ASCC3Q · Gene amplification in squamous cell carcinoma development: mapping of amplification regions on chromosome 3q in precancerous lesions of the upper aerodigestive tract by molecular copy number counting

Gene amplification in squamous cell carcinoma development: mapping of amplification regions on chromosome 3q in precancerous lesions of the aerodigestive tract ...

Gene amplification in squamous cell carcinoma development: mapping of amplification regions on chromosome 3q in precancerous lesions of the upper aero-digestive tract by molecular copy number counting. A major task required to initiate the work of this proposal was the collection of clinical material prior to its analysis. To achieve this, a research nurse and tissue collector, who were part of the research team, were trained by the Marie Curie Fellow. Dr Belvedere was also responsible for devising all the procedures, constructing the databases and identifying the measures for compliance with all the regulatory requirements necessary for these translational studies. This will leave a legacy of good practice that will still be in evidence many years into the future. As the imperative for translational research gathers momentum, creating and maintaining the infrastructure required for a tissue repository has become mandatory in large bioscience centres but it is not without its difficulties and pitfalls. Dr Belvedere has captured these in a manuscript entitled 'Lung cancer genomic profiling in a single centre: from specimen collection to quality-controlled DNA for next-generation sequencing submitted to the Journal of Thoracic Oncology. While accumulating samples from prospective collections from a number of clinics and operating theatres, through a collaboration with Professor Phil Quirke in our institute, it has also been possible to obtain access to a cohort of tumours from patients with lung cancer including 180 squamous cell carcinomas-the subtype in which we are most interested. These samples have been collected more than five years ago and preserved in paraffin embedded wax blocks. A database exists describing these patients in which the outcome of their disease, in particular survival, is documented. The tumour samples are available as individual fixed blocks from which DNA can be made and as tissue microarrays in which sections of each specimen as very small cores, are displayed in triplicate. The purpose of this proposal was to map the 3q amplification in pre-invasive lesions. Because the same amplification occurs in fully malignant squamous cell carcinoma, we used these as a surrogate benefiting from our large cohort, to provide markers that can be tested directly on our supply of upper aero-digestive tract pre-invasive lesions, available as both retrospective and prospective samples. The analysis was undertaken at both the level of gene expression and genome structure. For genome expression, six genes within the amplicon were selected and their protein expression evaluated by immuno-histochemistry across the 180 squamous cell carcinomas in the tissue micro arrays. The microarrays also include adenocarcinomas of the lung and they were analysed in parallel to indicate lung tumour subtype specific expression. Genome structure was analysed in two ways to provide gene copy number information. The same candidate genes whose protein products were examined by immuno- histochemistry were assessed by copy number by real time (quantitative) PCR. Recently, within our research group, we have developed a copy number counting method that relies on sequencing whole genomes but at a very low coverage such that chromosomes are essentially sampled along their length. This provides a whole genome copy number map as well as detailed information of gene copy in amplified regions. Since the patient outcomes is known, these data provide the opportunity of assessing these genomic parameters as candidate biomarkers for prognosis. The development of the copy number methodology using next generation sequencing by Drs Wood and Belvedere (Wood et al., NAR, 2010) has particular significance for molecular pathology as the method can be applied to the DNA isolated from the fixed material that forms part of routine diagnosis of tumour biopsies.

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