Preferential expression of potential markers for cancer stem cells in large cell neuroendocrine carcinoma of the lung. An FFPE proteomic study

Background Large cell neuroendocrine carcinoma (LCNEC) of the lung, a subtype of large cell carcinoma (LCC), is characterized by neuroendocrine differentiation that small cell lung carcinoma (SCLC) shares. Pre-therapeutic histological distinction between LCNEC and SCLC has so far been problematic, leading to adverse clinical outcome. We started a project establishing protein targets characteristic of LCNEC with a proteomic method using formalin fixed paraffin-embedded (FFPE) tissues, which will help make diagnosis convincing. Methods Cancer cells were collected by laser microdissection from cancer foci in FFPE tissues of LCNEC ( n = 4), SCLC ( n = 5), and LCC ( n = 5) with definite histological diagnosis. Proteins were extracted from the harvested sections, trypsin-digested, and subjected to HPLC/mass spectrometry. Proteins identified by database search were semi-quantified by spectral counting and statistically sorted by pair-wise G-statistics. The results were immunohistochemically verified using a total of 10 cases for each group to confirm proteomic results. Results A total of 1981 proteins identified from the three cancer groups were subjected to pair-wise G-test under p < 0.05 and specificity of a protein's expression to LCNEC was checked using a 3D plot with the coordinates comprising G-statistic values for every two group comparisons. We identified four protein candidates preferentially expressed in LCNEC compared with SCLC with convincingly low p -values: aldehyde dehydrogenase 1 family member A1 (AL1A1) ( p = 6.1 × 10 -4 ), aldo-keto reductase family 1 members C1 (AK1C1) ( p = 9.6x10 -10 ) and C3 (AK1C3) ( p = 3.9x10 -10 ) and CD44 antigen ( p = 0.021). These p -values were confirmed by non-parametric exact inference tests. Interestingly, all these candidates would belong to cancer stem cell markers. Immunohistochmistry supported proteomic results. Conclusions These results suggest that candidate biomarkers of LCNEC were related to cancer stem cells and this proteomic approach via FFPE samples was effective to detect them.