Figure 3

Discordance in SSP classification. (A) In-depth histopathological evaluation of a tumor initially classified as NSCLC-NOS that was re-classified as AC by a pathologist but SqCC by the SSP (top right panel) in validation cohort III. Hematoxylin staining of the tumor clearly demonstrates the tumor cell rich area (lower left IHC panel), while KRT5 IHC staining reveals positivity in bronchial epithelium with squamous metaplasia surrounding the tumor tissue (lower right IHC panel). The latter is most probably the cause of elevated KRT5 (and other squamous markers) expression in the RNA extracted from the tissue (top left expression panel). (B) Two misclassified LCNEC tumors in validation cohort IV. Due to low mRNA expression of LCNEC associated genes and high expression of AC genes, these tumors are classified as AC by the SSP (top expression panels), discordant to original histopathological assessment. In-depth review of these discordant cases revealed mixed AC and LCNEC histology, evident from IHC stains using AC and LCNEC immunomarkers respectively (lower panels). Further review of these cases revealed that RNA had been extracted and analyzed by RNAseq only from the AC component from these two tumors.