Performance of GeneXpert ultra in the diagnosis of Tuberculous Cervical lymphadenitis in formalin fixed paraffin embedded tissues.

The diagnosis of Tuberculous Cervical lymphadenitis (TCL) is challenging. The present study aimed to assess the performance of GeneXpert ultra (GXu) in the diagnosis of TCL on Formalin Fixed, Paraffin Embedded Tissues (FFPET). This study included 35 TCL cases confirmed by positive microbiology and/or positive GXu on Fresh Tissues (FT). The diagnostic performance parameters of GXu on FFPET were determined with reference to microbiology (positive Ziehl Neelsen and/or positive culture) and with reference to positive microbiology and/or positive GXu on FT. The GXu on FFPET was positive in 26/35 (74%) cases. With reference to positive ZN and or culture, the sensitivity, specificity, positive predictive value, and negative predictive value of GXu on FFPET were 63%, 100%, 100% and 71% respectively. With reference to positive microbiology and/or positive GXu on FT, these rates were 74%, 100%, 100% and 40% respectively. GXu on FFPET is a reliable tool for the detection of Mycobacterium tuberculosis complex particularly for cases where microbiological investigations have not been performed.

Molecular and immunohistochemical analysis of BRAF gene in primary cutaneous melanoma: Discovery of novel mutations.

BACKGROUND: Determination of BRAF status is mandatory for targeted therapy but it is not currently available in most developing countries. AIM: We aimed to analyze BRAF mutations in a series of cutaneous melanoma of Tunisian patients and to compare BRAF V600E mutation detection by immunohistochemistry to DNA sequencing. PATIENTS AND METHODS: Archival formalin fixed paraffin embedded tissues from 28 patients with primary cutaneous melanoma were evaluated for the BRAF mutations by Sanger sequencing and immunohistochemistry. RESULTS: BRAF mutations were detected in 19/28 (68%) of analyzed tumors. The sensitivity and specificity of immunohistochemistry compared to DNA sequencing for identification of the BRAF V600E mutation were 100%. We found five novel mutations, one of them had deleterious effect. CONCLUSION: The present study shows an intermediate frequency of mutations of the BRAF gene in cutaneous melanoma of Tunisian patients, and supports the use of immunohistochemistry as a screening test for the assessment of the BRAF V600E status in the management of melanoma in clinical practice.

ALK protein expression in pulmonary adenocarcinoma of Tunisian patients.

BACKGROUND: It is now necessary to determine ALK status in order to use targeted therapy. AIM: herein, we assess immunohistochemical profile of ALK protein in a series of Tunisian patients with pulmonary adenocarcinoma. MATERIALS AND METHODS: ALK protein expression was studied applying the D5F3 antibody with a fully automated Ventana CDx technique on a series of 19 patients. RESULTS: Positive ALK expression was found in one case (5.2%) corresponding to a papillary adenocarcinoma which showed a strong granular and homogenous cytoplasmic staining. The patient was a 30-years-old woman. CONCLUSION: The frequency of positive ALK expression based on immunohistochemistry in our series was similar to that reported in the world literature.

Prognostic value of immunohistochemical expression profile of epidermal growth factor receptor in urothelial bladder cancer.

We studied epidermal growth factor receptor (EGFR) expression profile in urothelial bladder carcinoma (UBC) which is a complex and heterogeneous disease with a large spectrum of histological aspects and deadly potential. Using immunohistochemistry (IHC), all GI tumors and pTa cases showed a low expression profile of EGFR. However, we note that when the stage of disease is advanced, tumors over-express EGFR. Indeed, 5% and 25% of GII and GIII tumors over-expressed EGFR, respectively. Further, 0% of pTa, 9,5% of pT1, 15% of pT2, 50% of pT3, and 90% of pT4 tumors were shown to be high EGFR expression (HEE). Moreover, we found a statistically significant correlation between the EGFR over-expression and grade and stage (P < 0.05). Thus, EGFR over-expression could be a potential prognostic marker to predict poor outcome in Tunisian patients with UBC.

MicroRNAs 146a and 147b biomarkers for colorectal tumor's localization.

The recently identified class of microRNAs (miRs) provided a new insight into cancer research, since abnormalities of members of microRNAs family have been found in various types of cancer. However, the relationship between five miRNAs (miR146a, miR155, miR21, miR135a, and miR147b) and colorectal cancer remains unclear. In the present study, we examined expression of these miRNAs in 25 pair-matched colon cancer tissues and normal colon mucosa. The expression levels of miR146a, miR155, miR21, miR135a, and miR147b were quantified by real-time PCR. We found that miR21, miR146a, and miR135a were all expressed at higher levels in colon tumors. On the other hand, miR146a and miR147b expressions are significantly higher in left colon compared to right colon. These two miRs, especially miR146a, seemed to be markers for the left colon tumors. Moreover, significant proportional and inverse correlations were found between miR expressions in tumor and healthy tissue, and the correlations profiles were different depending on cancer localization. Taken together, these results lead us to suggest the presence of different mechanisms regulating miRs expression and consequently their target genes in left and right colon. So the pathway of colorectal carcinogenesis would be different according to the site of the tumor.

