Introduction: DNA mutational profiling showed that atypical carcinoids (ACs) share alterations with large cell neuroendocrine carcinomas (LCNECs). Transcriptomic studies suggested that LCNECs are composed of two subtypes, one of which shares molecular anomalies with SCLC. The missing piece of information is the transcriptomic relationship between ACs and LCNECs, as a direct comparison is lacking in the literature. Methods: Transcriptomic and genomic alterations were investigated by next-generation sequencing in a discovery set of 14 ACs and 14 LCNECs and validated on 21 ACs and 18 LCNECs by using custom gene panels and immunohistochemistry for Men1 and Rb1. Results: A 58-gene signature distinguished three transcriptional clusters. Cluster 1 comprised 20 LCNECs and one AC harboring concurrent inactivation of tumor protein p53 gene (TP53) and retinoblastoma 1 gene (RB1) in the absence of menin 1 gene (MEN1) mutations; all cases lacked Rb1 nuclear immunostaining. Cluster 3 included 20

preoperative small biopsies. Moreover, in the same series, 426 patients and 369 patients, respectively, were evaluated for epidermal growth factor receptor and KRAS mutations, respectively. RESULTS: Of the total of 493 patients, 18 patients who were positive for a gene rearrangement (4.4%) demonstrated ALK FISH rearrangements, whereas 387 patients (95.6%) were negative. All other cases were classified as inadequate (7.7%) and insufficient (10.1%). A strong statistical association was found between the cytology and the small biopsy with respect to ALK rearrangements (P 5.0048). Fiftythree patients (12.4%) demonstrated an epidermal growth factor receptor mutation, whereas 90 patients (24.4%) were found to have KRAS mutations.

Our results show that IHC may be a useful technique for selecting NSCLC cases to undergo ALK FISH analysis.