival at 5 years was 75.3% and 59.3% at 10 years. Disease-free survival was 74% at 5 years and 58% at 10 years. CONCLUSION: Mucinous carcinoma consists of two distinct subtypes: pure and mixed with different prognosis. Larger data samples with longer follow-up are necessary to achieve an improved understanding of this particular tumor.

Comparative study of two complementary proliferation markers in 200 breast carcinomas: Ki67 and mitotic index.

BACKGROUND: The evaluation of the proliferation in the mammary carcinomas provides useful prognostic and predictive information for subsequent management. The purely morphological evaluation of proliferative activity was represented by the evaluation of mitotic index. New analytical methods were gradually developed and performed. Among these methods, evaluation of Ki67 by immunohistochemistry is particularly interesting. Its expression is significantly increased in the cell cycle. AIM: To correlate the mitotic index as a classic method of assessing cell proliferation and Ki 67 proliferation index detected by immunohistochemistry to identify the most reliable proliferative marker. METHODS: We studied 200 patients with invasive ductal carcinoma breast over a period of 12 months of 2014. We identified in each case the SBR grade, Ki67 proliferation index and the mitotic index. Correlation between the two parameters was identified using the Spearman test. A result is considered significant when p < 0.01. The distribution of these markers by SBR gradewas studied using the ANOVA method. RESULTS: Ki67 is significantly correlated to the mitotic index. Although these two methods are dependent, Ki67 is the most sensitive and bonded to SBR grade. Determination of Ki67 provides interesting information that could replace the mitotic account. It provides reliable and reproducible data that can be incorporated into a prognostic score. CONCLUSION: Ki67 is a more efficient marker mitotic index, reflecting the cell proliferation.

Screening for common mutations in BRCA1 and BRCA2 genes: interest in genetic testing of Tunisian families with breast and/or ovarian cancer.

BACKGROUND: In the Tunisian population, as yet a limited number of BRCA1/2 germline mutations have been reported in hereditary breast and/or ovarian cancer. These mutations are located in a few exons of BRCA1/2. The aim of the present study was to search for these mutations in 66 unrelated patients with hereditary breast and/or ovarian cancer in order to assess the interest in such a targeted approach for genetic testing in Tunisia. MATERIALS AND METHODS: Blood specimens from the 66 Tunisian patients, with family history of breast and/or ovarian cancer, were collected at the Salah Azaiz Cancer Institute of Tunis. The exons 5, 20 and part of exon 11 of BRCA1 as well as part of exons 10 and 11 of BRCA2 were analyzed by Sanger sequencing. RESULTS: 12 patients had deleterious mutations in the BRCA1 or BRCA2 genes (18%), including a novel frame-shift mutation of BRCA1 (c.3751dup; 3780insT). Four distinct BRCA1 mutations were detected eight patients: c.5266dup (5382insC) and c.211dup (330insA) each in three patients, c.3751dup (3870insT) and c.4041_4042del (4160delAG) each in one patient. The four remaining cases all carried the same BRCA2 mutation, c.1310_1313del (1538delAAGA). Besides these deleterious mutations, eight polymorphisms and unclassified variants were detected, one of them being never reported (BRCA1c.3030T>G, p.Pro1010Pro). CONCLUSION: In this study, we show that targeting relevant exons in BRCA1 and BRCA2 genes allows detection of a substantial percentage of mutations in the Tunisian population. Therefore such an approach may be of interest in genetic testing of high-risk breast and ovarian cancer families in Tunisia.

Descriptive analysis of molecular subtypes in Tunisian breast cancer.

AIM: The objective is to report the correlation between pathology and molecular subtype classifications of breast cancer in Tunisian women. METHODS: This retrospective study concerned data of 966 breast cancer cases collected from 2007 to 2009 at Salah Azaiz Institute of Tunis. These cases were classified by an immunohistochemistry test for estrogen and progesterone receptors and human epidermal growth factor receptor 2 (HER2) status in the four molecular subtypes, namely luminal A, luminal B, HER2+ and triple negative. The molecular classifications were correlated with the clinicopathological characteristics of the tumors. RESULTS: Luminal A (50.7% of cases) was the most common subtype, with triple negative subtype 22.5%, luminal B 13.4% and HER2+ 13.4%. Triple negative and HER2+ subtypes were significantly associated with large tumor size (>5 cm, P < 0.001), younger age (<40 years, P < 0.03) and high grade (P < 0.001). Conversely, there was no correlation with the lymph node status. CONCLUSION: Our data demonstrated that the luminal A subtype, associated with a favorable prognosis, was the most frequent subtype in the Tunisian population; however the triple negative subtype occurred at a high incidence in Tunisia compared to Western countries. The molecular subtypes are correlated to the tumor size, histological grade and patient's age.

