microRNAs expression profile in phyllodes tumors of the breast.

Proliferation of both stromal and epithelial components is a characteristic of fibroepithelial cancers of the breast. Certain fibroepithelial tumors of the breast, such as fibradenomas and phyllodes tumors, are challenging to distinguish and categorize. To find biomarkers for early diagnosis and improved disease management, it is crucial to deepen our understanding of the molecular pathogenesis pathways and tumor biology of PTs. It has been demonstrated that microRNAs (miRNAs) have significant roles in cancers; the expression pattern of miRNAs can help with cancer categorization and treatment. In contrast, little is understood about miRNAs in breast fibroepithelial cancers. This study was conducted retrospectively with the goal of assessing the expression of six mature miRNAs (hsa-miR-21, hsa-miR-155, hsa-miR-182, hsa-miR-34a, hsa-miR-148a, and hsa-miR-205) in breast fibroepithelial cancers using real-time PCR and predicting these miRNAs' targets using computational techniques. This study comprised 64 patients in total-55 with phyllodes tumors and 9 with fibroadenoma. The research was carried out at the Farhat Hached University Hospital's pathology department in Tunisia. These particular miRNAs expression levels were evaluated via qRT-PCR, and in silico techniques were utilized to predict potential miRNA targets. Analysis of miRNA expression in fibroadenoma and phyllodes tumor tissues revealed that miR-21, miR-155 and miR-182 were upregulated in PTs compared to fibroadenoma and normal tissues. We reported that miR-34a, miR-148a and miR-205 were downregulated in both borderline and malignant PTs compared to fibroadenoma and normal tissue. In silico miRNA target prediction suggested the involvement of these molecules in a wide context of cell signaling pathways.

Alterations in acetylcholinesterase and butyrylcholinesterase activities in chronic obstructive pulmonary disease: relationships with oxidative and inflammatory markers.

This work focused on finding a relationship between acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities and the development and severity of COPD. The possible link of these enzymes to oxidative and inflammatory processes was also investigated. The study included 229 healthy controls and 153 COPD patients. Erythrocyte AChE and plasma BChE activities were determined using spectrophotometric methods. Markers related to the oxidative status including thiobarbituric acid-reactive substances (TBARS), total protein carbonyls (PCs), advanced oxidation protein products (AOPP), reduced glutathione, nitric oxide, and peroxynitrite were measured. We also evaluated the activity of glutathione peroxidase, catalase, and superoxide dismutase in the plasma and erythrocytes. Serum levels of IL-6 and TNF-α were measured by the enzyme-linked immunosorbent assay. COPD patients showed increased AChE and BChE activities in comparison to healthy controls. Interestingly, AChE activity was higher in COPD smokers than in nonsmokers, while no difference was revealed for BChE. In addition, our results showed an inverse correlation between AChE activity and the levels of IL-6 in COPD smokers. Positive correlations were found, in COPD smokers, between plasma BChE activity and the levels of several biomarkers of protein oxidative damage including AOPP and PC. Our findings suggest that the alterations in AChE and BChE activities may be related to the oxidative and inflammatory processes in COPD patients rendering these enzymes as markers of COPD disease.

Clinicopathologic significance of DNA methyltransferase 1, 3a, and 3b overexpression in Tunisian breast cancers.

DNA methyltransferase 1, 3a, and 3b affect DNA methylation, and it is thought that they play an important role in the malignant transformation of various cancers. The current study was designed to analyze DNA methyltransferase expression by immunohistochemistry in a series of 94 Tunisian sporadic breast carcinomas. Results were correlated to clinicopathologic parameters and promoter methylation status of 8 tumor suppressor genes (BRCA1, BRCA2, RASSFA1, TIMP3, CDH1, P16, RARß2, and DAPK). Overexpression of DNA methyltransferase 1, 3a, and 3b was detected in 46.8%, 32%, and 44.7% of cases, respectively. A significant correlation was found between DNA methyltransferase 1 overexpression and Scarff-Bloom-Richardson histologic grade III (P = .01). DNA methyltransferase 3a overexpression was significantly associated with menopausal status (P = .01), Scarff-Bloom-Richardson histologic grade III (P = .0001), estrogen (P = .04) and progesterone (P = .007) receptor negativity, and HER2 overexpression (P = .004). However, DNA methyltransferase 3a overexpression was found less frequently in the luminal A intrinsic breast cancer subtype (9.7%) than in luminal B (53%), HER2 (41%), and triple-negative (50%) subtypes (P = .001). DNA methyltransferase 3b overexpression shows significant correlation with promoter hypermethylation of BRCA1 (P = .03) and RASSFA1 (P = .04) and with the hypermethylator phenotype (more than 4 methylated genes, P = .01). These data suggest that overexpression of various DNA methyltransferases might represent a critical event responsible for the epigenetic inactivation of multiple tumor suppressor genes, leading to the development of aggressive forms of sporadic breast cancer.

