Human papillomavirus genotypes and HPV-16 variants distribution among Tunisian women with normal cytology and squamous intraepithelial lesions.

BACKGROUND: Little is known about the epidemiological characteristics of papillomavirus (HPV) infection among North African countries. Herein, we conducted a molecular epidemiological study to investigate prevalence of HPV type and HPV-16 variants among cervical-screened unvaccinated Tunisian women. METHODS: Cross-sectional study was performed on 494 Tunisian women visiting Women's Healthcare Centers. HPV-DNA detection was carried out on cervical samples using real-time polymerase chain reaction. HPV genotyping and HPV-16 variants were characterized by direct sequencing of L1 viral capsid gene. RESULTS: The overall HPV prevalence was 34% (95% CI: 30-38%) with significantly higher prevalence among women with squamous intraepithelial lesions (SIL) than those with no intraepithelial lesions (NIL) 84% (95% CI: 76-92%) and 24.5% (95% CI: 20-29%) respectively. The distribution of HPV prevalence according to women's age shows a U-shaped curve and the highest HPV prevalence rates were observed among the youngest (≤25 years; 51.2%, 95% CI: 37-67%) and the oldest women (>55 years; 41.7%, 95% The HPV-16 prevalence was 32.8% (95% CI: 22-45%) among women with SIL and 9.2% (95% CI: 6-12%) among women with NIL. Whereas, the HPV-18 prevalence was 1.3% (95% CI: 0-5%) among women with SIL and 0.3% (95% CI: 0-1%) among women with NIL. Among HPV-16 positive women, European lineage (E) was identified as the predominant HPV-16 variant (85.7%, 95% CI: 76-95%). The frequency of E variant was lower among SIL than among NIL women (81%, 95% CI: 64-99%, and 88%, 95% CI: 77-100%, respectively). Conversely, the African-2 variant frequency was higher among SIL than among NIL women (18%, 95% CI: 1-36% and 6%, 95% CI: 2-14%, respectively). In multivariate analysis, young age was the only risk factor that is independently associated with HPV infection. Moreover, HPV infection and menopause were both found to be independently associated with SIL and HSIL. CONCLUSION: HPV DNA testing should be proposed to young and menopausal Tunisian women. Considering HPV prevalence, only 13% of the Tunisian women could be protected by the bivalent HPV vaccine. These results may be helpful for designing an adapted HPV testing and vaccination program in Tunisia.

Targeting proapoptotic protein BAD inhibits survival and self-renewal of cancer stem cells.

Emerging evidence suggests that the resistance of cancer stem cells (CSC) to many conventional therapies is one of the major limiting factors of cancer therapy efficacy. Identification of mechanisms responsible for survival and self-renewal of CSC will help design new therapeutic strategies that target and eliminate both differentiated cancer cells and CSC. Here we demonstrated the potential role of proapoptotic protein BAD in the biology of CSC in melanoma, prostate and breast cancers. We enriched CD44(+)/CD24(-) cells (CSC) by tumorosphere formation and purified this population by FACS. Both spheres and CSC exhibited increased potential for proliferation, migration, invasion, sphere formation, anchorage-independent growth, as well as upregulation of several stem cell-associated markers. We showed that the phosphorylation of BAD is essential for the survival of CSC. Conversely, ectopic expression of a phosphorylation-deficient mutant BAD induced apoptosis in CSC. This effect was enhanced by treatment with a BH3-mimetic, ABT-737. Both pharmacological agents that inhibit survival kinases and growth factors that are involved in drug resistance delivered their respective cytotoxic and protective effects by modulating the BAD phosphorylation in CSC. Furthermore, the frequency and self-renewal capacity of CSC was significantly reduced by knocking down the BAD expression. Consistent with our in vitro results, significant phosphorylation of BAD was found in CD44(+) CSC of 83% breast tumor specimens. In addition, we also identified a positive correlation between BAD expression and disease stage in prostate cancer, suggesting a role of BAD in tumor advancement. Our studies unveil the role of BAD in the survival and self-renewal of CSC and propose BAD not only as an attractive target for cancer therapy but also as a marker of tumor progression.

IRF-1 responsiveness to IFN-γ predicts different cancer immune phenotypes.

