Sadok Korbi

Biological significance of promoter hypermethylation of tumor-related genes in patients with gastric carcinoma.

BACKGROUND DNA promoter hypermethylation is a potential means of inactivating tumor-related genes in several types of cancers. METHODS We investigated aberrant promoter hypermethylation of eleven tumor-related genes in 68 gastric carcinomas and 53 adjacent non-tumor tissues using methylation-specific PCR, and we correlated the findings with clinico-pathological features. RESULTS In gastric carcinoma tissues, hypermethylation frequencies of the investigated genes were 61.8% for RASSFIA, 52.9% for APC, 36.8% for MGMT, 30.9% for DAPK, 29.4% for P16, 26.5% for P14, 25% for SHP1, 23.5% for RAR-beta2, 20.6% for GSTP1, 13.2% for TIMP3, and 8.8% for hMLH1. For adjacent non-tumor samples, the frequencies of methylation were respectively 5.7, 37.7, 5.7, 24.5, 3.8, 5.7, 20.8, 5.7, 1.9, 3.8, and 0%. Hypermethylation of P16 correlates with intestinal subtype and cardiac location (P = 0.044 and P = 0.004, respectively), whereas methylation of GSTP1 correlates with diffuse subtype (P = 0.050). Methylation of SHP1 was associated with EBV infection (P = 0.014). Methylation of APC and RAR-beta2 genes were significantly associated with improved patients outcome (P = 0.007 and P = 0.042, respectively). CONCLUSIONS Our data suggest that methylation of multiple genes may be involved in the pathogenesis and correlated with the prognosis of gastric carcinomas.

DNA methyltransferase DNMT3b protein overexpression as a prognostic factor in patients with diffuse large B-cell lymphomas

Diffuse large B‐cell lymphomas (DLBCL) are the most common type of aggressive lymphomas, with considerable heterogeneity in clinical presentation, molecular characteristics, and outcome. Previous studies have showed significant correlations between DNA methyltransferase (DNMT) overexpression and unfavorable prognosis in human cancers. Therefore, we investigated in this study the biological and prognostic significance of DNMT1, DNMT3a, and DNMT3b protein expression in DLBCL. DNA methyltransferase (DNMT) expression was analyzed by immunohistochemistry in 81 DLBCL cases and correlated with clinicopathological parameters. Kaplan–Meier curves were used to estimate survival rates, and the Cox proportional hazard regression model was used to evaluate the prognostic impact of DNMT expression. Our results showed that overexpression of DNMT1, DNMT3a, and DNMT3b were detected in 48%, 13%, and 45% of investigated cases, respectively. DNA methyltransferase 1 (DNMT1) and DNMT3b overexpression was significantly correlated with advanced clinical stages (P = 0.028 and P = 0.016, respectively). Moreover, concomitant expression of DNMT1 and DNMT3b was significantly correlated with resistance to treatment (P = 0.015). With regard to survival rates, although data was available only for 40 patients, DNMT3b overexpression was significantly correlated with shorter overall survival (P = 0.006) and progression‐free survival (P = 0.016). Interestingly, multivariate analysis demonstrated that DNMT3b overexpression was an independent prognostic factor for predicting shortened overall survival (P = 0.004) and progression‐free survival (P = 0.024). In conclusion, DNMT3b overexpression was identified as an independent prognostic factor for predicting shortened survival of patients with DLBCL and could be, therefore, useful in identifying patients who would benefit from aggressive therapy. (Cancer Sci 2010)

Prognostic significance of aberrant promoter hypermethylation of CpG islands in patients with diffuse large B-cell lymphomas

