Electronic supplementary materials:
Methods of the gene mutation analysis
Methods for DNA extraction from formalin-fixed and paraffin-embedded (FFPE) tissue: For LCNEC DNA extraction, five thin sliced sections of 5µm-thickness were serially prepared on glass slides from the formalin-fixed and paraffin-embedded (FFPE) tissue block including the LCNEC component, and one of them was subjected to immunohistochemistry (IHC) for synaptophysin. The thin sliced tissue corresponding to the synaptophysin-IHC positive areas on the rest unstained slides were stripped off from the slides and collected in a tube. DNA was extracted from the collected FFPE tissues with the GeneRead DNA FFPE kit (Qiagen,Hilden, Germany) precisely according to the provider’s protocol to avoid artificial C to T transition-mutations due to the formalin fixation. FFPE tissuescorresponding to G1 EC, non-neoplastic endometrium (as a control) and the metastatic lymphnode were also collected based not on IHC but on the morphology appeared in hematoxylin and eosin (HE) staining, and used for DNA extraction with the same way used for LCNEC.
Gene mutation analyses in the hotspots of 50 selected cancer-associated genes
First, the quality of extracted DNA was estimated by TaqMan RNase P Detection Reagents kit (short assay for 87 bp product) and TaqMan MGB gene expression detection kit for FFPE DNA QC assay (long assay for 256 bp product) (Thermofisher scientific, USA). The relative value of long assay / short assay (Ct value) for each sample DNA exceeded 0.2 which is good for the analysis.Ten ng of each DNA was used to examine mutation-hotspots of selected 50 cancer-associated genes with the Ion AmpliSeq™ Cancer Hotspot Panel v2 and Ion PGM semiconductor sequencer (Life Technologies, Guilford, CT). Mutation hotspots on the following genes were analyzed: ABL1, EGFR, GNAS, KRAS, PTPN11, AKT1, ERBB2, GNAQ, MET, RB1, ALK, ERBB4, HNF1A, MLH1, RET, APC, EZH2, HRAS, MPL, SMAD4, ATM, FBXW7, IDH1, NOTCH1, SMARCB1, BRAF, FGFR1, JAK2, NPM1, SMO, CDH1, FGFR2, JAK3, NRAS, SRC, CDKN2A, FGFR3, IDH2, PDGFRA, STK11, CSF1R, FLT3, KDR, PIK3CA, TP53, CTNNB1, GNA11, KIT, PTEN and VHL. The information of the examinedmutation hotspots can be found at the provider’s Web site ( The data obtained from eachsequencing run were analyzed by the Ion Torent softwares (Life Technologies) to call variants and mapping against the human GRCh37/hg19 assembly sequences. Variants with coverage x ≧20 that could change the encoded amino acid sequencefrom the referenced sequence were selected for further analyses. Reported single nucleotide polymorphisms (SNPs) in the dbSNPs were then omitted. Because no mutations were found in the non-neoplastic endometrium, all the variants found were considered as somatic mutations. PCR amplification of DNAs followed by the Sanger sequencing of the product was performed when needed and possible (with mutated allele frequency more than 10%) to confirm the variant sequences.