A 76-year-old woman was diagnosed with colon carcinoma and underwent preoperative examinations. Computed tomography coincidently revealed a well-demarcated tumor, 1.8 × 1.3 cm in size, located in the left lower lung (Figure 1). She did not have any respiratory symptoms. Laboratory data revealed no significantly abnormal findings. Endoscopic submucosal dissection of the colon carcinoma was performed. Bronchoscopy revealed an endobronchial mass, and transbronchial biopsy was performed. She was diagnosed with an adenocarcinoma, and thoracoscopic left lower lobectomy with hilar and mediastinal lymph node dissection was performed.
Pathological findings
Macroscopic findings
A specimen containing the tumor was obtained at surgery. Macroscopically, the tumor size was 2.7 × 1.9 × 1.8 cm, and the cut surface of the tumor was whitish–yellow to gray, shiny, and well-demarcated (Figure 2-a).
Histopathological and immunohistochemical findings
The tumor consisted of duct-like structures and polygonal and spindle cell features (Figure 2-b). The duct-like structures were composed of two distinct cell layers. The inner layer comprised cuboidal cells with eosinophilic cytoplasm and round nuclei, and the outer layer comprised cells with clear cytoplasm and oval nuclei (Figure 2-c). The duct-like structures contained eosinophilic material in the luminal spaces. The polygonal and spindle cells (black arrowhead) were similar to the outer-layer cells (black arrow) (Figure 2-d). There was no necrosis or hemorrhage. Mitotic activity was found (2 mitoses/10 high-power fields). Immunohistochemically, the inner-layer cuboidal cells were positive for pan-cytokeratin (Figure 3-a) and negative for vimentin, p63 (Figure 3-b), HHF35 (Figure 3-c), S-100 (Figure 3-d) and TTF-1, suggesting an epithelial phenotype. On the other hand, the outer-layer cells as well as polygonal and spindle cells were positive for p63 (Figure 3-b), HHF35 (Figure 3-c), and S-100 (Figure 3-d), and weakly positive for pan-cytokeratin (Figure 3-a), suggesting a myoepithelial phenotype. Overexpression of p53 protein was not found.
Mutation analysis
We conducted polymerase chain reaction (PCR) followed by Sanger sequencing and pyrosequencing to investigate the mutation status of the oncogenes associated with EMC of the salivary gland [7]. Briefly, DNA from formalin-fixed paraffin-embedded tissues was extracted using TaKaRa DEXPAT (Takara Bio Inc., Shiga, Japan). The tumor component on the slides was microdissected to increase the tumor cell proportion. The PCR products were purified using the NucleoSpin Gel and PCR Clean‑up, Mini kit (Marcherey-Nagel, Duren, Germany). Each purified product was directly sequenced using a forward primer and the BigDye Terminator version 3.1 cycle sequencing kit on the ABI 3730 instrument (Applied Biosystems Inc., Foster City, CA). Mutation analyses of AKT1 (exon 2), CTNNB1 (exon 3), HRAS (exons 2 and 3) and PIK3CA (exons 9 and 20) were performed based on the method described by Urano et al. [7]. The primer sequences are listed in Table 1. In addition, mutation analyses of BRAF (exon 15) and KRAS (exons 2 and 3) were performed using the BRAF Pyro Kit and KRAS Pyro Kit (Qiagen, Venlo, Netherlands), respectively, in real-time using pyrosequencing technology on the PyroMark Q24 System (Qiagen). No mutation in any of the six genes was detected.