BRCA1 Promoter Hypermethylation in Malignant Breast Tumors and in the Histologically Normal Adjacent Tissues to the Tumors: Exploring Its Potential as a Biomarker and Its Clinical Significance in a Translational Approach.

The hypermethylation status of the promoter region of the breast cancer 1 (BRCA1), a well-known tumor suppressor gene, has been extensively investigated in the last two decades as a potential biomarker for breast cancer. In this retrospective study, we investigated the prevalence of BRCA1 promoter methylation in 84 human breast tissues, and we correlated this epigenetic silencing with the clinical and histopathological parameters of breast cancer. We used methylation-specific PCR (MSP) to analyze BRCA1 promoter hypermethylation in 48 malignant breast tumors (MBTs), 15 normal adjacent tissues (NATs), and 21 benign breast lesions (BBLs). The results showed that BRCA1 promoter hypermethylation was higher in MBTs (20/48; 41.67%) and NATs (7/15; 46.67%) compared to BBLs (4/21; 19.05%). The high percentage of BRCA1 hypermethylation in the histologically normal adjacent tissues to the tumors (NATs) suggests the involvement of this epigenetic silencing as a potential biomarker of the early genomic instability in NATs surrounding the tumors. The detection of BRCA1 promoter hypermethylation in BBLs reinforces this suggestion, knowing that a non-negligible rate of benign breast lesions was reported to evolve into cancer. Moreover, our results indicated that the BRCA1 promoter hypermethylated group of MBTs exhibited higher rates of aggressive features, as indicated by the SBR III grade (14/19; 73.68%), elevated Ki67 levels (13/16; 81.25%), and Her2 receptor overexpression (5/20; 25%). Finally, we observed a concordance (60%) in BRCA1 promoter hypermethylation status between malignant breast tumors and their paired histologically normal adjacent tissues. This study highlights the role of BRCA1 promoter hypermethylation as a potential useful biomarker of aggressiveness in MBTs and as an early marker of genomic instability in both histological NATs and BBLs.

Pure squamous cell carcinoma of the pancreas: a rare and challenging diagnosis (case report).

Squamous cell carcinoma (SCC) of the pancreas is a rare exocrine ductal tumour with unknown pathophysiology and poor treatment options. We present a case of SCC in a 59-year-old male patient who presented with epigastric pain, vomiting, anorexia, asthenia, and weight loss. Abdomino-pelvic CT revealed a pancreatic mass with adenopathy satellites. Surgical biopsies were negative, and a trans-duodenal gastric endoscopy showed suspicious lymphadenopathies and a hypoechoic lesion invading the portal vein. An anapathological study confirmed SCC at the site of intense necrotic and inflammatory changes. The patient received radio-chemotherapy, but ultimately developed peritoneal carcinoma.

The Evaluation of Vascular Endothelial Growth Factor A (VEGFA) and VEGFR2 Receptor as Prognostic Biomarkers in Bladder Cancer.

Vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2) are the most important tissue factors involved in tumor growth and angiogenesis. The aim of this study was to evaluate the promoter mutational status of VEGFA and the expression levels of VEGFA, VEGFR1, and VEGFR2 in bladder cancer (BC) tissues and to correlate the results with the clinical-pathological parameters of BC patients. A total of 70 BC patients were recruited at the Urology Department of the Mohammed V Military Training Hospital in Rabat, Morocco. Sanger sequencing was performed to investigate the mutational status of VEGFA, and RT-QPCR was used to evaluate the expression levels of VEGFA, VEGFR1, and VEGFR2. Sequencing of the VEGFA gene promoter revealed the presence of -460T/C, -2578C/A, and -2549I/D polymorphisms, and statistical analyses showed a significant correlation between -460T/C SNP and smoking (p = 0.02). VEGFA and VEGFR2 expressions were significantly up-regulated in patients with NMIBC (p = 0.003) and MIBC (p = 0.03), respectively. Kaplan-Meier analyses showed that patients with high VEGFA expression had significantly longer disease-free survival (p = 0.014) and overall survival (p = 0.009). This study was very informative, showing the implication of VEGF alterations in BC, suggesting that VEGFA and VEGFR2 expressions could be promising biomarkers for the better management of BC.

