Objective: Epidermal growth factor receptor (EGFR) mutation detection is often based on lung cancer archival biopsies or cytological specimens. Pyrosequencing is a sensitive method to detect mutation presence and frequency even in DNA partially fragmented cases. The objective of the study was to assess the performance of pyrosequencing in EGFR mutation detection. Method: There were 202 selected lung adenocarcinomas tested (72 surgical specimens, 56 biopsies, 74 cytological samples, including 9 smears and 65 cell blocks). In microdissected paraffin sections and smears, exon 18-19- 21 mutations were screened using EGFR-TKI response (sensitivity) kit (Diatech), real-time-PCR (RotorGene,Qiagen) and pyrosequencing (PyroMarkQ96, Biotage). Results: Real-time-PCR and melting analyses showed abundant and specific amplification of each exon, irrespective of fixative and sample types. Pyrosequencing revealed 43/ 202 (21.3%) mutated samples [31 exon 19 in-frame deletions (72%) and 12 exon 21 point mutations (25.6% L858R and 2.4% L861Q)], with a respective frequency of 25.8% and 66.7% in surgical samples; 32.3% and 8.3% in biopsies; 41.9% and 8.3% in cell blocks; 0% and 16.7% in smears. Conclusion: Mutation frequencies are similar to those reported in the literature, with no marked differences among the various sample types. Pyrosequencing combined with microdissection and real-time-PCR appears a valuable method for molecular tests in different archival specimens.
Detection of EGFR mutations in archival lung cancer samples by pyrosequencing
CUCCURULLO, Alessandra;GIACHINO, Daniela Francesca;RIGHI, Luisella;ROVERE, Giulia;NOVELLO, Silvia;PAPOTTI, Mauro Giulio
2011-01-01
Abstract
Objective: Epidermal growth factor receptor (EGFR) mutation detection is often based on lung cancer archival biopsies or cytological specimens. Pyrosequencing is a sensitive method to detect mutation presence and frequency even in DNA partially fragmented cases. The objective of the study was to assess the performance of pyrosequencing in EGFR mutation detection. Method: There were 202 selected lung adenocarcinomas tested (72 surgical specimens, 56 biopsies, 74 cytological samples, including 9 smears and 65 cell blocks). In microdissected paraffin sections and smears, exon 18-19- 21 mutations were screened using EGFR-TKI response (sensitivity) kit (Diatech), real-time-PCR (RotorGene,Qiagen) and pyrosequencing (PyroMarkQ96, Biotage). Results: Real-time-PCR and melting analyses showed abundant and specific amplification of each exon, irrespective of fixative and sample types. Pyrosequencing revealed 43/ 202 (21.3%) mutated samples [31 exon 19 in-frame deletions (72%) and 12 exon 21 point mutations (25.6% L858R and 2.4% L861Q)], with a respective frequency of 25.8% and 66.7% in surgical samples; 32.3% and 8.3% in biopsies; 41.9% and 8.3% in cell blocks; 0% and 16.7% in smears. Conclusion: Mutation frequencies are similar to those reported in the literature, with no marked differences among the various sample types. Pyrosequencing combined with microdissection and real-time-PCR appears a valuable method for molecular tests in different archival specimens.
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
