Research Projects

Oesophageal Squamous Cell Carcinoma Research – Dr Pascale Willem

Oesophageal Squamous Cell Carcinoma Research – Dr Pascale Willem

Dr Pascale Willem

Medical School, Department of Haematology and Molecular Medicine, University of the Witwatersrand

Title of the project

DNA  Profiling of Oesophageal Squamous Cell Carcinomas in the South African Population

Highlights of the project

The aim of the project is to characterise the underlying common molecular signature of gene copy number variation associated with oesophageal squamous cell carcinomas (ESCC) in South Africa (SA).

It is expected to identify genes, and may be pathways, critical to the pathophysiology of this disease locally. These genes/pathways may represent potential therapeutic targets for future intervention.

To our knowledge this project is the first in South Africa studying cancer associated genome wide copy number variation using Affymetrix SNP arrays. It involved a learning curve and we have developed local competency in both the practical and importantly, in the analysis and interpretation of these results. This has already allowed for building capacity regarding this technical approach locally.

Peer-reviewed publications

  • A novel approach to simultaneously scan genes at fragile sites. BMC Cancer. 2006 Aug 8;6:205. Willem P, Brown J, Schouten Abstracts. Click here to read the article on Pub Med.
  • Oesophageal Carcinoma in South Africa: Possible involvement of FHIT J. Brown, A. Stafne, S. Engelbrecht, R. Veal and P.Willem  Chromosome Research, 2004, Vol 12, Supl 1,PO:08:55

Two publications are in preparation related to this project. The phase of the project regarding array data however will not be published before completion of both controls and patients samples ( N= 50 respectively) .

In Preparation: 
Characterization of Five South African Oesophageal Cancer Cell Lines by Cytogenetics,Multicolor Fluorescence in situ Hybridization and Array-Based Copy Number Analysis. Jacqueline Brown, Hannelie Bothma, Robert Veale, Pascale Willem

High frequency of FHIT and WWOX intragenic deletions in oesophageal squamous cell carcinoma from a high endemic region in South Africa. Willem P, Brown J, Veale R, Stepien A.

Other products forthcoming from the project

A control population group from the Eastern Cape, (Umtata) (N= 50) is being genotyped with the 500K SNP Affymetrix arrays as part of this project. This will provide a genotyping reference for the Eastern Cape population group that may be utilised by other researchers locally and /or internationally.

The SNP data on both patients and controls will also provide a resource for eventual ESCC genome association studies in the future by our or other groups. Demographic data from each patient and control specimens were recorded and although the magnitude of this type of analysis requires significantly larger numbers this resource will be valuable added information for future case control studies. 

How was this project of value in the struggle against cancer?

We started the third year of this study in May 2009. As stated above it is expected to identify molecular targets for future therapeutic developments. The results of this study may provide insights  into  the pathways involved in ESCC development and as such, may shed light on the possible role of environmental or genetic factors associated with this disease in SA.

What is your future plans with this project? 

The epidemiology of oesophageal squamous cell carcinoma is disconcerting with world wide geographic high endemic pockets, including in SA. Although the incidence tends to decrease it remains one of a major cause of death in both men and women in some regions of South Africa. This project attempts to identify molecular targets using a genome wide screening analysis for copy number variation. Similar approaches have recently allowed for the identification of the CDK8 gene in colorectal cancer as a promising therapeutic target. Our preliminary results points to such oncogenes /pathways and it is envisaged to verify the identified targets copy number variation using semi quantitative PCR methods, to assess their expression in ESCC cell lines and initiate functional studies. 

Due to the learning curve involved in applying the recent SNP array copy number technology and to the yearly budget limitations, this project can only produce publishable data on completion of the samples number (50 patients + 50 controls) ; the amount of data generated will however be highly valuable for both this project and other researchers. Two papers are currently being submitted. The array data will be published on completion of the sample size.

Brief summary of the research for the lay audience

Oesophageal squamous cell carcinoma (ESCC) has a peculiar epidemiology with worldwide geographical pockets of high incidence. In South Africa, ESCC is the second cause of death in black males and third in women in the Eastern Cape. The etiology of this cancer and the respective contribution of genetics and environmental factors remain unresolved.

The human chromosome complement contains nonrandom genomic regions that are prone to breaks and recurrently altered in tumors. These chromosomal “hot spots” are preferentially involved in early events of genomic instability. Amplicons and deletions are frequently generated at “hot spots”, especially at common fragile sites (CFS). These amplicons/deletions are thought to select cancer genes whose amplification or deletion drive cell proliferation and promote the initiation and, or progression of cancer.

Several environmental factors have been proposed to contribute to the development of ESCC. These include smoking, alcohol consumption, fumonisin exposure and infection with DNA viruses such as the Human Papilloma virus (HPV). Interestingly, these factors can all target chromosomal fragile sites either directly or indirectly; they are also prevalent in  South African regions showing a high incidence of ESCC.

Our group focuses on the study of copy number variation in ESCC specimens using two different approaches. In the first, we focus on the most commonly expressed fragile sites, FRA3B and FRA16D that both host a tumor suppressor gene, FHIT and WWOX respectively, in the second we screen tumor specimens for nonrandom regions of amplification and deletion likely to host cancer key genes.

Alterations in fragile site associated genes have indeed been reported in a variety of tumors including lung, esophageal, gastric, breast and cervical cancers most frequently as a result of submicroscopic deletions. Genomic deletions at CFS have been mostly investigated using loss of heterozygosity assays that do not necessarily inform on gene exon deletions. A new method was developed based on multiplex ligation-dependent probe amplification (MLPA) that screens for exon deletions/amplifications of genes at CFS. The assay was validated on five esophageal squamous carcinoma cell lines and showed deletions in the FRA3B-associated gene FHIT in four of the cell lines.

Two geographically distinct South African cohorts of esophageal squamous cell carcinoma were then screened for FHIT/WWOX exon deletions and a visual basic (VBA) encoded program was written to automate MPLA products analysis. A high frequency of intragenic deletions in FHIT and/or WWOX (over 70%) was observed in the Eastern Cape cohort. FHIT deletions were seen in 27% of specimens from the Gauteng cohort, which by contrast did not show WWOX deletions. This difference may however reflect a difference in sampling collection.

Our second approach consists in a whole genome analysis for copy number variation using microarray technology with the aim to identify key genes in regions of genomic amplifications and deletions. The Affymetrix 500K SNP arrays are being used to screen 50 ESCC tumor specimens and five ESCC cell lines, population matched specimens are used as a control . It is hoped to identify key genes involved in the pathogenic pathway of ESCC.


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