Over-expression of EGFR is closely correlated to poor prognosis in Tunisian patients with non-small cell lung adenocarcinoma.

We studied epidermal growth factor receptor (EGFR) expression profile with the aim of an individualized therapy for patients with non-small cell lung cancer (NSCLC) from whom tumor materials are not sufficient for molecular investigations. Using immunohistochemistry, we found a markedly increased EGFR expression with significant difference in term of intensity and distribution from normal mucosa to primary tumors (p < 0.05). Furthermore, patients with EGFR positive tumors had significantly shorter survival than those with EGFR negative tumors (p = 0.0001). Thus, EGFR over-expression is a valuable prognostic marker to predict poor outcome in Tunisian patients with NSCLC.

[Immunohistochemical analysis of mismatch repair proteins in colorectal adenomas]. / Analyse immunohistochimique des protéins de réparation de I'ADN dans les adénomes colorectaux.

BACKGROUND: The deficiency of mismatch repair system is one of the main pathways in colorectal cancer. This system consists mainly of four proteins: MLH1, MSH2, MSH6 and PMS2. Colorectal cancer develops in the majority of cases from precancerous lesions called adenomas. Only few studies have reported on the deficiencies of these proteins in adenomas. AIM: In this study we used immunohistochemistry staining in colorectal adenomas to assay functional status of MLH1, MSH2, MSH6, and PMS2 proteins. METHODS: 102 adenomas from 93 patients were collected in our institution during six years (2007-2012). The immunohistochemical technique was performed with 4 antibodies: MLH1, MSH2, MSH6 and PMS2. The loss of expression was retained if adenomatous cells were not stained with positive internal control. Staining was considered as abnormal if nucleus of adenomatous cells showed low nuclear staining and / or heterogeneous one, while positive internal control had normal staining. RESULTS: Loss of expression of MSH2 and MSH6 in adenomatous cells was found in only 1 case which was a tubular adenoma 3mm high-grade dysplasia. Abnormal staining of the adenomatous cells was noted in 23 cases (22.5%) for MSH2 and in 8 cases (7.8%) for MSH6. No cases showed loss of expression of MLH1 and PMS2. Abnormal expression of MSH2 and MSH6 was not correlated with sex of patients, the location of the adenoma, its grade of dysplasia and its histological type. CONCLUSIONS: Loss of Mismatch repair proteins expression is a rare event in adenomas. However, the abnormal expression levels are higher in our study compared to those reported in the literature. This could reflect a higher rate of microsatellite instability in our patients. Multicenter and larger studies with molecular biology techniques are needed.

Evaluation of microsatellite instability, MLH1 expression and hMLH1 promoter hypermethylation in colorectal carcinomas among Tunisians patients.

BACKGROUND: About 10% to 15% of sporadic colorectal cancers demonstrate high level of microsatellite instability that is generally associated with aberrant methylation of hMLH1 promoter. AIM: To investigate the association between MSI status, hMLH1 protein expression and methylation status of the hMLH1 promoter in a cohort of Tunisian sporadic colorectal cancer. METHODS: Expression of MLH1 and MSH2 was determined by immunohistochemistry and the MSI status was analysed by microfluid-based on-chip electrophoresis. Methylation of the hMLH1 gene promoter was determined by methylation-specific PCR. RESULTS: Of the 150 colorectal cancers 57% were MSS, 28% were MSI-L and 15%were MSI-H. MSI-H tumors were more frequently right-sided, exhibited a stage III of TNM and tended more to be mucinous. The MSI status had no effect on overall patient survival. Most of the MSS/MSI-L 79% cancers were unmethylated at the hMLH1 promoter, while 26% MSI-H cancers were unmethylated. 84% of MSS and MSI-L expressed MLH1 and 52% of MSI-H expressed MLH1. Of the methylated MSI-H cases, 35% expressed MLH1 protein while 100% of the unmethylated MSI-H were positive for MLH1 staining. Of 11 MSI-H cancers with loss of MLH1 expression, all cases were also methylated while 50% MSI-H cancers with positive immunostaining for MLH1 were methylated at the hMLH1 promoter. CONCLUSION: Our study showed that MSI-H phenotype was mucinous, right-side and exhibit stade III of TNM. The relative correlation of MLH1 expression and promotor hypermethylation of hMLH1 for the MSI status is similar to that reported for several study.

The prognostic value of the immunohistochemical expression and mutational pattern of the key mediator of Wnt signaling: beta-catenin in Tunisian patients with colorectal carcinoma.