BACKGROUND: Several lines of evidence suggest a dichotomy between immune active and quiescent cancers, with the former associated with a good prognostic phenotype and better responsiveness to immunotherapy. Central to such dichotomy is the master regulator of the acute inflammatory process interferon regulatory factor (IRF)-1. However, it remains unknown whether the responsiveness of IRF-1 to cytokines is able to differentiate cancer immune phenotypes. METHODS: IRF-1 activation was measured in 15 melanoma cell lines at basal level and after treatment with IFN-γ, TNF-α and a combination of both. Microarray analysis was used to compare transcriptional patterns between cell lines characterised by high or low IRF-1 activation. RESULTS: We observed a strong positive correlation between IRF-1 activation at basal level and after IFN-γ and TNF-α treatment. Microarray demonstrated that three cell lines with low and three with high IRF-1 inducible translocation scores differed in the expression of 597 transcripts. Functional interpretation analysis showed mTOR and Wnt/ß-cathenin as the top downregulated pathways in the cell lines with low inducible IRF-1 activation, suggesting that a low IRF-1 inducibility recapitulates a cancer phenotype already described in literature characterised by poor prognosis. CONCLUSION: Our findings support the central role of IRF-1 in influencing different tumour phenotypes.

[The therapeutic contribution of proteomic approaches in cancers]. / L'apport thérapeutique des approches protéomiques dans les cancers.

Up to now, there are no protein tumor markers with a specificity and sensitivity sufficient to have a utility in prognosis and early diagnosis of cancer. Recent advances in proteomics approaches have led to the identification of novel tumor markers of cancer that may have a utility in screening strategies and treatment. The purpose of the current review is to describe the major advances in cancer proteomics, especially those related to the study of serum biomarkers, immune-related responses (autoantibodies) and alterations in cellular proteins.

Proteomics approaches: new technologies and clinical applications in breast carcinomas.

UNLABELLED: Breast cancer is the most diagnosed cancer in women, accounting for approximately 40,000 deaths annually in the USA. In Tunisia, the incidence of breast cancer is approximately 19 new cases per 100,000 women per year. Significant advances have been made in the areas of detection and treatment, but a significant number of breast cancers are detected late. The advent of proteomics provides the hope of discovering novel biological markers that can be used for early detection, prognosis, diagnosis, and therapy. Several proteomics technologies have been used to uncover molecular mechanisms associated with breast. INTRODUCTION: Breast cancer is a major health problem and one of the leading causes of death among women worldwide. Its incidence is steadily rising in developing countries. In Tunisia, the incidence of breast cancer is approximately 19 new cases per 100,000 women per year(1). Invasive carcinomas represent 70-80% of all breast cancer and among these, infiltrating ductal carcinomas (IDCA) are the most aggressive forms and have a poor prognosis(2). Histopathologically identical breast cancers show a different biological behavior in terms of aggressiveness, progression, and response to therapy. Thus, there is a great need for new breast cancer biomarkers that might help detect this cancer at an earlier stage, to uncover prognostically distinct subclasses, and to provide best individual treatment(2). Currently, the search for specific cancer-related alterations are largely carcinoma at the global level to discover protein patterns that distinguish disease and disease-free states with high sensitivity and specificity. Two dimensional gel electrophoresis coupled with mass spectrometry constitute a new proteomics based paradigm for detecting disease in pathology specimens and monitoring disease response to therapy. This review describes these proteomics technologies and their application in the analysis of breast carcinoma.

Lack of effect of tumor necrosis factor-alpha -308 G/A polymorphism on severity of liver fibrosis in Tunisian hepatitis C virus (HCV)-infected patients.

