NKX2­1 copy number alterations are associated with oncogenic, immunological and prognostic remodeling in non­small cell lung cancer.

NK2 homeobox 1 (NKX2-1) copy number alterations (CNAs) are frequently observed in lung cancer. However, little is known about the complete landscape of focal alterations in NKX2-1 copy number (CN), their clinical significance and their therapeutic implications in non-small cell lung cancer (NSCLC). The correlations between NKX2-1 expression and EGFR driver mutations and programmed death ligand 1 (PD-L1) co-expression were studied using immunohistochemistry and PCR from the tumors of recruited Filipino patients (n=45). Clinical features of NSCLC with NKX2-1 CNAs were resolved at the tumor and clonal levels using the molecular profiles of patients with lung adenocarcinoma and lung squamous cell carcinoma from The Cancer Genome Atlas (n=1,130), and deconvoluted single-cell RNA-seq data from the Bivona project (n=1,654), respectively. Despite a significant and positive correlation between expression and CN (r=0.264; P<0.001), NKX2-1 CNAs exerted a stronger influence on the combined EGFR and PD-L1 status of NSCLC tumors than expression. NKX2-1 CN gain was prognostic of favorable survival (P=0.018) and a better response to targeted therapy. NKX2-1 CN loss predicted a worse survival (P=0.041). Mutational architecture in the Y-chromosome differentiated the two prognostic groups. There were 19,941 synonymous mutations and 1,408 genome-wide CN perturbations associated with NKX2-1 CNAs. Tumors with NKX2-1 CN gain expressed lymphocyte markers more heterogeneously than those with CN loss. Higher expression of tumor-infiltrating lymphocyte gene signatures in CN gain was prognostic of longer disease-free survival (P=0.005). Tumors with NKX2-1 CN gain had higher B-cell (P<0.001) and total T-cell estimates (P=0.003). NKX2-1 CN loss was associated with immunologically colder tumors due to higher M2 macrophage infiltrates (P=0.011) and higher expression of immune checkpoint proteins, CD274 (P=0.025), VTCN1 (P<0.001) and LGALS9 (P=0.002). In conclusion, NKX2-1 CNAs are associated with tumors that exhibit clinically diverse characteristics, and with unique oncogenic, immunological and prognostic signatures.

The differential prognostic implications of PD-L1 expression in the outcomes of Filipinos with EGFR-mutant NSCLC treated with tyrosine kinase inhibitors.

Background: The tumor immune microenvironment influences tumor evolution in non-small cell lung cancer (NSCLC). Yet, the prognostic value of programmed death-ligand 1 (PD-L1) in epidermal growth factor receptor (EGFR)-mutant NSCLC remains controversial. Additionally, prognostic studies in Filipinos with EGFR-mutant NSCLC remain unexplored to this day. Methods: We prospectively studied the outcomes of EGFR-mutant NSCLC in Filipino cohort, and retrospectively verified the survival trend using The Cancer Genome Atlas (TCGA) cohort. Kaplan-Meier method and generalized linear regression were used to assess survival. Expression and DNA methylation of cluster of differentiation 274 (CD274, gene that codes for PD-L1) were examined from TCGA tumor profiles. Pearson's correlation was used to correlate PD-L1 expression with outcomes associated with occurrence of EGFR mutations, tyrosine kinase inhibitor (TKI) types, and programmed cell death protein 1 (PD-1) expression. Proteome network analysis was used to examine the correlation between drug resistance and PD-L1. Results: PD-L1 positivity was associated with significantly longer progression-free survival (PFS; P=0.0096) but had a significantly contrasting influence in the overall survival (OS; P=0.0011). PD-L1 positivity (in both protein and RNA) was associated with longer median OS (mOS) in exon21 L858R, whereas, negativity was associated with longer mOS in exon19 deletion (exon19del). Stratification (high, low, negative) of PD-L1 expression lacked significant prognostic value (all P>0.05). PD-L1/CD274 expression (P<0.05) and DNA methylation (P<0.001) vary significantly among NSCLC subtypes and in different disease stages. Erlotinib treatment produced the longest median progression-free survival (mPFS; 874 days) relative to other EGFR-TKIs (137-311 days). PD-L1 lacked a significant correlation with EGFR-TKIs. Consistent with the immune-regulation activities of PD-1, higher expression leads to relatively shorter mOS. PD-1 correlated positively with PD-L1 expression and occurrence of exon21 L858R. Conclusions: PD-L1 differentially influenced the outcomes of Filipinos with EGFR-mutant NSCLC. NSCLC subtypes, disease stage, and PD-1 expression may impact the collective outcomes associated with PD-L1 and EGFR-sensitizing mutations.