AKT1 and PIK3CA activating mutations in Moroccan bladder cancer patients´ biopsies and matched urine.

Introduction: in cancer cells, activating mutations in PIK3CA and AKT1 genes, major players of PI3K-AKT-mTOR signalling pathway, are widely reported in many cancers and present attractive targets for the identification of new therapeutics and better cancer management. The present study was planned to evaluate the mutational status of PIK3CA and AKT1 genes in bladder cancer patients and to assess the association between these mutations and patients´ clinico-pathological features. Methods: in this prospective study, bladder cancer biopsies and matched urine sediments samples were collected form 70 patients. Mutations were assessed by deoxyribonucleic acid (DNA) sequencing and correlation with clinico-pathological data was performed using SPSS software. Results: AKT1 alterations were poorly detected. Only one patient with pT1 stage and high-grade tumour carried the E17K mutation. In PIK3CA exon 9, 2 point mutations, E545K and Q546E, and a SNP (E547E) were reported, whereas in exon 20, 2 point mutations (L989V and H1047R) and 2 SNPs (I1022I and T1025T) were detected. PIK3CA mutations were mainly observed in early stages and high-grade tumours. Statistical analysis showed no significant association between the studied AKT1 and PIK3CA mutations and patients´ clinico-pathological parameters (p > 0.05). Detection of these mutations in voided urine samples showed a high specificity (100%) for both genes and a moderate sensitivity: 100% for AKT1 and 66.7% for PIK3CA genes. Conclusion: this study shows clearly that mutations in AKT1 and PIK3CA are rare events and could not be considered as valuable biomarkers for bladder cancer management.

Evaluation of DNA methylation in promoter regions of hTERT, TWIST1, VIM and NID2 genes in Moroccan bladder cancer patients.

Promoter hypermethylation have been reported to play a key role in bladder cancer development and progression. The aim of this study is to evaluate the methylation status of hTERT, TWIST1, VIM and NID2 genes in bladder cancer. The methylation status was evaluated using the Methylation-Specific PCR (MSP) approach on 70 tumour biopsies from Moroccan bladder cancer patients. Overall, methylation frequencies of hTERT, TWIST1, VIM and NID2 genes, were 90%, 85.71%, 67.14% and 67.14%, respectively. Hypermethylation of all studied genes was found in all pathological grades and stages of bladder cancer. Nevertheless, statistical analysis showed no significant association between promoter methylation of hTERT, TWIST1, VIM and NID2 genes and tumours stage/grade (p value >0.05). Moreover, we have investigated the association between the methylation pattern of selected genes and the treatment outcome in a sub-group of non-muscle-invasive bladder cancer cases (52/70). Hypermethylation of hTERT, TWIST1, VIM and NID2 was detected in 83.34%; 66.67%; 83.34% and 58.34% of recurrent cases, respectively, and in 80%; 80%; 80% and 60% of progressive cases, respectively. Statistical analysis highlighted a significant association between TWIST1 hypermethylation and tumour recurrence (p = 0.041<0.05). Our results indicate that hypermethylation of hTERT, TWIST1, VIM and NID2 genes is a frequent epigenetic event in bladder cancer and could be a promising therapeutic target to prevent bladder cancer progression and metastasis.

Correlation of immunohistochemical expression of HIF-1alpha and IDH1 with clinicopathological and therapeutic data of moroccan glioblastoma and survival analysis.

