Materials and Methods
Eight hundred and fourteen surgically resected cases of mucosal cancer of the head and neck regions were identified from 2002 to 2009 in the pathology files of Shizuoka Cancer Center Hospital, Shizuoka, Japan. All were primary surgically resected Japanese patients. Twenty-four cases of basaloid squamous cell carcinoma (BSCC) and 73 cases of poorly differentiated squamous cell carcinoma (PDSCC) were selected; 24 BSCC and 32 PDSCC were extracted according to the histological criteria of pulmonary LCNEC in the WHO classification. Clinical data of all cases were obtained. Immunohistochemical examinations for CD56 (neural cell adhesion molecule, NCAM), ChA and Syn as NE markers were performed with an EnVision kit (DakoCytomation, Carpinteria, California, USA). The antibodies used in this study are summarised in Table 1. Peroxidase activity was detected using 3-3'-daminobenzidine as the chromogen. After the sections were counterstained with haematoxylin, they were dehydrated and mounted in the synthetic medium. Immunopositivity of all tumour cells examined in a single section of <10%, 10–19%, 20–49% and >50% was defined as focally +, partially +, + and ++, respectively. Staining evaluation was performed using semiquantitative optical analysis by two independent observers (KK and MA). Cases that were immunopositive for two or three NE markers were defined as M-LCNEC and were subsequently examined in detail for their clinical, histological and immunohistochemical characteristics.
Transmission electron microscopy was performed in one patient (case 3) using formalin-fixed specimens with decalcification. Small blocks (approximately 1 mm) of resected specimens were post-fixed in 2% glutaraldehyde in phosphate buffered saline at 4°C overnight and then for 2 h at 4°C with 1% osmium tetraoxide in phosphate buffered saline. Following dehydration, each specimen was transferred to propylene oxide and embedded in Quetol 812 epoxy resin (Nisshin EM, Tokyo, Japan). Ultrathin sections were cut with an Ultracut microtome (Leica, Weltzlar, Germany) and stained with uranyl acetate and lead citrate. Sections were observed with a JEM-1230 electron microscope (JEOL, Tokyo, Japan) using standard operating conditions.