Grape seed and skin extract alleviates high-fat diet-induced renal lipotoxicity and prevents copper depletion in rat.

Obesity is a public health problem that contributes to morbidity and mortality from diabetes, heart disease, stroke, and cancers. The purpose of this investigation was to analyse the link between obesity-induced oxidative stress, renal steatosis, and kidney dysfunction, as well as the protective effect of grape seed and skin extract. Rats were fed a standard diet or a high-fat diet for 6 weeks and were either treated or not treated with grape seed and skin extract. Fat-induced oxidative stress was evaluated in the kidney with a special emphasis on transition metals. High-fat diet induced triglyceride deposition and disturbances in kidney function parameters, which are linked to an oxidative stress status and depletion of copper from the kidney. Grape seed and skin extract abrogated almost all fat-induced kidney disturbances. Grape seed and skin extract exerted potential protection against fat-induced kidney lipotoxicity and should find potential application in other kidney-related diseases.

Complementary determination of Epstein-Barr virus DNA load and serum markers for nasopharyngeal carcinoma screening and early detection in individuals at risk in Tunisia.

Because nasopharyngeal carcinoma (NPC) has a close association with Epstein-Barr virus (EBV), measuring serum EBV DNA and anti-EBV serum marker concentrations could be a feasible method for NPC diagnosis, monitoring and probably screening especially in a community at risk. The aim of this study was to determine the EBV pattern in sporadic NPC and in high risk NPC Tunisian families in order to evaluate their risk factors and help for NPC screening. The rates of anti-EBV antibodies and EBV DNA were determined in the serum of 47 healthy members randomly selected from 23 NPC multiplex families with two or more affected members, 93 healthy Tunisian community controls chosen with the same age, sex and geographic origin as unaffected individuals and 66 EBV positive sporadic NPC patients whose serum was available before and after treatment. Unexpectedly, significant lower concentrations of anti-EA (Early Antigen) IgG and anti-VCA (Viral Capsid Antigen) IgG were found in unaffected members from NPC families than in healthy controls while viral loads were negative in all the tested sera. For sporadic NPC patients, anti-EA IgG and anti-VCA IgA concentrations were significantly higher than in healthy controls and these rates decreased after treatment. The level of EBV DNA load varied according to the condition of the tumour. This study suggests that in the Tunisian NPC families, screening for malignancy is based on serum concentrations but not on EBV DNA load while in the sporadic NPC group, serologic markers and EBV DNA load are complementary for diagnosis and follow-up.

[Inflammatory myofibroblastic tumor of the larynx: a case report]. / Tumeur myofibroblastique inflammatoire du larynx: a propos d'un cas.

BACKGROUND: Inflammatory myofibroblatic tumor is a recently caracterized solid mesenchymal tumor. They are frequently localised in the lung. However, the head and neck forms are uncommon. AIM: To report a new case of inflammatory myofibroblastic tumor of the larynx. CASE REPORT: This 47-year old man presented with dysphonia without dyspnea. Laryngoscopy showed a tumor of anterior vocal cord. CT scan of larynx confirmed diagnosis. The patient had a cordectomy. Histology concluded to the diagnosis of inflammatory myofibroblastic tumor. No relapses have been noted after 4 months. CONCLUSION: Only a few cases of laryngeal inflammatory myofibroblastic tumor have been reported in the literature. Clinical presentation depends of tumor location. Radiological images are not specific and diagnosis is based on histology. The expected course is essentially marked by the risk of local recurrences. Surgical excision seems to be the treatment of choice.

Protective effects of crude garlic by reducing iron-mediated oxidative stress, proliferation and autophagy in rats.

The impact of garlic, known for its antioxidant activities, on iron metabolism has been poorly investigated. The aim of this work was to study the effect of crude garlic pre-treatment on iron-mediated lipid peroxidation, proliferation and autophagy for 5 weeks. Rats were fed distilled water or garlic solution (1 g/kg body weight) by gavage for the first 3 weeks as pre-treatment and received a basal diet supplemented or not with ferrous sulfate (650 mg Fe/kg diet) for the last 2 weeks of treatment. Immunohistochemistry labeling and ultrastuctural observations were used to evaluate the iron deleterious effects in the liver. Iron supplementation induced cell proliferation predominantly in non parenchymal cells comparing to hepatocytes, but not apoptosis. In addition, iron was accumulated within the hepatic lysosomes where it triggers autophagy as evidenced by the formation of autophagic vesicles detected by LC3-II staining. It also induced morphologic alterations of the mitochondrial membranes due to increased lipid peroxidation as shown by elevated iron and malondialdehyde concentrations in serum and tissues. Garlic pre-treatment reduced iron-catalyzed lipid peroxidation by decreasing the malondialdehyde level in the liver and colon and by enhancing the status of antioxidants. In addition, garlic reduced the iron-mediated cell proliferation and autophagy by lowering iron storage in the liver and protected mitochondrial membrane. Based on these results, garlic treatment significantly prevented iron-induced oxidative stress, proliferation and autophagy at both biochemical and histological levels due to its potent free radical scavenging and antioxidant properties.