INTRODUCTION: Glioblastomas are aggressive primary intracranial tumours of the central nervous system causing significant mortality and morbidity worldwide. OBJECTIVE: This study aims to evaluate the prognostic value of tissue expression by immunostaining of hypoxia-inducible factor (HIF-1α), isocitrate dehydrogenase 1 (IDH1), and tumour protein p53 in glioblastoma in Moroccan patients. The association of HIF-1α, IDH1, and p53 expression with the clinicopathological data and overall patient survival (OS) was also evaluated. MATERIALS AND METHODS: Confirmed glioblastomas were included in this study. Twenty-two tissue samples were obtained by neurosurgical intervention resulting from total resection, and subtotal resection or biopsy. Karnofsky index, histological type of tumour, and the status of IDH1, p53 protein, and HIF-1α expression by immunostaining were reported. RESULTS: The majority of the patients were males (64%) with a sex ratio of 1.75. The average age was 54 ± 13. Median follow-up was 10.10 months and median overall survival was 10 months. The expression of HIF-1α was high in 10 samples (45%) and low in 12 (55%). There was a statistically significant difference in OS of 85% at 12 months for the subgroup of patients "HIF-1α negative IDH1 positive" p = 0.038, the unadjusted analysis showed that the group "HIF-1α positive, IDH1 positive" was a poor prognostic factor, the HR was 0.08 (95% CI: 0.009-0.756, p = 0.027). CONCLUSION: Patients with negative HIF-1α expression and positive IDH1 expression have a better prognosis, suggesting that these two biomarkers may be useful in the search for new approaches for targeted therapy in glioblastoma.

Immune Checkpoint and Telomerase Crosstalk Is Mediated by miRNA-138 in Bladder Cancer.

BACKGROUND: Tumor recurrence and progression in non-muscle invasive bladder cancer (NMIBC), therapy failure, and severe side effects in muscle invasive bladder cancer (MIBC) are the major challenges in the clinical management of bladder cancer (BC). Here, we identify new molecular targetable signatures to improve BC patients' stratification and the outcome of current immunotherapies. MATERIAL AND METHODS: In a prospective cohort of 70 BC patients, we assessed the genetic and molecular regulation of TERT in maintaining telomere length in parallel to immune checkpoint and microRNA expression. RESULTS: TERT was undetectable in healthy bladder tissues but upregulated in invasive BC stages and high tumor grade. Its expression was linked with the combined effect of the C250T mutation and THOR hypermethylation, associated with progressing tumors and maintaining of telomere length. In the same cohort, PD-L1 scored highest in NMIBC, while PD-L2 was upregulated in MIBC. We also show that miR-100-5p and 138-5p were highly expressed in healthy bladder specimens and cell line, while expression decreased in the BC tissues and BC cell lines. In line with the binding prediction for these miRNAs on target genes, miRs 100-5p and 138-5p expression strongly inverse correlated with TERT, PD-L1, and PD-L2 expression, but not PD1. CONCLUSION: We identify a loop involving TERT, PD1-ligands, and miR-138-5p in BC, that might represent not only a useful biomarker for improved diagnosis and patients' stratification but also as a promising axis that might be therapeutically targeted in situ.

BRAFV600E hot spot mutation in thyroid carcinomas: first Moroccan experience from a single-institution retrospective study.

BACKGROUND: The incidence of thyroid cancer is increasing worldwide at an alarming rate. BRAFV600E mutation is described to be associated with a worse prognostic of thyroid carcinomas, as well as extrathyroidal invasion and increased mortality. OBJECTIVE: To our knowledge, there are no reported studies neither from Morocco nor from other Maghreb countries regarding the prevalence of BRAFV600E mutation in thyroid carcinomas. Here we aim to evaluate the frequency of BRAFV600E oncogene in Moroccan thyroid carcinomas. METHODS: In this Single-Institution retrospective study realized in the Anatomic Pathology and Histology Service in the Military Hospital of Instruction Mohammed V 'HMIMV' in Rabat, we report, using direct genomic sequencing, the assessment of BRAFV600E in 37 thyroid tumors. RESULTS: We detected BRAFV600E mutation exclusively in Papillary Thyroid Carcinomas 'PTC' with a prevalence of 28% (8 PTC out 29 PTC). Like international trends, Papillary Thyroid Carcinomas 'PTC' is more frequent than Follicular Thyroid Carcinomas 'FTC' and Anaplastic Thyroid Carcinomas 'ATC' (29 PTC, 7 FTC and 1 ATC). CONCLUSION: Our finding gives to the international community the first estimated incidence of this oncogene in Morocco showing that this prevalence falls within the range of international trends (30% to 90%) reported in distinct worldwide geographic regions.

Urinary schistosomiasis: report of case diagnosed in bladder biopsy.

