EGFR activating mutations detected by different PCR techniques in Caucasian NSCLC patients with CNS metastases: short report

Abstract

EGFR mutation testing has become an essential determination to decide treatment options for NSCLC. The mutation analysis is often conducted in samples with low percentage of tumour cells from primary tumour biopsies. There is very little evidence that samples from metastatic tissues are suitable for EGFR testing. We had evaluated the frequency of EGFR mutations with three highly sensitive PCR techniques in formalin-fixed, paraffin-embedded samples of 143 NSCLC patients with central nervous system (CNS) metastases. 32 corresponding primary tumours were also examined. We used PCR followed by DNA fragments length analysis (FLA), ASP–PCR and PNA–LNA PCR clamp techniques. We found 9 (6.29 %) EGFR gene mutations in CNS samples: 3 (2.1 %) in exon 19 and 6 (4.2 %) in exon 21. The full concordance between CNS metastases and primary tumour samples was observed. PCR followed by DNA–FLA and PNA–LNA PCR clamp were sensitive enough to detect exon 19 deletions. Two mutations in exon 21 were detected by ASP–PCR only, one L858R substitution was detected only by PNA–LNA PCR clamp. With respect to sensitivity, PCR followed by DNA–FLA achieved a level of detection of at least 10 % of mutated DNA for exon 19 deletion, as for ASP–PCR it was at least 5 % of mutated DNA for L858R substitution. Higher sensitivity of 1 % of mutated DNA was achieved by PNA–LNA PCR clamp technique for both mutations. The use of different methodological techniques authenticates the negative result of molecular tests.

Tumor heterogeneity and resistance to EGFR-targeted therapy in advanced nonsmall cell lung cancer: challenges and perspectives

“… 104 , 105 The high wild-type EGFR detection rate in the former studies was thought to be explained by normal tissue contamination, as well as technical variations (eg, the minor presence of EGFR mutations that may be neglected by less-sensitive methods). 104 , 106 , 107 Moreover, although some groups reported a conversion from EGFR mutant to wild-type after TKI treatment in occasional cases, 108 most of the studies show highly consistent EGFR mutations in tumors before and after the treatment. 57 , 104 , 109 It is hard to imagine why the conversion rate of mutant to wild-type after TKI treatment is so low if intratumor heterogeneity of EGFR mutation is not rare.…”

Sensitive methods for detection of the S768R substitution in exon 18 of the DDR2 gene in patients with central nervous system metastases of non-small cell lung cancer

“…Moreover, the low quality of DNA isolated from FFPE tissue samples and sub-clonality of DDR2 mutations in the metastases could have affected on the results of direct sequencing analysis. All three methods have different sensitivity of mt DNA detection that had been previously described (direct sequencing >40 %, real-time PCR >1 %, ASP-DNA-FLA PCR >5 %) [ 8 – 10 ]. Taking into account a low purity of the analyzed samples, the next generation sequencing method had not been used to verify positive results.…”

Risk of brain metastasis reduced after erlotinib treatment in advanced pulmonary adenocarcinoma patients with sensitive EGFR mutation

“…Currently, EGFR mutation genotype is routinely determined in pulmonary adenocarcinoma. Furthermore, for lung cancer patients with BM, highly sensitive polymerase chain reaction techniques provide the feasibility of determination of EGFR mutation genotype in BM, 25 which provides detail biological information of BM and thus guide treatment decisions in clinical practice.…”