Germline Predisposition and Copy Number Alteration in Pre-stage Lung Adenocarcinomas Presenting as Ground-Glass Nodules

Abstract

Objective: Synchronous multiple ground-glass nodules (SM-GGNs) are a distinct entity of lung cancer which has been emerging increasingly in recent years in China. The oncogenesis molecular mechanisms of SM-GGNs remain elusive. Methods: We investigated single nucleotide variations (SNV), insertions and deletions (INDEL), somatic copy number variations (CNV), and germline mutations of 69 SM-GGN samples collected from 31 patients, using target sequencing (TRS) and whole exome sequencing (WES). Results: In the entire cohort, many known driver mutations were found, including EGFR (21.7%), BRAF (14.5%), and KRAS (6%). However, only one out of the 31 patients had the same somatic missense or truncated events within SM-GGNs, indicating the independent origins for almost all of these SM-GGNs. Many germline mutations with a low frequency in the Chinese population, and genes harboring both germline and somatic variations, were discovered in these pre-stage GGNs. These GGNs also bore large segments of copy number gains and/or losses. The CNV segment number tended to be positively correlated with the germline mutations (r = 0.57). The CNV sizes were correlated with the somatic mutations (r = 0.55). A moderate correlation (r = 0.54) was also shown between the somatic and germline mutations. Conclusion: Our data suggests that the precancerous unstable CNVs with potentially predisposing genetic backgrounds may foster the onset of driver mutations and the development of independent SM-GGNs during the local stimulation of mutagens.

Keywords: copy number variation; driver mutations; ground-glass nodule; lung cancer; whole-exome sequencing.

Figure 1

Overview of genomic variant analyses of 69 GGN cases. The Targeted sequencing data and whole-exome sequencing data were analyzed separately for the variants. SNV, single nucleotide variations; INDEL, insertions and deletions; CNV, copy number variations; SM-GGN, Synchronous multiple ground-glass nodules.

Figure 2

Somatic variant detections. Somatic SNVs were detected by MuTect and INDELs were identified by StrelKa. Variants in genes with missense/truncated or in splicing sites (patho-variants) were identified, ranked, and displayed by paired samples. (A) Patho-variants detected from Targeted sequencing. (B) Patho-variants detected from whole-exome sequencing.

Figure 3

Somatic mutational signatures deconstructed from GGN samples. (A) The average somatic mutation spectra of the AAH, AIS, MIA, and AD groups were obtained from variants (SNV/INDELs) of 25 AAH, 13 AIS, 5 MIA, and 8 AD TRS samples (left). The somatic signatures of AIS (mean of 9 AIS), MIA (mean of 5), and AD (mean of 4) detected from 18 WES data are displayed (right). (B) The bar chart represents the proportions of the signatures in each group.

Figure 4

Lolliplots showing the distribution of germline and somatic variants in the top two genes, FLG (A,B) and MUC4 (C,D). For those germline variants that had >0.01 Chinese MAF, only those variants that were predicted to be deleterious by PROVEAN or damaging by SIFT are displayed (red star). The X-axis represents the exon and chromosome location. The Y-axis represents the occurrence of variants in GGNs (somatic) or patients (germline). The distribution of variants in AAH GGNs (upper panel) (A,C) was compared with that in other GGNs (bottom panel) (B,D).

Figure 5

Unsupervised hierarchical clustering of copy number variations among GGNs and correlations between CNV and mutations. (A) CNVs of 51 SM-GGNs from TRS data were clustered by Pearson correlation. (B) Eighteen triple GGNs from the WES data also showed correlation patterns within each patient by clustering. Pearson correlation analysis was performed. (C) Pearson correlation between total CNV segment number and germline nonsynonymous mutations of WES data. (D) Correlation between total CNV size and all somatic mutations of WES data. (E) Correlation between all germline and somatic mutations of WES data.

Figure 6

Phylogenic tree view of the triple GGN evolution structure. All somatic variants (SNV/INDELs) detected from whole-exome sequencing were compared among the three GGNs of the same patients. The key potential driver mutations acquired at a particular point are indicated. The trees showed genetic similarity (trunk) and dissimilarity (branch) of the SM-GGNs. Six patients of WES cohort: (A) patient M1; (B) M2; (C) M3; (D) M4; (E) M5; (F) M6.

Figure 7

Proposed SM-GGN origination models. Five models plus an unknown process were hypothesized. These different originations could occur in different cases, or even mixed in one patient. There are evidence supporting the lymph metastasis SM-GGNs, aero metastasis SM-GGNs, Convergent SM-GGNs (CVG), and the inherent sporadic SM-GGNs (ISG). The independent clonal SM-GGNs (ICG) we hypothesize is prone to the deficient local immune microenvironment or presumably to the biochemical substances released from the primary tumor lesion.