OBJECTIVES: Tumor necrosis factor alpha (TNF-alpha) plays a key role in the immune response. An elevated plasma level of TNF-alpha was repeatedly observed in patients with active liver injury or cirrhosis regardless of the aetiology. The G/A transition at position -308 in the promoter region have been shown to influence TNF-alpha expression. In this study, we aimed to evaluate the impact of TNF-alpha -308 G/A functional polymorphism on fibrosis severity in Tunisian Hepatitis C Virus (HCV)-infected patients. METHODS: TNF-alpha -308 G/A polymorphism was evaluated by polymerase chain reaction (PCR) amplification followed by Restriction Fragment Length Polymorphism (RFLP) method in 53 chronic hepatitis C patients. Single-nucleotide polymorphism (SNP) frequencies were compared with regard to liver fibrosis severity as assessed by the METAVIR scoring system (F1-F2; n=22 versus F3-F4; n=31). RESULTS: The genotype distribution of the TNF-alpha -308 G/A polymorphism among the HCV-infected patients was as follows : GG : 67.9%, GA : 32.1%, AA : 0%. With regard to fibrosis score, no significant differences in TNF-alpha genotype distribution were observed between F1-F2 and F3-F4 patients (p=0.15). CONCLUSION: No significant association between TNF-alpha -308 polymorphism and and the severity of liver fibrosis was found in our Tunisian cohort.

Dimorphism of TAP-1 gene in Caucasian with juvenile myoclonic epilepsy and in Tunisian with idiopathic generalized epilepsies.

Juvenile myoclonic epilepsy (JME) is the most common form of idiopathic generalized epilepsies (IGE) that account for about 5-10% of all types of epilepsies. The first putative locus termed EJM1 is on the human leucocyte antigen (HLA-II) region of chromosome 6p21.3. Interestingly, the EJM1 region includes the Transporter associated with antigen processing 1 (TAP-1) gene encoding the TAP-1, and previous studies have reported associations between HLA-II polymorphisms and different types of epilepsy. In this study, we report an association between two TAP-1 functional polymorphisms the I333V and the D637G and most common IGE in Tunisian population, but we fail to find significant results in Caucasian with JME.

NS5A(ISDR-V3) region genetic variability of Tunisian HCV-1b strains: Correlation with the response to the combined interferon/ribavirin therapy.

In the non-structural protein 5A (NS5A) of hepatitis C virus (HCV), mutations within the interferon sensitivity-determining region (ISDR), the PKR-binding domain (PKR-BD), the variable region 3 (V3), and the interferon/ribavirin resistance-determining region (IRRDR) have been correlated with the IFN-based therapy response. In Tunisia, where a high prevalence of HCV-1b has been found, no data regarding the implication of NS5A in treatment response were available. The current study examined the relationship between the pre-treatment mutation number within ISDR, PKR-BD, V3, IRRDR, as well as in the entire ISDR-V3 region of NS5A (aa 2209-2379) and the response to the 48-week course of combined IFN plus ribavirin therapy in 15 HCV-1b-infected Tunisian patients. Referring to HCV-J sequence, a significant high genetic variability was observed within PKR-BD in the sustained virological responder patients compared to non-responders (P = 0.040). More importantly, when considering the entire region from ISDR to V3, referred to as NS5A(ISDR-V3), a clear difference in the mutation number was observed between sustained virological responders (19.6 +/- 3.16) and non-responders (15.0 +/- 1.41) (P = 0.002). Additionally, a more detailed analysis of NS5A(ISDR-V3) region revealed an elevated degree of mutation rate within the region located between amino acids 2282 and 2308 (P = 0.0006). Interestingly, an analysis of specific amino acid variations defined proline and serine at position 2300 as signature patterns for sensitive and resistant strains, respectively. The genetic variability within the NS5A region of HCV-1b strains was associated with the response to the combined IFN plus ribavirin therapy in our Tunisian cohort.

Cannabis, tobacco and domestic fumes intake are associated with nasopharyngeal carcinoma in North Africa.

BACKGROUND: The lifestyle risk factors for nasopharyngeal carcinoma (NPC) in North Africa are not known. METHODS: From 2002 to 2005, we interviewed 636 patients and 615 controls from Algeria, Morocco and Tunisia, frequency-matched by centre, age, sex, and childhood household type (urban/rural). Conditional logistic regression was used to evaluate the association of lifestyles with NPC risk, controlling for socioeconomic status and dietary risk factors. RESULTS: Cigarette smoking and snuff (tobacco powder with additives) intake were significantly associated with differentiated NPC but not with undifferentiated carcinoma (UCNT), which is the major histological type of NPC in these populations. As demonstrated by a stratified permutation test and by conditional logistic regression, marijuana smoking significantly elevated NPC risk independently of cigarette smoking, suggesting dissimilar carcinogenic mechanisms between cannabis and tobacco. Domestic cooking fumes intake by using kanoun (compact charcoal oven) during childhood increased NPC risk, whereas exposure during adulthood had less effect. Neither alcohol nor shisha (water pipe) was associated with risk. CONCLUSION: Tobacco, cannabis and domestic cooking fumes intake are risk factors for NPC in western North Africa.

