Molecular Profiling reveals Tumor Immune Microenvironment Heterogeneity of Concurrent Lung Adenocarcinoma and Squamous Cell Carcinoma in Multiple Primary Lung Cancers

Abstract

Background: Over the past decade, the annual incidence of multiple primary lung cancers (MPLC) has increased. There is a group of pathologically different patients with MPLC presenting as mostly having concurrent lung adenocarcinoma (ADC) and squamous cell carcinoma (SQCC). Their molecular profiles and tumor immune microenvironment (TIME) remain unknown. We aimed to clarify these factors in this patient group. Methods: We performed mutational, biomarker, and neoantigen analyses using whole-exome sequencing (WES) and differential, enrichment,and TIME analyses using RNA sequencing (RNA-Seq) for five patients with concurrent ADC and SQCC to analyze their mutational profile, expression profile, and TIME features. Results: WES of 10 lesions (five ADC and five SQCC) in five concurrent ADC and SQCC patients showed that the most frequently mutated genes in both groups were TTN, TP53, WDFY3, EGFR, ZFHX4 and TTC40, and there were no significantly different mutated genes in the comparative analysis of mutation frequencies between both groups. There were no significant differences inneoantigen analysis, including tumor mutational burden, tumor neoantigen burden, and mutant allele tumor heterogeneity. HLA genotype analysis indicated that three of five (60%) patients carried the HLA-I class B62 supertype, and seven of 10 (70%) lesions had loss of HLA heterozygosity (HLA-LOH). RNA-Seq revealed that 2231 genes were differently expressed between the ADC and SQCC groups. GSVA based on the molecular signature database (MsigDB) showed that 12 gene sets were significantly differentially expressed between the ADC and SQCC groups (p < 0.05), with four gene sets relevant to squamous cell features upregulated in the SQCC group and eight metabolism-related gene sets upregulated in ADC. GO/KEGG analysis of immune function-related pathways showed that the interleukin-12 family-related signaling pathways, TP53 and TLR2/4 signaling pathways were upregulated in the ADC group. Co-expression analysis of TIME revealed that tumor infiltrating immune cells were significantly more enriched and diverse, with tumor-infiltrating lymphocytes scoring significantly higher (p = 0.047) in ADC. Specifically, CD8+ T cells, exhausted CD8+ cells, and macrophages infiltrated significantly more frequently in ADC (p < 0.05). Moreover, immunocostimulator expression (p = 0.034) and tertiary lymphoid structure score (p = 0.017) were significantly higher in ADC than in SQCC. Conclusion: In patients with concurrent ADC and SQCC MPLC, the transcriptomic profiles and TIME features were quite different between ADC and SQCC lesions. ADC lesions exhibited a more active TIME than SQCC lesions.

From Morphology to Molecules: Advances in the Distinction of Multiple Primary Lung Cancers From Intrapulmonary Metastases in Non–Small Cell Lung Cancer

“…17 The debate over NGS panel size (number of genes) is ongoing. 40,41 Although larger panels are suggested to yield higher accuracy, they do not necessarily guarantee higher benefits. For instance, similar results were obtained with a 48 genes test and a panel covering only 4 driver genes.…”