BACKGROUND: Urinary schistosomiasis is a common parasitic disease in endemic countries. CASE PRESENTATION: We report the case of a patient who was on a working trip to Mauritania. This parasitosis, suspected in the presence of hematuria and the notion of stay in an endemic zone, was confirmed by the presence of Schistosoma heamatobium eggs during the histological examination of the bladder biopsy performed after cystoscopy, highlighting a bilharzial granuloma and of course, the diagnosis was confirmed by the presence of eggs during the direct examination of the freshly collected urine. CONCLUSIONS: It should be pointed out that the diagnosis of schistosomiasis must be evoked with the association of hematuria and the particular inflammatory aspect of the vesical mucosa and, of course, the notion of stay in an endemic zone.

Evaluation of glutathione S-transferase pi 1 expression and gene promoter methylation in Moroccan patients with urothelial bladder cancer.

BACKGROUND: Glutathione S-transferase pi 1 (GSTP1) is a cytosolic detoxifying enzyme that protects cells against deleterious effects of oxidative stress. Deregulated expression of GSTP1 protein and aberrant promoter methylation of GSTP1 gene were reported in various human tumors and were shown to be involved in the molecular pathway for cancer development. AIMS AND METHODS: In this study, we aimed to determine the expression status of GSTP1 in relation to its gene promoter methylation in Moroccan population of 30 bladder cancer (BC) patients and in two noncancerous bladder tissues used as controls. GSTP1 expression was assessed by immunohistochemistry and GSTP1 gene promoter methylation status was studied by methylation-specific PCR (MS-PCR). RESULTS: Glutathione S-transferase pi 1 was expressed in the two normal tissues. In BC cases, GSTP1 expression was strong in 23.33% (7/30), moderate in 60% (18/30), and weak in 13.33% (4/30) of cases, while GSTP1 was not expressed in one cancer case (3.33%). Variability of GSTP1 expression does not correlate with high-grade cancer or invasive-stage (p > 0.05). No GSTP1 gene promoter methylation was detected in all control and cancer cases. CONCLUSION: Our data suggest that GSTP1 expression is not associated with BC development, limiting its use as a biomarker for BC management in Morocco. Moreover, difference in GSTP1 expression among BC cases is not due to GSTP1 promoter methylation.

Inflammatory myofibroblastic tumor of the lacrimal gland: case report of an exceptional location.

BACKGROUND: Inflammatory myofibroblastic tumour (IMT) is a mesenchymal neoplasm of intermediate biological potential that may affect a wide range of anatomic sites but has a particular predilection for the lung and intra-abdominal soft tissues. CASE PRESENTATION: We report here an exceptional case of inflammatory myofibroblastic tumor arising in the lacrimal gland and presenting as an orbital mass in a 24-year-old male. CONCLUSION: This report aims to discuss the importance of histopathological and immunohistochemical findings in arriving at the diagnosis, which helps dictate the management, treatment and prognosis of the patient.

Expression of human epidermal growth factor receptor 2 in bladder urothelial carcinoma.

BACKGROUND: Urothelial bladder carcinoma (UBC) is one of the most prevalent cancers in men worldwide. Human epidermal growth factor receptor 2 (HER2) expression has been detected in a wide range of urothelial carcinoma. Despite many reports in the literature, the prognostic significance of this overexpression remains unclear. The aim of this study was to assess the expression of HER2 in urothelial bladder carcinomas and its association with clinical and pathological parameters. METHODS: 103 cases of UBC were diagnosed in our department between January 2014 and December 2015. The tumor specimens obtained by transurethral resection or cystectomy were evaluated by immunohistochemistry using HER2 antibody. RESULTS: HER2 protein overexpression was present in 11.7% of cases and associated with tumor grade (p = 0.003) and pathological stage (p = 0.015). In multivariate analysis, HER2 overexpression was associated only with tumor grade (P = 0.04). CONCLUSION: HER2 protein overexpression is noted in patients with high grade cancer. This expression may select patients for anti HER2 targeted therapy. Future larger and prospective studies will verify the frequency of HER2 alteration and the role of HER2 in the aggressive behavior.

NADPH Oxidase NOX4 Is a Critical Mediator of BRAFV600E-Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas.

