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Emerging molecular diagnostic methods are more sensitive and objective, which can overcome the intrinsic failings of morphological diagnosis. Here, a RT-PCR-based  in vitro  diagnostic test kit (LungMe ® ) was developed and characterized to simultaneously quantify the DNA methylation of  SHOX2  and  RASSF1A  in FFPE tissue specimens. The clinical manifestations were evaluated in 251 FFPE samples with specificity and sensitivity of 90.4 and 89.8%, respectively. Furthermore, the quantitative analysis shows that the degree of  SHOX2  methylation was correlated with the stages of lung cancer, but not in the case of  RASSF1A . Our observation indicated that the DNA methylation of  SHOX2  and  RASSF1A  may play different roles in cancer development. Comparison of the methylation levels of  SHOX2  and  RASSF1A  between cancer and cancer-adjacent specimens (n = 30), showed they have “epigenetic field defect”. As additional clinical validation, the hypermethylation of  SHOX2  and  RASSF1A  was detected not only in surgical operative specimens, but also in histopathological negative puncture biopsies.  SHOX2  and  RASSF1A  methylation detection can be used to increase sensitivity and NPV, which provide us with a more accurate method of differential diagnosis and are likely to be rapidly applied in clinical examinations.

Performance Evaluation of SHOX2 and RASSF1A Methylation for the Aid in Diagnosis of Lung Cancer Based on the Analysis of FFPE Specimen