BACKGROUND Diffuse large B-cell lymphoma (DLBCL) exhibits heterogeneous clinical features and a marked variable response to treatment. PATIENTS AND METHODS We investigated the prognostic significance of the methylation status of DAPK, GSTP1, P14, P15, P16, P33, RB1, SHP1, CDH1, APC, BLU, VHL, TIMP3, and RASSF1A genes in 46 DLBCL specimens from Tunisian patients. Methylation status of each gene was correlated with clinicopathological parameters including the International Prognostic Index (IPI), the germinal center immunophenotype, and response to treatment and survival. Overall survival (OS) and disease-free survival (DFS) rates were calculated by the Kaplan-Meier method and differences were compared with the log-rank test. RESULTS Hypermethylation of SHP1 was associated with elevated lactate dehydrogenase level (P = 0.031). P16 and VHL were frequently hypermethylated in patients with high IPI scores (P = 0.006 and 0.004) and a performance status of two or more (P = 0.007 and 0.047). In addition, hypermethylation of P16 was significantly associated with advanced clinical stages and B symptoms (P = 0.041 and 0.012). Interestingly, hypermethylation of DAPK was significantly correlated with resistance to treatment (P = 0.023). With regard to survival rates, promoter hypermethylation of DAPK, P16, and VHL were significantly associated with shortened OS (P = 0.003, 0.001, and 0.017, respectively) and DFS (P = 0.006, 0.003, and 0.046, respectively). In multivariate analysis, hypermethylation of DAPK remains an independent prognostic factor in predicting shortened OS (P = 0.001) and DFS (P = 0.024), as well as the IPI and the germinal center status. CONCLUSIONS This study demonstrates that DLBCLs with hypermethylated P16, VHL, DAPK, and SHP1 commonly show a biologically aggressive phenotype and worse prognosis. Interestingly, hypermethylation of DAPK was found to be an independent prognostic factor that may be used in conjunction with the conventional prognostic factors such as the IPI and the germinal center status.

Poly(ADP-ribose) polymerase-1, a novel partner of progesterone receptors in endometrial cancer and its precursors

Endometrial carcinomas are the most common malignancy of the female genital tract. Although the downregulation of the progesterone receptor (PR) in the progression of endometrioid carcinomas (ECs) has been well documented, the mechanism of PR alteration in endometrioid carcinogenesis is poorly understood. Recently, biochemical studies have shown that the DNA strand break‐sensing molecule poly(ADP‐ribose) polymerase (PARP‐1) was associated with the DNA binding domain of PR. In our present study, we show that in normal endometrial epithelium, the expression level of PARP‐1 protein is high in the proliferative phase but markedly decreases during the secretory phase. Interestingly, PARP‐1 expression gradually increases in nonatypical and atypical endometrial hyperplasia, reaching its highest level in grade I, and decreases significantly toward grade III ECs. Notably, PARP‐1 and PR expressions, in each stage, are positively correlated (p < 0.0001), with the exception of nonendometrioid carcinomas. Thus, these data suggest that PARP‐1 is substantially involved in the regulation of progesterone action in the development of ECs.

Proteomics-based identification of α1-antitrypsin and haptoglobin precursors as novel serum markers in infiltrating ductal breast carcinomas

BACKGROUND The identification of pathological markers of breast cancer for either diagnosis, treatment response or for survival is of critical importance. METHODS Serum protein profiling using 2-DE separations coupled to matrix-assisted laser desorption ionization mass spectrometry has been used to explore protein alterations in patients with infiltrating ductal breast carcinomas (IDCA). Sera from 39 breast cancer patients and 40 healthy controls were selected for screening study using 2-DE combined with MS. The protein expression patterns obtained after the depletion of high abundance proteins was determined by coomassie blue G-250 stain after 2-DE electrophoresis. RESULTS Six proteins that expressed differentially in the IDCA group were found. The expression levels of four isoforms corresponding to haptoglobin precursor and two isoforms of alpha1-antitrypsin precursor (alpha1-AT) were upregulated in sera from breast cancer patients. There was an increased expression of both proteins in the sera of patients with various tumor stages (I, II, III) in comparison to healthy women. Applying immunohistochemistry, we further validated alpha1-AT immunoreactivity in 51 formalin-fixed paraffin-embedded sections of breast tumors. Enhanced expression of alpha1-AT like activity has been found in IDCA breast tumors, as well as, in different histological types of breast cancer. No significant association has been found with lymph node occurrence, while in high tumor categories a tendency to an increased expression of alpha1-AT has been found, thereby suggesting a possible role of this protein in tumor growth. CONCLUSIONS These proteins may constitute new and useful markers of breast cancer that offer a clue to a better understanding of inflammatory pathways and carcinogenesis events linked to breast cancer progression.

