title: Detection of nasopharyngeal carcinoma in Morocco (North Africa) using a multiplex methylation-specific PCR biomarker assay.
abstract: BACKGROUND
  Silencing of tumor suppressor genes (TSGs) or activation of oncogenes by, e.g., aberrant promoter methylation, may be early events during carcinogenesis. The methylation status of such genes can be used for early detection of cancer. We are pursuing this approach in our efforts to develop markers for early detection and follow-up of nasopharyngeal carcinoma (NPC). We set out to develop this approach to allow identification of NPC from Morocco and then also compared with NPC samples from different geographical locations and different ethnicity with different NPC incidences, Epstein-Barr virus (EBV) prevalence, and environments.
  
  
  RESULTS
  By multiplex methylation-specific PCR (MMSP), multiple relevant genes can be detected simultaneously, to achieve high sensitivity and specificity. The strong association of EBV with NPC is also very useful in such an approach. We have initially screened for 12 potential marker genes including EBV genes coding for EBV nuclear antigen 1 (EBNA1) and latent membrane protein-1 (LMP1) and ten potential TSGs obtained from previously published data. The resulting assay included EBNA1, LMP1, and three cellular TSGs: ITGA9, RASSF1A, and P16. We evaluated this assay on 64 NPC patient biopsies from Morocco, Italy, and China compared to deoxyribonucleic acid (DNA) from 20 nasopharyngeal control tissues. In the Moroccan NPC cohort (n = 44), prevalence of the EBNA1 gene showed the highest sensitivity (36/44; 82 %) with 94 % specificity. Out of eight (18 %) EBNA1 negative Moroccan samples, only three were positive for at least one methylated cellular gene. By detection of cellular marker genes, the sensitivity increased from 82 to 89 % (39/44). In the whole material of 64 biopsies from three geographical locations, at least any one marker (viral or cellular) could be detected in 91 % of biopsies with 90 % specificity. In a pilot evaluating assay performance on serum DNA from NPC and controls including samples from Italy (n = 11) and China (n = 5), at least any one marker from the MMSP assay could be detected in 88 %, but the specificity was only 50 %.
  
  
  CONCLUSIONS
  An MMSP assay has the potential for detection of NPC by screening in high-risk populations. Serum-derived DNA seems not as good as earlier published NPC swab DNA for screening purpose.
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box 280, Stockholm, SE-17177 Sweden.|Department of Microbiology, Faculty of Life Sciences, University of Balochistan, Quetta, Pakistan.
authors: Imran
authors: I
authors: Nawaz
authors: Department of Molecular Genetic Epidemiology, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany.|Oncovirology Laboratory, Institut Pasteur du Maroc, 20360 Casablanca, Morocco.
authors: Khalid
authors: K
authors: Moumad
authors: Cancer Bio-Immunotherapy Unit Centro di Riferimento Oncologico IRCCS - National Cancer Institute, Via Franco Gallini, 233081 Aviano, PN Italy.
authors: Debora
authors: D
authors: Martorelli
authors: University Hassan II, Faculty of Sciences and Techniques, Mohammedia - Casablanca, Laboratory of Virology, Microbiology and Quality/ETB, Mohammedia, , BP 146, 20650 Morocco.
authors: Moulay Mustapha
authors: MM
authors: Ennaji
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box 280, Stockholm, SE-17177 Sweden.|Department of Orolaryngology - Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Guangxi, People's Republic of China.
authors: Xiaoying
authors: X
authors: Zhou
authors: Department of Orolaryngology - Head and Neck Surgery, First Affiliated Hospital of Guangxi Medical University, Guangxi, People's Republic of China.
authors: Zhe
authors: Z
authors: Zhang
authors: Cancer Bio-Immunotherapy Unit Centro di Riferimento Oncologico IRCCS - National Cancer Institute, Via Franco Gallini, 233081 Aviano, PN Italy.
authors: Riccardo
authors: R
authors: Dolcetti
authors: Oncovirology Laboratory, Institut Pasteur du Maroc, 20360 Casablanca, Morocco.
authors: Meriem
authors: M
authors: Khyatti
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box 280, Stockholm, SE-17177 Sweden.
authors: Ingemar
authors: I
authors: Ernberg
authors: Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Box 280, Stockholm, SE-17177 Sweden.
authors: Li-Fu
authors: LF
authors: Hu
keywords: Biomarker; Bisulfite conversion; DNA methylation; Early diagnosis; MMSP; Morocco; NPC; Screening; TSGs