A limited number of patients with lung squamous cell carcinoma (SCC) benefit clinically from molecular targeted drugs because of a lack of targetable driver alterations. We aimed to understand the prevalence and clinical significance of lysine-specific demethylase 5D (KDM5D) copy number loss in SCC and explore its potential as a predictive biomarker for ataxia-telangiectasia and Rad3-related (ATR) inhibitor treatment. We evaluated KDM5D copy number loss in 173 surgically resected SCCs from male patients using fluorescence in situ hybridization. KDM5D copy number loss was detected in 75 of the 173 patients (43%). Genome-wide expression profiles of the transcription start sites (TSSs) were obtained from 17 SCCs, for which the cap analysis of gene expression assay was performed, revealing that upregulated genes in tumors with the KDM5D copy number loss are associated with 'cell cycle', whereas downregulated genes in tumors with KDM5D copy number loss were associated with 'immune response'. Clinicopathologically, SCCs with KDM5D copy number loss were associated with late pathological stage (p = 0.0085) and high stromal content (p = 0.0254). Multiplexed fluorescent immunohistochemistry showed that the number of tumor-infiltrating CD8+ /T-bet+ T cells was lower in SCCs with KDM5D copy number loss than in wild-type tumors. In conclusion, approximately 40% of the male patients with SCC exhibited KDM5D copy number loss. Tumors in patients who show this distinct phenotype can be 'cold tumors', which are characterized by the paucity of tumor T-cell infiltration and usually do not respond to immunotherapy. Thus, they may be candidates for trials with ATR inhibitors.

There are multiple transcription start sites (TSSs) in agreement with multiple transcript variants encoding different isoforms of NKX2-1/TTF-1 (thyroid transcription factor 1); however, the clinicopathological significance of each transcript isoform of NKX2-1/TTF-1 in lung adenocarcinoma (LAD) is unknown. Herein, TSS-level expression of NKX2-1/TTF-1 isoforms was evaluated in 71 LADs using bioinformatic analysis of cap analysis of gene expression (CAGE)-sequencing data, which provides genome-wide expression levels of the 5'-untranslated regions and the TSSs of different isoforms. Results of CAGE were further validated in 664 LADs using in situ hybridisation. Fourteen of 17 TSSs in NKX2-1/TTF-1 (80% of known TSSs in FANTOM5, an atlas of mammalian promoters) were identified in LADs, including TSSs 1-13 and 15; four isoforms of NKX2-1/TTF-1 transcripts (NKX2-1_001, NKX2-1_002, NKX2-1_004, and NKX2-1_005) were expressed in LADs, although NKX2-1_005 did not contain a homeodomain. Among those, six TSSs regulated NKX2-1_004 and NKX2-1_005, both of which contain exon 1. LADs with low expression of isoforms from TSS region 11 regulating exon 1 were significantly associated with poor prognosis in the CAGE data set. In the validation set, 62 tumours (9.3%) showed no expression of NKX2-1/TTF-1 exon 1; such tumours were significantly associated with older age, EGFR wild-type tumours, and poor prognosis. In contrast, 94 tumours, including 22 of 30 pulmonary invasive mucinous adenocarcinomas (IMAs) exhibited exon 1 expression without immunohistochemical TTF-1 protein expression. Furthermore, IMAs commonly exhibited higher exon 1 expression relative to that of exon 4/5, which contained a homeodomain in comparison with EGFR-mutated LADs. These transcriptome and clinicopathological results reveal that LAD use at least 80% of NKX2-1 TSSs and expression of the NKX2-1/TTF-1 transcript isoform without exon 1 (NKX2-1_004 and NKX2-1_005) defines a distinct subset of LAD characterised by aggressive behaviour in elder patients. Moreover, usage of alternative TSSs regions regulating NKX2-1_005 may occur in subsets of LADs.