Head and neck liposarcomas: a 32 years experience.

OBJECTIVE: The aim of this review is to study natural history, presentation, treatment and prognosis of head and neck liposarcomas. METHODS: We carry a retrospective study about 15 patients diagnosed and treated in our department between 1969 and 2001. Therapeutic modalities included surgery, radiotherapy and chemotherapy. RESULTS: Tumors were mostly localized at the neck (4 cases) and the scalp (3 cases). Cervical lymphadenopathy was found in 1 case. Chest X-ray found showed metastases in 2 cases. Histological types of liposarcomas were the following: myxoid (5 cases), well differentiated (4 cases), pleomorphic (4 cases), round cell (1 case) and dedifferentiated (1 case). Twelve patients underwent surgery. Complete excision was performed in 7 cases. In the other 5 cases, exeresis was incomplete and 4 of them received postoperative radiotherapy. Three patients were not operated because of vascular involvement (1 case) or metastasis (2 cases), and they all received radiotherapy alone. After initial treatment, complete remission was achieved in 7 cases (46.7%). A recurrence occurred in 8 cases (53.3%). Two of these patients were reoperated and another one received chemotherapy. In the other 5 cases, the tumor was uncontrollable and no additional treatment has been proposed. Five-year survival rate was 87% for patients who underwent surgery alone, 75% for those who underwent surgery and postoperative radiotherapy, and 0% for those treated by radiotherapy alone. CONCLUSION: The mainstay of treatment of head and neck liposarcomas is surgical excision and the prognosis is largely determined by the histological grade and the clinical stage.

Prognostic evaluation of p53, heat shock protein 70, Ki67, and CD34 expression in cancer of the tongue in Tunisia.

OBJECTIVES: Oral tongue cancer remains an aggressive tumour in Tunisia, and patients often present with locally advanced disease, leading to high mortality. The molecular pathway of its tumorigenesis is not yet understood. This study aimed to assess the biologic significance of p53, heat shock protein 70 (Hsp70), Ki67, and CD34 and their influence on survival in patients with tongue cancer. METHOD: Archival tissues from 68 patients with squamous cell carcinoma of the tongue were examined for p53, Hsp70, Ki67, and CD34 by immunohistochemistry and correlated with 5-year survival. RESULTS: p53 and Ki-67 were detected in 55% and 77% of tumours, respectively; a significant association between p53 and Ki67 expression was found (p = .038). Forty-seven percent of cases of tongue cancer expressed Hsp70, and only 9% of tumours expressed CD34 angiogenic factor. No significant correlation was noted between survival and expression of p53, Hsp70, Ki67, and CD34 in patients. CONCLUSION: None of the markers p53, Hsp70, Ki67, and CD34 demonstrated prognostic significance for 5-year survival in patients with squamous cell carcinoma of the tongue.

Breast cancer in Tunisia in 2004: a comparative clinical and epidemiological study.

The aim of this study was to determine the epidemiological, clinical and anatomopathological aspects of breast cancer in Tunisia. We censored and analyzed all cases of breast cancer newly diagnosed in Tunisia during the year 2004. During that year, 1437 new cases of invasive breast cancer were diagnosed and 35 cases of non invasive breast cancer. There were 1408 women and 29 men. The accrual incidence was 27.1 and the standardized incidence was 28.5. The mean age for women was 51 years. The mean tumour size was 40.8 mm (32.3 mm and 42.5 mm for private and public sectors respectively). Tumour stage was T1 in 12.2% cases, T2 in 46.9% cases, T3 in 11.2% cases and T4 in 24.7% cases. Invasive ductal carcinoma was the most frequent (86,6%) with SBR II grade representing 54.5%. 27.7% of the patients had undergone conservative treatment. There was an unexpected increase of the incidence since 1994 where the standardized incidence was 16.9. We have noticed a moderate decrease of the tumour size of 8 mm during the last decade. This national epidemiological study on breast cancer showed an increase in the incidence of this cancer with a moderate decrease in the clinical tumour size. The relative young mean age of our patients may be explained by the age distribution in our population or by risk factors that may be particular to our country.