Complete mutation screening and haplotype characterization of BRCA1 gene in Tunisian patients with familial breast cancer.

Breast cancer, the most commonly diagnosed cancer in women, is the second leading cause of cancer death in women worldwide. To investigate the contribution of BRCA1 gene mutations to familial breast cancer in Tunisia, 32 unrelated patients who had at least one first degree relative affected with breast and/or ovarian cancer were analysed. BRCA1 mutation analysis was performed by DNA sequencing of all BRCA1 exons. We identified four different BRCA1 frameshift mutations: c.4041delAG, c.2551delG and c.5266dupC already been described and one novel mutation, c.211dupA, observed in two unrelated families. C.5266dupC has previously been found among Jewish Ashkenazi and Eastern European populations. Our study describes it in Arabic/Berber population. Five out of thirty two familial cases had deleterious BRCA1 mutations. Fifteen additional cases carried unclassified variants (UV) or single nucleotide polymorphisms (SNPs). Our study is the first molecular investigation on the role of BRCA1 in hereditary breast cancer in North Tunisia.

[Human genome project: a federator program of genomic medicine]. / Le projet génome humain: programme fédérateur de la médecine génomique.

The Human Genome Project improves our understanding of the molecular genetics basis of the inherited and complex diseases such as diabetes, schizophrenia, and cancer. Information from the human genome sequence is essential for several antenatal and neonatal screening programmes. The new genomic tools emerging from this project have revolutionized biology and medicine and have transformed our understanding of health and the provision of healthcare. Its implications pervade all areas of medicine, from disease prediction and prevention to the diagnosis and treatment of all forms of disease. Increasingly, it will be possible to drive predisposition testing into clinical practice, to develop new treatments or to adapt available treatments more specifically to an individual's genetic make-up. This genomic information should transform the traditional medications that are effective for every members of the population to personalized medicine and personalized therapy. The pharmacogenomics could give rise to a new generation of highly effective drugs that treat causes, not just symptoms.

Interleukin-10 and interferon-gamma gene polymorphisms in patients with nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a multifactorial disease. Cytokines driving the immune response seem to be disturbed in NPC patients. Since interleukin-10 (IL-10) is known to reduce the production of interferon-gamma (IFN-gamma), we supposed that genetic differences in IL-10 and IFN-gamma expression could be a mechanism by which NPC cells escape antitumour immune response. As the production of each cytokine is affected by the genetic background, we investigated the possible association between single nucleotide polymorphisms in genes of IL-10 and IFN-gamma with NPC. Different IL-10 -1082 G/A and IFN-gamma+874 Tau/Alpha genotypes were determined in 160 patients with nasopharyngeal carcinoma and 197 healthy controls. No association was found either for each SNP studied alone or for the combined analysis for both IL-10 and IFN-gamma polymorphisms among NPC patients in comparison with controls. Compared with individuals from high incidence countries, we noted huge significant differences in genotype distribution between individuals from low and intermediate NPC incidence countries. Polymorphisms of the IL-10 and IFN-gamma do not appear to be associated with NPC risk in the Tunisian population. Nevertheless, we strongly believe that the relationship between cytokines polymorphisms and NPC susceptibility deeply depends on the ethnicity.

Association of interleukin-18 polymorphisms and plasma level with the outcome of chronic HCV infection.

