DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer（NSCLC） therapy,detecting mutation status of the epidermal growth factor receptor（EGFR） gene constitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor（TKI） therapy.Now,the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded（FFPE） tissues.DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction（PCR） are two key steps for detecting EGFR gene mutation.We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene.Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100（pH=10.0） was performed by heating at 95 °C for 30 min.Meanwhile,a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison.DNA extracted products were used as template for amplifying the exons 18,19,20 and 21 of EGFR by PCR for different amplified fragments.Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250―500 base pairs（bp）.However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp.The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit.For about 500 bp fragment,four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit.However,for about 200 bp fragment.This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR,further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.

Cetuximab Combination with Chemotherapy in Advanced Non-Small Cell Lung Cancer

Objective： To observe the efficacy and safety of cetuximab combined with chemotherapy in advanced non-small-cell lung cancer （NSCLC）, and to investigate the association of status of K-RAS gene mutation and epidermal growth factor receptor （EGFR） genotype with clinical outcome. Methods： Between Jan. 2006 and Sep. 2009, nineteen patients with advanced NSCLC received cetuximab （〉4 weeks） combined with chemotherapy in Department of Thoracic Oncology at Beijing Cancer Hospital. Response, survival and toxicity were retrospectively assessed, epidermal growth factor receptor （EGFR） protein expression was evaluated by ELISA Kit. The status of K-RAS gene mutation was tested by PCR-RFLP and EGFR gene amplification was measured by EGFR fluorescence in situ hybridization （FISH）. Results： Partial response（PR） was observed in 26.3%（5/19） of the patients and stable disease（SD） in 52.6%（10/19）. Median progression free survival（PFS） was 6 months （95% CI： 3.6-8.4）. Median overall survival （MST） and 1-year survival rate（SR） were 10.6 months （95% CI： 6.6-14.6） and 47.6%, respectively. Mild or moderate skin rash was the most common toxicity related with cetuximab. K-RAS gene mutation, EGFR protein level and amplification have little correlation with prognosis. Conclusion： Cetuximab combined with chemotherapy was tolerable and the skin rash related with cetuximab was mild to moderate. Cetuximab may prolong survival of the patients who failed to previous chemotherapy.