Investigation of human JC and BK polyomaviruses in breast carcinomas.

We have previously showed the presence of the simian virus 40 (SV40) and the mouse mammary tumor virus (MMTV)-like in a significant proportions of Tunisian breast carcinomas. However, to date there are no published studies concerning evaluation of the possible implication of the human polyomaviruses JC (JCV) and BK (BKV) in breast carcinomas. The presence of JCV and BKV DNA was investigated by PCR in a 123 primary breast carcinomas and matched adjacent non-tumor breast tissues. The results were correlated to clinicopathological and virological parameters. JCV T-antigen DNA was detected in 23% of breast carcinoma cases; however, all cases were negative for BKV. JCV T antigen PCR products were further confirmed as authentic JCV genome by direct sequencing. JCV was found in invasive ductal carcinomas (28/112 cases) but not in invasive lobular carcinomas (0/5) or medullary carcinomas (0/6). JCV DNA presence correlates inversely with the expression of estrogen (P = 0.022) and progesterone (P = 0.008) receptors. JCV DNA presence correlates also with "triple negative" phenotype (P = 0.021). With regard to virological data, a trend toward an inverse correlation was noted between the presence of JCV and SV40 (P = 0.06). Moreover, significant correlation was found between multiple viral infection (JCV, and/or SV40, and/or MMTV-like in the same tumor) and "triple negative" phenotype (P = 0.001) and also with p53 accumulation (P = 0.028). To the best of our knowledge, this is the first study demonstrating the presence of JCV in a subset of breast carcinomas. Also our results suggest that "triple negative" breast carcinomas are viral-related tumors.

Investigation of Epstein-Barr virus in breast carcinomas in Tunisia.

Breast carcinoma is a major cause of death among women, and the potential implication of viruses in its pathogenesis remains worth a hypothesis. The potential role of Epstein-Barr virus (EBV) in its pathogenesis is still a subject of continued discussion and investigation. The aim of this study was to evaluate the prevalence of EBV in sporadic breast cancers in Tunisia, and to determine the clinicopathological characteristics of virus-positive cases. Viral presence has been evaluated by polymerase chain reaction (PCR), in situ hybridization, and immunohistochemistry investigated on tumor tissues and their corresponding normal breast tissues collected from 123 Tunisian women with sporadic breast carcinomas. Viral status in tumors was then correlated with various clinicopathological parameters. Using specific PCR assays, EBV DNA was found in 33 (27%) out of 123 breast carcinoma cases. EBV-encoded small RNAs (EBERs) in situ hybridization was negative in the neoplastic cells, but stomal lymphocytes were positive in 4 cases. Immunohistochemistry for latent membrane protein 1 (LMP1) was negative in all cases. None of the normal breast tissues showed positive results for EBV using PCR, in situ hybridization, and immunohistochemistry. A correlation was found between EBV DNA presence and the negativity of estrogen receptor (P=0.008). However, no significant correlation was found for the other parameters investigated, including patient age, Scarff-Bloom-Richardson (SBR) histological grade, tumor size, and histological node involvement. With regard to survival data, overall and disease-free survivals were shorter in EBV-positive breast carcinoma cases than in EBV-negative ones, but this difference did not reach statistical significance. Our study indicates the presence of EBV DNA in a significant proportion of breast cancer in Tunisia. Further studies are required to elucidate the role of this virus in breast carcinogenesis.

Investigation of human papillomavirus in bladder cancer in a series of Tunisian patients.

The association between human papillomavirus (HPV) infection and development of bladder cancer is variable. Furthermore, the prevalence of HPV DNA in bladder carcinoma subtypes varies from study to study. To clarify the impact of HPV infection on the development of bladder carcinoma, we performed a retrospective study on Tunisian patients to determine the status of HPV infection in urothelial carcinoma, squamous cell carcinoma, and adenocarcinoma. A total of 125 formalin-fixed, paraffin-embedded archival tissue specimens of bladder carcinoma were reviewed and classified according to the World Health Organization (WHO) classification of tumors (119 urothelial carcinomas, five squamous carcinomas, and one adenocarcinoma). Anogenital HPV DNA detection was performed using three different polymerase chain reaction (PCR) techniques: the first one used primers pU-2R/pU-1M specific to high-risk oncogenic HPV; the second one used primers PU-2R/PU-31B specific to low-risk oncogenic HPV; and the third one employed consensus primers (E1-547R/E1-350L). No evidence of HPV infection was detected by morphological examination and PCR in any case of bladder carcinoma. Our study shows that the anogenital HPVs investigated are not associated with the pathogenesis of bladder cancer in Tunisia; however, the question of whether other subtypes of HPV contribute to bladder carcinogenesis remains to be clarified.

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.

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.

Prevalence and characteristics of Epstein-Barr virus-associated gastric carcinomas in Tunisia.