Presence of simian virus 40 DNA sequences in diffuse large B-cell lymphomas in Tunisia correlates with aberrant promoter hypermethylation of multiple tumor suppressor genes.

The simian virus SV40 (SV40), a potent DNA oncogenic polyomavirus, has been detected in several human tumors including lymphomas, mainly in diffuse large B‐cell type (DLBCL). However, a causative role for this virus has not been convincingly established. Hypermethylation in promoter regions is a frequent process of silencing tumor suppressor genes (TSGs) in cancers, which may be induced by oncogenic viruses. In this study, we investigated the relationship between the presence of SV40 DNA sequences and the methylation status of 13 TSGs in 108 DLBCLs and 60 nontumoral samples from Tunisia. SV40 DNA presence was investigated by PCR assays targeting the large T‐antigen, the regulatory and the VP1 regions. Hypermethylation was carried out by methylation‐specific PCR. SV40 DNA was detected in 63/108 (56%) of DLBCL and in 4/60 (6%) of nontumoral samples. Hypermethylation frequencies for the tested TSGs were 74% for DAPK, 70% for CDH1, SHP1, and GSTP1, 58% for p16, 54% for APC, 50% for p14, 39% for p15, 19% for RB1, 15% for BLU, 3% for p53, and 0% for p300 and MGMT. No hypermethylation was observed in nontumoral samples. Hypermethylation of SHP1, DAPK, CDH1, GSTP1 and p16 genes were significantly higher in SV40‐positive than in SV40‐negative DLBCL samples (p values ranging from 0.0006 to <0.0001). Our findings showed a high prevalence of SV40 DNA in DLBCLs in Tunisia. The significant association of promoter hypermethylation of multiple TSGs with the presence of SV40 DNA in DLBCLs supports a functional effect of the virus in those lymphomas.

p16INK4A overexpression and HPV infection in uterine cervix adenocarcinoma.

Human papillomaviruses (HPVs) are causally involved in the genesis of cervical carcinomas and their precursors, and there is a strong relationship between the cyclin-dependant kinase inhibitor p16INK4A and HPV infection. This study was carried out to assess the correlations between p16INK4A expression as an early biomarker of the endocervical adenocarcinoma and HPV infection. p16INK4A expression and HPV typing were performed on 46 samples including 5 normal endocervix, 9 benign lesions of the endocervix, 25 endocervical adenocarcinomas, and 7 endometrioid adenocarcinomas of the uterine corpus. A semiquantification of the p16INK4A immunostaining was realized (using both the staining intensity and the percentage of positive cells) and was graded from 0 to 15. All of the 25 endocervical adenocarcinomas overexpressed p16INK4A; the adjacent epithelium and the connective tissue were strictly negative. No p16INK4A was detected in nine benign endocervical lesions and in five normal endocervix. Few endometrioid adenocarcinomas of the uterine corpus that infiltrate the endocervix exhibited a low immunoreactivity (score 0/15 or 1/15). This pattern of expression is significantly associated with HPV infection (p<10−3), mainly high-risk HPV types (p=0.02). Our results suggest that p16INK4A is a putative molecular biomarker that consistently discriminates uterine cervix adenocarcinomas from benign lesions and from endometrioid adenocarcinomas of the uterine corpus .

Trends in the incidence of cancer in the Sousse region, Tunisia, 1993-2006.