AIMS: The BRAFV600E oncogene, reported in 40%-60% of papillary thyroid cancer (PTC), has an important role in the pathogenesis of PTC. It is associated with the loss of thyroid iodide-metabolizing genes, such as sodium/iodide symporter (NIS), and therefore with radioiodine refractoriness. Inhibition of mitogen-activated protein kinase (MAPK) pathway, constitutively activated by BRAFV600E, is not always efficient in resistant tumors suggesting that other compensatory mechanisms contribute to a BRAFV600E adaptive resistance. Recent studies pointed to a key role of transforming growth factor ß (TGF-ß) in BRAFV600E-induced effects. The reactive oxygen species (ROS)-generating NADPH oxidase NOX4, which is increased in PTC, has been identified as a new key effector of TGF-ß in cancer, suggestive of a potential role in BRAFV600E-induced thyroid tumors. RESULTS: Here, using two human BRAFV600E-mutated thyroid cell lines and a rat thyroid cell line expressing BRAFV600E in a conditional manner, we show that NOX4 upregulation is controlled at the transcriptional level by the oncogene via the TGF-ß/Smad3 signaling pathway. Importantly, treatment of cells with NOX4-targeted siRNA downregulates BRAFV600E-induced NIS repression. Innovation and Conclusion: Our results establish a link between BRAFV600E and NOX4, which is confirmed by a comparative analysis of NOX4 expression in human (TCGA) and mouse thyroid cancers. Remarkably, analysis of human and murine BRAFV600E-mutated thyroid tumors highlights that the level of NOX4 expression is inversely correlated to thyroid differentiation suggesting that other genes involved in thyroid differentiation in addition to NIS might be silenced by a mechanism controlled by NOX4-derived ROS. This study opens a new opportunity to optimize thyroid cancer therapy. Antioxid. Redox Signal. 26, 864-877.

Helicobacter pylori Primary Antibiotic Resistance in 2015 in Morocco: A Phenotypic and Genotypic Prospective and Multicenter Study.

Knowledge of local antibiotic resistance is crucial to adaption of the choice of effective empirical first-line treatment for Helicobacter pylori infection. The aim of this study was to evaluate, for the first time in Morocco, the prevalence of the primary resistance of H. pylori to clarithromycin, metronidazole, amoxicillin, levofloxacin, tetracycline, and rifamycin. We conducted a 1-year prospective study (2015), including 255 Moroccan patients referred for gastro-duodenal endoscopy to two hospitals of Rabat (Morocco) and never previously treated for H. pylori infection. Three gastric biopsies were collected: one for histology, one for culture, and one for molecular detection of H. pylori and the mutations in 23S rRNA genes that confer resistance to clarithromycin. Antimicrobial susceptibility testing was performed on isolated strains by Etest and disk diffusion methods. One hundred seventy-seven patients were infected (69.4%). The prevalence of primary resistances of H. pylori to clarithromycin was 29%, 40% to metronidazole, 0% to amoxicillin, tetracycline, and rifamycin, and 11% to levofloxacin. Only four isolates (2%) were resistant to both clarithromycin and metronidazole. The high level of primary clarithromycin resistance in the H. pylori strains infecting the Moroccan population leads us to recommend the abandonment of the standard clarithromycin-based triple therapy as a first-line treatment in Morocco and to prefer a concomitant quadruple therapy.

Primary Burkitt lymphoma of the thyroid gland: case report of an exceptional type of thyroid neoplasm and review of the literature.

BACKGROUND: Primary thyroid lymphoma is an uncommon pathological entity that accounts for only 1 to 5 % of all thyroid malignancies. Primary Burkitt lymphoma of the thyroid gland is very rare. This article presents the first Moroccan case of a primary BL of the thyroid to be reported in the literature to date. CASE PRESENTATION: We describe here a case of a 70-year-old male who developed a rapidly enlarging thyroid gland with progressive symptoms of compression. Core biopsy confirmed the diagnosis of Burkitt lymphoma. The patient died of septic shock, 2 weeks after the first cycle of appropriate therapeutic chemotherapy. CONCLUSIONS: This presentation emphasizes the importance of considering lymphoma when dealing with a thyroid mass, as its management is different from that of other thyroid pathologies, and affords an opportunity to review a very rare type of primary thyroid lymphoma.