OBJECTIVE: Epstein-Barr virus (EBV) has been linked to gastric carcinoma (GC) with worldwide geographical variations of prevalence ranging from 1 to 18% of cases. Investigations carried out in north Africa have shown that some EBV-associated types of cancers are common in this area. This study was taken to determine the prevalence of EBV-associated GC in Tunisia. METHODS: Ninety-six nonselected GC cases (male/female ratio 1.7/1, mean age 60.9 years, range: 20-88 years) were evaluated for the presence of EBV by polymerase chain reaction as well as by in-situ hybridization for EBV-encoded small RNAs (EBERs) and immunohistochemistry for LMP-1 and EBNA-2 expression. RESULTS: EBV was detected by polymerase chain reaction in 36% of cases, whereas EBERs were detected in the tumor cells in only four cases (4.1%). Immunohistochemistry for LMP-1 and EBNA-2 was negative in all cases. The mean age for patients harboring EBERs-positive GC was 55.7 years (range: 52-59 years). All EBERs-positive GC cases were males of advanced clinical stage (pT3-pT4). According to Lauren's classification, two cases were of diffuse histological type and two cases were of intestinal type. In three cases, the tumors have a proximal location and in the remaining case the tumor arises in the antrum. All EBV strains detected from EBV-associated GC were exclusively of type A and D, prototype F, and XhoI-maintained variant. CONCLUSION: We conclude that the prevalence of EBV-associated GC in Tunisia is low (4.1%), suggesting that this virus is not an important etiological factor in GC arising in north African populations. The clinicopathological profile of EBV-associated GC in Tunisia did not differ markedly from that found elsewhere.

Investigation of allelic imbalances on chromosome 3p in nasopharyngeal carcinoma in Tunisia: high frequency of microsatellite instability in patients with early-onset of the disease.

Tunisia is one of the world's intermediate risk areas for nasopharyngeal carcinoma (NPC). Loss of heterozygosity (LOH) on the short arm of chromosome 3 (3p) is the most frequent genetic change reported in NPC from endemic areas. In the present study, we investigate the incidence of LOH and microsatellite instability (MSI) on chromosome 3p in 49 microdissected primary NPC specimens and corresponding non-cancerous tissues from Tunisian patients using six microsatellite polymorphic markers. LOH at one or more markers was observed in 40 out of 48 informative cases (83.3%). The markers D3S1038 at 3p25.2-26.1 and D3S1076 at 3p21.1-21.2 have showed the highest frequency of LOH (51.3%), followed by D3S1067 at 3p14.3-21.1 (48.7%), D3S1568 at 3p21.3 (47.4%), D3S659 at 3p13 (15.3%), and D3S1228 at 3p14.1-14.2 (11%). Interestingly, MSI at one or more microsatellite markers was observed in 15 cases (31.2%). The highest frequency of MSI was presented by D3S1568 (18.4%), D3S1067 (17.9%), and D3S1038 (12.8%). With regard to clinicopathological features, LOH was found to be less common in young patients (under 25 years) than in adults (p=0.04), whereas MSI was found to be more frequent in patients under 45 years than in older patients (p=0.006). No significant correlation was found between LOH or MSI and the other clinicopathological features investigated including, gender, tumor size, lymph node metastasis, UICC clinical stage, and histological subtype. This study revealed different patterns of allelic imbalance on chromosome 3P in NPC between age groups in Tunisia, and suggests an alteration in the DNA mismatch repair machinery that may be, in part, responsible of the early age onset form of this disease in North African populations. More attention should be given to the mismatch repair system in the juvenile form of this disease in future studies.

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.

CD10 expression by fusiform stromal cells in nasopharyngeal carcinoma correlates with tumor progression.

CD10 is a cell surface zinc metalloprotease expressed through a variety of normal cell types, including lymphoid precursor cells, germinal center B lymphocytes, and some epithelial cells. Many studies showed that CD10 expression is associated with the tumor progression of a large variety of cancers, such as breast and colorectal carcinomas. The aim of this study was to investigate the expression of CD10 in nasopharyngeal carcinoma (NPC). The expression of CD10 was immunohistochemically examined in 47 paraffin embedded NPC biopsies from Tunisian patients compared with 16 reactional nasopharyngeal mucosas. A significant expression of CD10 was observed in stromal fusiform cells in 46.8% of NPC cases but was not in malignant and normal epithelial cells. There was no significant expression of CD10 in control group. The stromal expression of CD10 was more frequently detected in advanced clinical stage than early stage (56 vs 23%; p=0.04) and in patients older than 25 years than in patients under 25 years (56.2 vs 26.5%; p=0.05). Our study is the first in investigating CD10 expression in nasopharyngeal carcinoma and showed that CD10 expression by stromal cells in this malignancy play an important role in tumor progression, particularly in older patients.