Hepatitis C virus (HCV) infection is the main cause of chronic liver disease throughout the world, and may progress to cirrhosis and hepatocellular carcinoma (HCC). Immunological factors, especially cytokines and some host genetic variations, rather than direct HCV action, seem to play an important role in the pathogenesis of HCV infection. Elevated levels of interleukin-18 (IL-18) were described previously for chronically (HCV)-infected patients. This study is aimed at investigating IL-18 promoter polymorphisms (-607C/A and -137G/C) in HCV-infected patients with different disease severities (chronic hepatitis C, liver cirrhosis and HCC) and establishing an association between these polymorphisms and IL-18 plasma concentration with the outcome of chronic HCV infection. The carriage of at least one C allele at position -607 (CC + CA) was associated with a higher risk of cirrhosis and HCC (P = 0.032). Compared with controls, HCV-infected patients had significantly higher levels of IL-18 (P = 0.0001) that correlate with disease severity (P = 0.01, P = 0.001, P = 0.0006, respectively). In conclusion, we supposed a possible implication of IL-18 promoter polymorphisms in the pathogenesis of chronic HCV infection.

Comparative clinical and transcriptomal profiles of breast cancer between French and South Mediterranean patients show minor but significative biological differences.

BACKGROUND: In Western countries, breast cancer incidence and mortality are higher than in Mediterranean countries. These differences have been ascribed to environmental factors but also to late-stage diagnostic and biological specific characteristics. PATIENTS AND METHODS: Between September 2002 and September 2005, we collected clinical data by phone counselling 180 French and Mediterranean breast cancer patients and performed microarray experiments. RESULTS: Characteristics of breast cancer in patients from Lebanon, Tunisia and Morocco were more aggressive (more SBR grade III and positive node invasion) and patients were 10 years younger at diagnosis. Sixteen differentially expressed genes such as MMP9, VEGF, PHB1, BRCA1, TFAP2C, GJA1 and TFF1 were also found. Additionally, an up-regulation of cytokeratins KRT8 and KRT18 may indicate a luminal B subtype in "South" (Lebanon, Tunisia and Morocco) tumors while "North" (France) tumors may more frequently be luminal A type. CONCLUSION: This study allowed the identification of specific clinical and transcriptomic parameters in patients from South Mediterranean countries.

Immunolocalization of BRCA1 protein in tumor breast tissue: prescreening of BRCA1 mutation in Tunisian patients with hereditary breast cancer?

BRCA1 is a tumor suppressor gene which is inactivated by mutation in familial breast and ovarian cancers. Over 300 different disease causing germ-line mutations have been described; 60% are unique to an individual family. This diversity and the large size of the gene lead us to search for a prescreening method for BRCA1 mutations. Since BRCA1 is a nuclear protein in normal cells, but reported by some authors to be cytoplasmic in breast tumor cells of patients with BRCA1 mutation, we evaluated immunohistochemistry as a prescreening technique to identify BRCA1 mutations in patients with familial presentation of breast cancer. Using a monoclonal antibody against the carboxy-terminal region of BRCA1, we performed immunohistochemistry on 18 tumor samples from patients with hereditary breast cancer. Cytoplasmic staining of BRCA1 was observed in 10 cases. Of the 18 tumors, 12 (66%) showed either BRCA mutation or BRCA1 accumulation or both, indicating that BRCA1 function might be lost in breast tumor cells not only through mutation, but also via abnormal cytoplasmic location. The immunohistochemical test used in this study would not be efficient as a pre-screening method of deleterious mutations, but it appeared useful to investigate tumor physiology.

Contribution of the BRCA1 and BRCA2 mutations to breast cancer in Tunisia.

Hereditary breast cancer accounts for 3-8% of all breast cancers, with mutations in the BRCA1 and BRCA2 genes responsible for up to 30% of these. To investigate the prevalence of BRCA1 and BRCA2 gene mutations in breast cancer patients with affected relatives in Tunisia, we studied 36 patients who had at least one first degree relative with breast and/or ovarian cancer Thirty-four 34 patients were suggestive of the BRCA1 mutation and two were suggestive of the BRCA2 mutation, based on the presence of male breast cancer detected in their corresponding pedigrees. Four mutations in BRCA1 were detected, including a novel frame-shift mutation (c.211dupA) in two unrelated patients and three other frameshift mutations--c.4041delAG, c.2551delG and c.5266dupC. Our study is the first to describe the c.5266dupC mutation in a non-Jewish Ashkenazi population. Two frameshift mutations (c.1309del4 and c.5682insA) were observed in BRCA2. Nineteen percent (7/36) of the familial cases had deleterious mutations of the BRCA1 or BRCA2 genes. Almost all patients with deleterious mutations of BRCA1 reported a family history of breast and/or ovarian cancer in the index case or in their relatives. Our data are the first to contribute to information on the mutation spectrum of BRCA genes in Tunisia, and we give a recommendation for improving clinical genetic testing policy.

