ANALISI COMPARATIVA DI METODICHE MOLECOLARI PER LA DETERMINAZIONE DELLO STATO MUTAZIONALE DI EGFR IN PAZIENTI CON CARCINOMA DEL POLMONE NON A PICCOLE CELLULE

The epidermal growth factor receptor (EGFR) is a transmembrane receptor with tyrosine-kinase activity that can be activated by different ligands. This event triggers a cascade of signals downstream with effects on cell survival and proliferation. The mutational event of EGFR is able to induce the constitutional activation that can be effectively blocked by the administration of drugs with anti-receptor activity. The evaluation of EGFR mutational profile is a fundamental survey to select patients affected by advanced non-small cell lung cancer (NSCLC), which can undergo treatment with specific drugs. In fact, EGFR mutations are reported in a subpopulation of patients ( 8-20 %) with NSCLC, which grows in non-smokers, being female and belonging to Asian race. The most frequent mutations (90 % of the mutated samples) are located in Exon 19 (in-frame deletions: E746 - A750 ) and Exon 21 of the EGFR (point substitutions: L858R). Different technologies are used to evaluate the EGFR mutational status, including Sanger Sequencing, Pyrosequencing and Real Time PCR. Immunohistochemistry has also been proposed, even if not validated as a possible investigation complementary to those of molecular type. The purpose of the present work is to carry out a comparative analysis of these technologies (Sanger Sequencing, Pyrosequencing and Immunohistochemistry) in a series of patients with primary non-small cell lung cancer. The evaluation of the results took into account both the accuracy of the methods and other factors such as execution time, complexity of the procedure and cost/benefit ratio. Using tumor cell lines containing scalar dilutions of EGFR mutated alleles (1, 10, 20, 50%) we preliminarily stated that the Pyrosequencing in our hands, within a tumor population, is able to detect 10% of mutated alleles compared to 20% of mutated alleles detected by Sanger Sequencing. Then, we validated the diagnostic accuracy of these methods in our laboratory as part of two programs of quality controls developed at the national level on external occurences. The comparative research between Sanger Sequencing and Pirosequencing was carried out on 258 primary lung carcinomas, including 229 adenocarcinomas, using archival histological material formalin-fixed and paraffine-included which comprised materials of surgical origin both from therapeutic procedures (lung resections) and from diagnostic procedures (biopsies). The frequency of mutation detected by Pyrosequencing was 13,1% , higher than the one observed using Sanger Sequencing of 11,4%. Over the entire population of primary lung cancer with EGFR mutation (n=26), the gain in sensitivity of Pyrosequencing compared to Sanger Sequencing was 15,4% (4/26 cases). It is interesting to note that the cases identified as mutated by Pyrosequencing (not by Sanger Sequencing) came from samples characterized by reduced tumor cells (2 biopsies and 2 surgical microdissections). This finding is particularly significant taking into account the fact that EGFR mutation study is at present almost exclusively carried out on bioptic material taken from patients with lung cancer in advanced phase and thus inoperable. We then evaluated the diagnostic accuracy of immunohistochemistry in identifying typical mutations of EGFR. For this purpose, 73 adenocarcinomas with mutational profile related to Pyrosequencing [41 cases mutated for Exon 19 of EGFR (E746–A750del) and 31 mutated for Exon 21 (L858R)] were immunocolored using antibodies anti-E746-A750del for Exon 19 and anti-L858R for Exon 21. The diagnostic accuracy of immunohistochemistry was found to be between 80,3% (antibody anti-E746- A750del) and 90,7% ( antibody anti-L858R) with absolute specificity and sensitivity ranging from 67,7% to 69,0%. Overall, the immunohistochemical technique, even if limited to the identification of the two most frequent mutational events, turned out to be accurate enough and with much lower costs and execution times faster than molecular investigations. To sum up, our research proves both the suitability and the superiority of Pyrosequencing compared to Sanger Sequencing as for the ability to detect the presence of EGFR mutations in a consecutive series of primary non-small cell lung carcinomas. On the contrary, immunohistochemistry turned out to be a technique less sensitive in determining mutational arrangements of the same cases. Since this technique proved to get the same specificity and to be less expensive and faster than the first two, it is possible to use it in clinical diagnostics as preliminary screening investigation of the most frequent mutational events.