Archived cases from the Department of Pathology, Samsung Medical Center, Seoul, Korea were evaluated. All cases were diagnosed by one experienced pulmonary pathologist. The tumor sections were evaluated after being stained with hematoxylin and eosin. Histologic type, subtype, size, pleural invasion, lymphovascular invasion, perineural invasion, and lymph node metastasis were assessed according to the international tumour, node, and metastasis (TNM) classification system. Clinical data, including age, sex, smoking history, treatment, and clinical course were retrieved from the patients' electronic medical records retrospectively. Patient clinicopathologic parameters are summarized in Table 1.

Representative formalin-fixed, paraffin-embedded (FFPE) tissue sections were used for IHC. These sections were incubated with primary antibodies against CD56 (1:200; Novocastra, Newcastle-upon-Tyne, UK) and ALK (Clone 5A4; Leica, Wetzlar, Germany). Immunohistochemical staining using a biotin-avidin-peroxidase method with BOND-MAX autostainer (Leica) was performed on 3-µm-thick sections from each patient after retrieval with T/E buffer. Nuclei were counterstained with hematoxylin.

Tumor tissues were microscopically dissected from FFPE tissue sections for EGFR mutation detection. Using a DNeasy tissue kit (Qiagen, Helden, Germany), DNA was extracted from the tissue sections according to the manufacturer's instructions. The EGFR mutation status of tumor samples was assessed with the PNA Clamp EGFR Mutation Detection Kit (Panagene, Inc., Daejeon, Korea). For determining ALK gene rearrangement, ALK fluorescence in situ hybridization (FISH) testing was performed using the Vysis ALK Break-Apart Probe kit (Abbott Molecular, Des Plaines, IL, USA).

The present study protocol was reviewed and approved by the Institutional Review Board of Samsung Medical Center (IRB File No. 2017-08-110). Informed consent was waived for individual participants included in the study given the retrospective nature of this work.

Case 1 was a 73-year-old female never-smoker who presented with an incidentally found lung mass. She underwent a lobectomy of the right upper lobe without neoadjuvant treatment. The lung mass was 2.8 cm and appeared to be comprised of acinar-pattern adenocarcinoma (30%) and large cell neuroendocrine carcinoma (70%) (Fig. 1A and B). The perihilar lymph node was involved in the tumor (pT1bN1 by 7th edition American Joint Committee on Cancer/International Union Against Cancer (AJCC/UICC) cancer staging system). Clinically distant metastasis was not present. On immunohistochemical evaluation, the adenocarcinoma component of the tumor was positive for thyroid transcription factor-1 (TTF-1) (image not included) and ALK (Fig. 1C), but negative for CD56 (Fig. 1D). The large cell neuroendocrine component of the tumor was positive for TTF-1 (image not included), ALK (Fig. 1C), and CD56 (Fig. 1D). Break-apart signals were detected on an ALK FISH test (Fig. 1E). An EGFR mutation was not detected. After 157 days without adjuvant therapy, the patient had multiple lymph node metastases, peritoneal seeding, adrenal metastasis, and bone metastasis in the right humerus, as shown on positron emission tomography-computed tomography (PET-CT). Endobronchial ultrasound-guided transbronchial needle aspiration biopsy (EBUS-guided TBNA) was performed and metastatic carcinoma, mainly consisting of large cell neuroendocrine carcinoma, was present. After diagnosis, the patient received chemoradiation therapy and crizotinib administration and a partial response was noted after two months. She was then lost to follow-up due to personal reasons.

Case 2 was a 73-year-old male patient who was diagnosed with idiopathic pulmonary fibrosis (IPF). He was an ex-smoker with a 40-pack-year history and was diagnosed with interstitial lung disease on low-dose chest computed tomography (CT) during a health examination. During follow-up, a 2.5-cm mass and a nodular lesion were found in the right middle and lower lobes, respectively. He underwent bilobectomy of the right middle and lower lobes. Two masses were noted and the larger one measured 3.5 cm at its greatest diameter. Hilar, interlobar, and lobar lymph nodes were involved in the tumor (pT3N1 by 7th edition AJCC/UICC cancer staging system). Clinically distant metastasis was not observed. On microscopic examination, the tumor was poorly differentiated, solid-pattern adenocarcinoma, which displayed diffusely large cell neuroendocrine differentiation (Fig. 2A and B). The poorly differentiated adenocarcinoma components of the tumor were positive for ALK and focally positive for CD56 (Fig. 2C and D). The neuroendocrine differentiated component showed ALK- and CD56-positivity (Fig. 2C and D). Break-apart signal was not detected on an ALK FISH test (Fig. 2E). No EGFR mutation was detected. While there was no evidence of disease recurrence noted on follow-up, the patient died three months later due to IPF progression.

Case 3 was a 64-year-old male who was a current-smoker with a 60-pack-year smoking history. A mass in the left upper lobe was identified during a low-dose chest CT. He underwent a lobectomy of the left lower lobe. The resected specimen was a 2.2-cm mass with multiple satellite small nodules that were comprised of combined adenocarcinoma (10%) and SCLC (90%) components. The left lower paratracheal and subaortic lymph nodes were involved in the tumor (pT3N2 by 7th edition AJCC/UICC cancer staging system). Clinically distant metastasis was not observed. On microscopic examination, the tumor appeared to be composed mostly of SCLC (90%) with focal acinar- and solid-pattern adenocarcinomas (Fig. 3A-C). Tumor cells were positive for ALK (Fig. 3D) and CD56 (Fig. 3E). Break-apart signal was not shown on an ALK FISH test (image not included). No EGFR mutation was shown. After surgery, the patient was given a standard SCLC chemotherapy regimen. The tumor metastasized in multiple T- and L-spine bones after 243 days, and to the brain thereafter. He was eventually lost to follow-up.

Case 4 was a 35-year-old female, a never-smoker who presented with left chest pain and dyspnea. A left pleural effusion and pleural multifocal nodularity were discovered with no definite lung parenchymal lesions on a chest CT taken during her initial work-up. Metastatic non-SCLC (NSCLC) was present on pleural fluid aspiration cytology. Palliative chemotherapy with pemetrexed and cisplatin was initiated. After showing a partial response and subsequent progression, palliative target therapy with gefitinib was administered, but the same combined response, partial response and subsequent progression was observed. Re-biopsy of the left axillary lymph node was done, and the lymph node revealed metastatic adenocarcinoma (Fig. 4A). The tumor cells were positive for ALK immunohistochemically (Fig. 4B), and an ALK FISH test was performed. The FISH test revealed break-apart signals (Image not shown). An EGFR mutation was not detected. Thereafter, target therapy using ALK-TKI with crizotinib was administrated. After partial response and progression, palliative radiotherapy was begun. Re-biopsy of the right supraclavicular lymph node was done, and the tumor cells displayed neuroendocrine morphology without typical adenocarcinoma (Fig. 4C). This tumor cells were positive for CD56 (Fig. 4D). The possibility of transformation to neuroendocrine carcinoma as a resistance mechanism to ALK-TKI was suspected. She underwent radiation therapy and was administrated another ALK-TKI-ceritinib regimen. She displayed a partial response to the ceritinib treatment, and was still alive six years later.