A polymorphism in FAS gene promoter correlated with circulating soluble FAS levels.

The -670FAS (A/G) polymorphism and elevated concentrations of the soluble form of FAS (sFAS) have been associated with neoplasic and autoimmune diseases. This polymorphism in the Fas promoter gene could modulate the transcription of Fas expression and therefore, contribute to these pathologies. The in vivo functional significance of the FAS polymorphism was investigated by assessing the correlation between FAS genotypes and the serum-circulating FAS (sFAS) levels. We determined the FAS polymorphism distributions by restriction fragment-length polymorphism-polymerase chain reaction in 170 normal subjects. We used enzyme-linked immunosorbent assay to evaluate the sFAS levels in 44 of these individuals. Assessment of the concentration of sFAS indicated that the level of sFAS in subjects carrying the FAS-A/A genotype was significantly higher than that of those carrying the G/G genotype (3.90 ng mL(-1) vs. 3.12 ng mL(-1), P = 0.035). Our results demonstrated that FAS promoter polymorphism was significantly associated with the level of soluble FAS production in normal subjects.

[Detection of EBV by PCR in fresh and paraffin embedded samples of cavum tumour]. / Détection de l'EBV par PCR dans des biopsies fraîches et enrobées en paraffine du cancer du cavum.

The nasopharyngeal carcinoma (NPC) is frequent in Tunisia. It's the second ORL cancer of men after the larynx one. To analyse the NPC characteristics in our population, we determined the frequency of EBV infection in 47 paraffin-embedded and 6 fresh NPC biopsies. We first extracted the DNA from tumoral tissus and then amplified viral sequences by PCR to detect and to type the infecting virus (EBV-A or ABV-B). Our results showed that amplifiable DNA has been obtained from 34/47 paraffin-embedded NPC biopsies while 13/47 of the others biopsies contained degraded and not amplifiable DNA. All the fresh biopsies allowed to obtain DNA with good quality. The EBV infection frequency in paraffin-embedded NPC biopsies is 35% while EBV is detected in all fresh biopsies (6/6). Our analyse also showed that the EBV-A is predominant in our population compared to EBV-B as it was shown in most countries of the world. This study clearly shows that PCR results obtained with paraffin-embedded NPC biopsies are divergeant from those obtained with fresh biopsies. Because of DNA degradation in paraffin-embedded NPC biopsies, the biology molecular results from that kind of samples is criticable. Moreover the results obtained from fresh NPC biopsies confirmed the quasi-constant association of EBV with undifferenciated carcinoma nasopharyngeal type.

The peroxisome proliferator activated receptorgamma2 (PPARgamma2) Pro12Ala variant: lack of association with type 2 diabetes in obese and non obese Tunisian patients.

OBJECTIVES: Peroxisome proliferator activated receptorgamma2 (PPARgamma2) is a nuclear receptor that regulates adipocyte differentiation, lipid metabolism and probably insulin sensitivity. There have been several reports on the relationship between the PPARgamma2 Pro12Ala genotype and the development of obesity or type 2 diabetes. We designed a case-controlled study to investigate the potential association of the genetic variation of the PPARgamma2 gene with type 2 diabetes in Tunisians. METHODS: We used the polymerase chain reaction and restriction enzyme digestion to characterize the variation of the Pro12Ala polymorphism of the PPARgamma2 gene in 242 unrelated Tunisian patients with type 2 diabetes and 246 healthy control subjects. RESULTS: Analysis of the Pro12Ala polymorphism of the PPARgamma2 gene in patients with type 2 diabetes and in control subjects revealed no significant differences in the PPARgamma2 allele frequencies between diabetic patients and control subjects. However the PPARgamma2 Ala12 allele was found significantly associated with a high level of systolic blood pressure in diabetic patients. Stratification of diabetic patients on obese and non obese subjects showed non significant differences in the PPARgamma2 Ala12 frequency between the two groups. CONCLUSION: These results suggest that the PPARgamma2 gene is unlikely a major gene for type 2 diabetes mellitus or obesity in Tunisian subjects.