Detection of early genetic and epigenetic alterations in NSCLC by using mass spectrometry and pyrosequencing analysis

Background

Complete surgical resection remains the best curative treatment for patients with stage I non small cell lung cancer (NSCLC), but despite tumor resection, a high proportion of patients is still at high risk for cancer related death. Thus, there is a strong need of reliable biomarkers for providing information about the molecular events that occur in early stage of NSCLC.

Methods

We analyzed a ten-year series of 167 consecutive formalin fixed stage I NSCLCs. We included 67 squamous cell carcinomas (SCC) and 100 adenocarcinomas (ADC). Mutation analysis of 10 genes involved in NSCLC (EGFR, KRAS, BRAF, PIK3CA, NRAS, ALK, ERBB2, DDR2, MAP2K1 and RET) was performed by MALDI-TOF Mass Spectometry (MassARRAY, Agena Bioscience) using the Myriapod Lung Status Kit (Diatech Pharmacogenetics). Tumor LINE-1 methylation status was determined by PCR-pyrosequencing on bisulfite-treated DNA and compared with normal lung tissue.

Results

In ADCs we identified 29 KRAS (29%), 17 EGFR (17%), two BRAF (2%) and one PIK3CA mutations (1%). Considering the smoking habit, we observed that all the KRAS mutations clustered in NSCLC from smoker patients. The never-smoker group only showed EGFR mutations. In SCCs, we identified four non-canonical mutations in EGFR gene (6%), and four mutation in PIK3CA (6%). NSCLC showed methylation levels ranging from 14.8% to 78.8% while normal tissue had percentages from 66% to 76.4%. The mean LINE1 methylation value was significantly lower in NSCLC than in normal lung (p = 0.0025). A strong LINE-1 hypomethylation was observed in SCC compared with ADC samples (p 

Conclusions

The mutation pattern typical of advanced disease is observed also in stage I NSCLC patients who may deserve tailored adjuvant target therapy. LINE-1 hypomethylation occurs early in NSCLC and is specifically associated with smoking habit and with SCC histology. Genetic and epigenetic events represents two complementary mechanisms in cancer and the knowledge of both types of alterations in NSCLC opens the possibility of new combinations of therapeutic agents.

Clinical trial identification

No clinical trial

Legal entity responsible for the study

University of Insubria

Funding

University of Insubria

Disclosure

All authors have declared no conflicts of interest.