Beta-catenin plays a critical role with E-cadherin in cell-cell adhesion and is also a key molecule of the highly conserved Wnt signaling pathway that regulates cell proliferation and differentiation. Abrogation of this pathway is implicated in the carcinogenesis of several malignancies, especially colorectal cancer. The objective of this study was to determine the prognostic value of ß-catenin/E-cadherin complex in Tunisian patients with colorectal cancer. Matched primary tumors from 150 patients with sporadic colorectal adenocarcinomas were stained for ß-catenin and E-cadherin by using immunohistochemistry. Deletion of exon 3 of CTNNB1 gene was performed by polymerase chain reaction. Our results showed that ß-catenin and E-cadherin expressions were related inversely to tumor differentiation. Furthermore, the nuclear expression of ß-catenin was considerably increased in advanced colorectal adenocarcinomas and was highly associated with shorter survival of patients. Deletion of exon 3 of CTNNB1 was identified in 2 cases by using polymerase chain reaction and was significantly related to tumor invasion and aberrant expression of E-cadherin. The major finding of this study is that activation of ß-catenin gene by deletions involving exon 3 may be considered as an advanced event in colorectal tumorigenesis in Tunisian patients, in contrast to some worldwide studies. Moreover, disruption of ß-catenin/E-cadherin complex may be considered as a dependent predictor of disease outcome.

Loss of galectin-3 expression in mucinous colorectal carcinomas is associated with 5'CpG island methylation in Tunisian patients.

The ß-galactoside-binding protein galectin-3 (gal-3) has pleitropic biological functions and has been implicated in cell growth, differentiation, adhesion, RNA processing, apoptosis, and malignant transformation. To investigate the pattern of inactivation of the gal-3 gene (LGALS3) in colorectal cancers (CRC), we studied a series of Tunisian patients with CRC to identify abnormal methylation in LGALS3 promoter using a methylation-specific PCR. We also examined the gal-3 gene expression by reverse transcription-PCR and the expression of gal-3 protein by immunohistochemistry. Analysis of DNA methylation in nonmucinous colorectal carcinomas expressing gal-3 protein showed an unmethylated profile of LGALS3 promoter, whereas gal-3 was aberrantly methylated in mucinous colorectal carcinomas. Complete loss of the gal-3 expression both at mRNA and the protein level was associated with the gal-3 methylation in the mucinous colorectal carcinomas. Our results show that methylation of the gal-3 promoter could be an important mechanism in the regulation of the expression of this gene in mucinous CRCs.

Relationship between p73 polymorphism and the immunohistochemical profile of the full-length (TAp73) and NH2-truncated (ΔNp73) isoforms in Tunisian patients.

BACKGROUND: We examined the association of one linked GC/AT polymorphism at p73 with the risk of colorectal cancer. AIM: In this study, we investigated whether this polymorphism was related to the risk of colorectal cancer, and whether there were relationships between the polymorphism and loss of heterozygosity, protein expression, or clinicopathologic variables. MATERIALS AND METHODS: The p73 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism in 150 Tunisian patients with colorectal cancer and in 204 healthy control subjects. Immunohistochemistry was performed on normal mucosa, primary tumor, and metastasis. RESULTS: The frequencies of the genotypes were 52% for wild-type (GC/GC), 31% for heterozygotes (GC/AT), and 17% for variants(AT/AT) in patients, and 54%, 35%, and 11% in controls, respectively. There were no significant differences of the frequencies of the 3 genotypes between the patients and controls (P=0.11). We did not find any relationship of the genotypes with clinicopathologic features of patients. We found that patients with the GC/GC genotype had a significantly more favorable clinical outcome than the patients with the AT variants (AT/AT or GC/AT genotype). There were no significant difference between tumoral immunostaining and p73 polymorphism (P=0.16) but we found that the samples carrying the AT allele showed a tendency to be more stained in tumor. No loss of heterozygosity was observed at p73 locus. Our results suggest that the AT/AT genotype is significantly associated with poor prognosis in colorectal cancer. All these findings suggest that p73 polymorphism analysis may provide useful prognostic information for colorectal cancer patients.

Association of a p73 exon 2 GC/AT polymorphism with colorectal cancer risk and survival in Tunisian patients.

We examined the association of one linked GC/AT polymorphism at p73 with the risk of colorectal cancer. In the present study, we investigated whether this polymorphism was related to the risk of colorectal cancer, and whether there were relationships between the polymorphism and LOH, protein expression or clinicopathological variables. The p73 genotypes were determined by PCR-restriction fragment length polymorphism in 150 Tunisians patients with colorectal cancer and in 204 healthy control subjects. Immunohistochemistry was performed on normal mucosa, primary tumour and metastasis. The frequencies of the genotypes were 52% for wild-type (GC/GC), 31% for heterozygotes (GC/AT) and 17% for variants (AT/AT) in patients, and 54%, 35% and 11% in controls, respectively. There were no significant differences of the frequencies of the three genotypes between the patients and controls (p = 0.11). We did not find any relationship of the genotypes with clinicopathological features of patients. We found that patients with the AT/AT genotype had a significantly worse clinical outcome than those with the GC/AT and GC/GC genotype. There were no significant differences between tumoural immunostaining of the total p73 and p73 polymorphism (p = 0.16). However, we found a significant difference between the expression profile of DeltaNp73 isoform and frequencies of the three genotypes (p = 0.0001). No LOH was observed at p73 locus. Our results suggest that the AT/AT genotype is significantly associated with poor prognosis in colorectal cancer. All these findings suggest that p73 polymorphism analysis may provide useful prognostic information for colorectal cancer patients.