Evaluation of Combined Quantification of PCA3 and AMACR Gene Expression for Molecular Diagnosis of Prostate Cancer in Moroccan Patients by RT-qPCR

Prostate cancer (PCa) remains one of the most widespread and perplexing of all human malignancies. Assessment of gene expression is thought to have an important impact on cancer diagnosis, prognosis and therapeutic decisions. In this context, we explored combined expression of PCa related target genes AMACR and PCA3 in 126 formalin fixed paraffin embedded prostate tissues (FFPE) from Moroccan patients, using quantitative real time reverse transcription-PCR (RT-qPCR). This quantification required data normalization accomplished using stably expressed reference genes (RGs). A panel of twelve RG was assessed, data being analyzed using GenEx V6 based on geNorm, NormFinder and statistical methods. Accordingly, the hnRNP A1 gene was identified and selected as the most stably expressed RG for reliable and accurate gene expression quantification in prostate tissues. The ratios of both PCA3 and AMACR gene expression relative to that of the hnRNP A1 gene were calculated and the performance of each target gene for PCa diagnosis was evaluated using receiver-operating characteristics. PCA3 and AMACR mRNA quantification based on RT-qPCR may prove useful in PCa diagnosis. Of particular interesting, combining PCA3 and AMACR quantification improved PCa prediction by increasing sensitivity with retention of good specificity.

A rare association--amelogenesis imperfecta, platispondyly and bicytopenia: a case report.

INTRODUCTION: Amelogenesis imperfecta is an inherited disease characterized by generalized structural abnormalities of the enamel on all teeth, including both primary and permanent dentition. To the best of our knowledge, this is the first case report of a rare association of amelogenesis imperfecta, platyspondyly, and bicytopenia. CASE PRESENTATION: A 5-year-old Moroccan boy was examined in the Centre for Dental Consultation and Treatment, Faculty of Dentistry, Rabat. He was a child of consanguineous parents (first degree). The child failed to thrive (-4 standard deviation score) and displayed delayed overall development. A dental examination revealed a hypoplastic amelogenesis imperfecta with a bacterial biofilm deposit on tooth surfaces. A complete blood count revealed bicytopenia (normocytic-normochromic anemia with thrombocytopenia). A radiographic examination of the spinal column showed a deviation of the spine in the frontal plane in the form of thoracolumbar scoliosis. The interpedicular distance was not expanded; but a mild platyspondyly exists, especially pronounced in T11 and T12. CONCLUSIONS: No other family members presented amelogenesis imperfecta, bicytopenia, or platyspondyly. The consanguineous marriage suggested an autosomal recessive mode of inheritance. Further studies are necessary to clarify the genetic defect producing this syndrome, and the symptomatic associations of amelogenesis imperfecta, platyspondyly and bicytopenia.

BRCA genetic screening in Middle Eastern and North African: mutational spectrum and founder BRCA1 mutation (c.798_799delTT) in North African.

BACKGROUND: The contribution of BRCA1 mutations to both hereditary and sporadic breast and ovarian cancer (HBOC) has not yet been thoroughly investigated in MENA. METHODS: To establish the knowledge about BRCA1 mutations and their correlation with the clinical aspect in diagnosed cases of HBOC in MENA populations. A systematic review of studies examining BRCA1 in BC women in Cyprus, Jordan, Egypt, Lebanon, Morocco, Algeria, and Tunisia was conducted. RESULTS: Thirteen relevant references were identified, including ten studies which performed DNA sequencing of all BRCA1 exons. For the latter, 31 mutations were detected in 57 of the 547 patients ascertained. Familial history of BC was present in 388 (71%) patients, of whom 50 were mutation carriers. c.798_799delTT was identified in 11 North African families, accounting for 22% of total identified BRCA1 mutations, suggesting a founder allele. A broad spectrum of other mutations including c.68_69delAG, c.181T>G, c.5095C>T, and c.5266dupC, as well as sequence of unclassified variants and polymorphisms, was also detected. CONCLUSION: The knowledge of genetic structure of BRCA1 in MENA should contribute to the assessment of the necessity of preventive programs for mutation carriers and clinical management. The high prevalence of BC and the presence of frequent mutations of the BRCA1 gene emphasize the need for improving screening programs and individual testing/counseling.