In this article, we analyzed trends in incidence rates of the major cancer sites for a 14‐year period, 1993–2006, in the Sousse region localized in the centre of Tunisia. Five‐year age‐specific rates, crude incidence rates (CR), world age‐standardized rates (ASR), percent change (PC) and annual percent change (APC) were calculated using annual data on population size and its estimated age structure. A total of 6,975 incident cases of cancer were registered, with a male to‐female sex ratio of 1.4:1. ASRs showed stable trends (−0.1% in males, and +1.0% in females). The leading cancer sites in rank were lung, breast, lymphoma, colon‐rectum, bladder, prostate, leukemia, stomach and cervix uteri. For males, the incidence rates of lung, bladder and prostate cancers remained stable over time. While, cancers of colon‐rectum showed a marked increase in incidence (APC: +4.8%; 95% CI: 1.2%, 8.4%) and non‐Hodgkins lymphoma (NHL) showed a notable decline (APC: −4.4%; 95% CI: −8.2, −0.6). For females, cancers of the breast (APC: +2.2%; 95% CI: 0.4%, 4.0%) and corpus uteri (APC: +7.4%; 95% CI: 2.8%, 12.0%) showed a marked increase in incidence during the study period, while the cervix uteri cancer decreased significantly (APC: −6.1%; 95% CI: −9.2%, −3.0%). The results underline the increasing importance of cancer as a cause of mortality and morbidity in Tunisia. Our findings justify the need to develop effective program aiming at the control and prevention of the spread of cancer amongst Tunisian population.

Prevalence and characteristics of the MMTV-like associated breast carcinomas in Tunisia

The involvement of a retrovirus homologous to the mouse mammary tumor virus (MMTV) in the pathogenesis of human breast cancer (BC) has long been assumed, but has never been proven. Previous studies have reported the detection of MMTV-like env sequences in variable proportions that did not exceed 40% of BC cases in several countries. However, these viral sequences have been found in higher proportion (74%) in Tunisian diagnosed with BC during the seventies. This study is an attempt to evaluate the current prevalence of MMTV-like env gene in BC in Tunisian women. We used semi-nested PCR that amplify a 190-bp MMTV-like env sequence, followed by direct sequencing to screen a series of 122 cases of BC randomly selected. The findings were correlated to clinicopathological data and immunohistochemical expression status of progesterone and oestrogen receptors, HER2, and P53. Specific MMTV-like env sequences were found in 17 (13.9%) cases of breast carcinomas, whereas the same sequences were not detected in matched normal breast tissues. The presence of the viral sequences correlates inversely with progesterone receptor expression (6.8% versus 20.3%; P=0.03) and HER2 overexpression (3.1% versus 17.7%; P=0.04). This present study confirms the presence of MMTV-like env sequences in BC in Tunisian women but describes an important decrease in the prevalence of the viral sequences compared with previous studies. This reduction may be due to some changes in the virological characteristics or exposure to the virus.

No evidence of human papillomavirus DNA in breast carcinoma in Tunisian patients

The aim of this study was to evaluate the prevalence of broad range of anogenital HPVs in a series of 123 Tunisian breast carcinoma cases. PCR assays were performed to amplify regions within the L1, E1, E6 and E7 open reading frames of a broad range of anogenital HPVs and specific types HPV16, 18, 31 and 33. In addition, we performed an in situ hybridization analysis using HPV biotinylated DNA probes for the detection of broad spectrum of anogenital HPV types, high-risk HPV types (16 and 18), intermediate-risk HPV types (31 and 33) and low-risk HPV types (6 and 11). None of the 123 breast carcinoma samples showed PCR amplification of HPV DNA using the broad spectrum consensus primer-pairs E1-350L/E1-547R and GP5+/GP6+ primers. Furthermore, neither high risk nor low-risk HPV types were detected in any of these cases. Moreover, using in situ hybridization for the detection of HPVs, we failed to detect a positive signal in neoplastic cells in any case. Our results suggest that anogenital papillomaviruses are unlikely to play a role in the development of breast carcinomas in Tunisian patients.