Identification of candidate genes in clinical samples
This clinical part will be the key element in pathway discovery and is divided into four main steps:
- Creation of a prospective biobank for gene and pathway discovery. The aim is to obtain tumour material before (day 0) and early after treatment start (day 21) in order to assess biological response, before clinical response, which will occur later (3 to 6 months). We will thus obtain paired matched samples from the same patients, as well as data on response to therapy. According to the clinical situation, the samples will be either histological for accessible metastases (for instance cutaneous metastases) or cytological in the other situations. The sequential cytological samplings will follow the procedures established and demonstrated in the FP-5 funded TUBAFROST project.
For the specific studies, tumour samples, serum/plasma and leukocyte DNA will be obtained from equal numbers of responders and non-responders in NSCLC and melanoma, while SCLC samples will be collected before therapy and at relapse. In total, the goal is to obtain paired samples from 240 patients with NSCLC (participating in clinical trials of cisplatin/carboplatin + gemcitabine/ vinorelbine therapy), 120 patients with SCLC receiving chemotherapy and 240 patients with melanoma (participating in a randomized trial of temozolomide vs. DTIC). In addition, a large number of serum/plasma samples from patients participating in randomized trials of adjuvant interferon therapy will be assembled for analysis of auto antibodies and for proteomic studies.
- Functional genomics investigations for discovery of key genes and pathways involved in chemoresistance will be performed through direct comparisons of the matched pairs of biopsies, before and after treatment, for each patient. These will consist of:
- Transcriptome analysis and identification of splice variants. Bioinformatics analysis will be performed with an expected output of 100 top key candidate genes and assessment of underlying pathways
- MicroRNA profiling, using a set of 180 mature specific microRNAs validated by Applied Biosystems by quantitative RT-PCR. The expected output is a list of microRNAs with potential role in regulation of mRNAs. The relationship between mRNAs and microRNAs will be assessed through sequence targeting profiling
- DNA promoter methylation studies. The results will be correlated to mRNA expression of the corresponding genes.
- Structural genomics to assess fine structure-function relations will be carried by sequencing of PCR products representing a limited number of key genes in lung cancer and melanoma. A list of 20 amplicons will be selected on the basis of existent knowledge. Such genes will contain at least the following list of candidates: p53, K-Ras, N-Ras, H-Ras, B-Raf, Src, CDKN2A, Jak, MEKK, p15, PTEN. A list of mutations will be provided.
- Bioinformatics. An independent analysis and validation of the pathways will be performed integrating gene expression, microRNA profiles, DNA promoter methylation, but also mutation status. The list of key candidate genes and of pathways of interest will be refined, integrating mutation data.