Assessment of the feasibility of frozen sections for the detection of spread through air spaces (STAS) in pulmonary adenocarcinoma.

Spread through air spaces (STAS) is reportedly associated with worse prognosis in sublobar resections of lung adenocarcinoma. Recently, it was proposed that STAS detected on frozen sections can be an indication for lobectomy instead of sublobar resection. We undertook this study to evaluate the reliability of STAS assessment on frozen sections compared to permanent sections, as well as the associations among STAS, tumor grade, and recurrence-free survival (RFS) after sublobar resection. A total of 163 stage I lung adenocarcinoma resections with frozen sections were identified retrospectively. For each case, and for frozen and permanent sections separately, the presence or absence of STAS, as well as the tumor grade, were recorded. Compared to permanent sections, STAS detection on frozen sections had low sensitivity (55%), low positive predictive value (48%), and fair agreement (K = 0.34), whereas there was higher specificity (80%) and negative predictive value (85%). Accuracy was 74%. Tumor grade assessment on frozen sections showed higher sensitivity (77%), positive predictive value (90%), agreement (K = 0.72), specificity (94%), and accuracy (87%), and the same negative predictive value (85%). High-grade histology on frozen sections was associated with shorter RFS (p = 0.02), whereas STAS on frozen sections was not (p = 0.47). Our results suggest that the intraoperative detection of STAS has low sensitivity and positive predictive value. False-positive results may lead to overtreatment of patients with lung cancer. The determination of tumor grade on frozen sections offers better sensitivity and specificity, plus it is associated with RFS, whereas STAS on frozen sections is not. Further study is needed to explore the utility of assessing tumor grade on frozen sections.

Accuracy and Reproducibility of Intraoperative Assessment on Tumor Spread Through Air Spaces in Stage 1 Lung Adenocarcinomas.

INTRODUCTION: Tumor spread through air spaces (STAS) is associated with worse prognosis in early-stage lung adenocarcinomas, particularly in sublobar resection. Intraoperative consultation for STAS has been advocated to guide surgical management. However, data on accuracy and reproducibility of intraoperative assessment of STAS remain limited. We evaluated diagnostic yield, interobserver agreement (IOA), and intraobserver agreement (ITA) for STAS detection on frozen section (FS). METHODS: A panel of three pathologists evaluated stage 1 lung adenocarcinomas (n = 100) for the presence or absence of STAS and artifacts as reference. Five pulmonary pathologists independently reviewed all cases in two rounds, detecting STAS and artifacts in FS and the corresponding FS permanent and non-FS permanent, with a consensus conference between rounds. RESULTS: The FS had low sensitivity (44%), high specificity (91%), relatively high accuracy (71%), and overall area under the receiver operating characteristic curve of 0.67 for detecting STAS. The average ITA was moderate for both STAS (κmean: 0.598) and artifact (κmean: 0.402) detection on FS. IOA was moderate for STAS (κround-1: 0.453; κround-2: 0.506) and fair for artifact (κround-1: 0.300; κround-2: 0.204) detection on FS. IOA for STAS improved in FS permanent and non-FS permanent, whereas ITA was similar across section types. On multivariable logistic regression, the only significant predictor of diagnostic discordance was the presence of artifacts. CONCLUSIONS: FS is highly specific but not sensitive for STAS detection in stage 1 lung adenocarcinomas. IOA on STAS is moderate in FS and improved only marginally after a consensus conference, raising concerns regarding global implementation of intraoperative assessment of STAS and warranting more precise criteria for STAS and artifacts.