Differential pathogenesis of lung adenocarcinoma subtypes involving sequence mutations, copy number, chromosomal instability, and methylation

Abstract

Background: Lung adenocarcinoma (LAD) has extreme genetic variation among patients, which is currently not well understood, limiting progress in therapy development and research. LAD intrinsic molecular subtypes are a validated stratification of naturally-occurring gene expression patterns and encompass different functional pathways and patient outcomes. Patients may have incurred different mutations and alterations that led to the different subtypes. We hypothesized that the LAD molecular subtypes co-occur with distinct mutations and alterations in patient tumors.

Methodology/principal findings: The LAD molecular subtypes (Bronchioid, Magnoid, and Squamoid) were tested for association with gene mutations and DNA copy number alterations using statistical methods and published cohorts (n = 504). A novel validation (n = 116) cohort was assayed and interrogated to confirm subtype-alteration associations. Gene mutation rates (EGFR, KRAS, STK11, TP53), chromosomal instability, regional copy number, and genomewide DNA methylation were significantly different among tumors of the molecular subtypes. Secondary analyses compared subtypes by integrated alterations and patient outcomes. Tumors having integrated alterations in the same gene associated with the subtypes, e.g. mutation, deletion and underexpression of STK11 with Magnoid, and mutation, amplification, and overexpression of EGFR with Bronchioid. The subtypes also associated with tumors having concurrent mutant genes, such as KRAS-STK11 with Magnoid. Patient overall survival, cisplatin plus vinorelbine therapy response and predicted gefitinib sensitivity were significantly different among the subtypes.

Conclusions/ significance: The lung adenocarcinoma intrinsic molecular subtypes co-occur with grossly distinct genomic alterations and with patient therapy response. These results advance the understanding of lung adenocarcinoma etiology and nominate patient subgroups for future evaluation of treatment response.

Figure 1. Lung adenocarcinoma molecular subtype expression characteristics.

LAD subtype gene expression is displayed for all cohorts (Bhattacharjee et al., Chitale et al., Ding et al., Shedden et al., Tomida et al., UNC, Zhu et al.) in which columns are tumors and rows are genes and shading indicates gene expression level (A). Exemplar genes are displayed separately for visualization ease (B).

Figure 2. Molecular subtypes compared by chromosomal instability and regional DNA copy number.

Chromosomal instability (CIN) grouped by molecular subtypes is displayed for discovery cohorts with CN (Chitale et al., Ding et al.) and the validation cohort (UNC) (A). Subtype CIN was compared by a Kruskal-Wallis test on three subtypes of the discovery cohorts (two-sided P′). A Wilcoxon rank-sum test evaluated the null hypothesis that Magnoid had not greater CIN than other subtypes in the validation cohort (one-sided P″). Normal lung specimens' CIN are shown for reference. The subtype regional DNA copy number (CN) medians of the discovery cohort are displayed (B). CN values below zero indicate copy number deletion, at zero indicate normal copy number, and above zero indicate copy number amplification. Subtype-associated CN regions from the discovery cohorts are displayed (C), in which the subtype with greatest absolute copy number is indicated by colored rectangle. For independent confirmation, these regions and associated-subtypes were tested in the validation cohort, results of which are displayed (D). For example in discovery cohorts, CN of 1q21–23 was significantly different among subtypes with Magnoid having the greatest CN (C), and in the validation cohort, Magnoid had significantly greater absolute CN compared to other subtypes (D). Afterwards, subtypes were compared by all regions in the validation cohort and new regions are marked by a dot (D).

Figure 3. Coordination of DNA copy number and gene expression among subtypes.

Each point represents one of the 26 subtype-associated copy number (CN) regions, which are colored by the subtype having the greatest absolute copy number (GACN). The vertical axis is the difference in median CN between the GACN subtype and other subtypes. For the genes in each region, the differences in median expression between the GACN subtype and other subtypes were calculated and the median of these differences is the value on the horizontal axis. The association of CN difference and gene expression difference across these regions was compared by a Spearman correlation test (two-sided P). Two example DNA regions are circled. Region 1q21–23 had GACN in Magnoid. Hepatoma-derived growth factor (HDGF) was one of the most Magnoid overexpressed genes in this region. CN and gene expression for HDGF is displayed in which each point is one tumor (B). Region 7p22-12 had GACN in Squamoid. Fascin (FSCN1) was the most Squamoid overexpressed gene in this region (C). For reference, black lines in (B, C) indicate median gene expression and DNA CN. LAD tumors with copy number and expression arrays from all cohorts were used (n = 362).

Figure 4:. Molecular subtypes compared by DNA methylation.

Genomewide DNA methylation among the three LAD subtypes were compared by a Kruskal-Wallis test (two-sided P) in all cohorts with methylation (UNC n = 33) (A). Normal lung specimens are shown for reference. Regional variation in DNA methylation is displayed (B). For each chromosome arm, the proportion of sites hypermethylated in a subtype with respect to normal lung is plotted. Chromosome arms with at least 4 methylation sites are displayed. Tumors' DNA methylation and DNA copy number values for chromosome 5 p are displayed (C).

Figure 5. Integrated gene alterations compared among molecular subtypes.

Tumors are depicted as columns and genetic features as rows from cohorts with CN and gene sequencing (Chitale et al., Ding et al., UNC). Gene CN's were defined by the gene's genomic position. The percentages of tumors within a subtype having a given integrated combination of alterations are displayed in grey. Fisher's exact tests on each integrated combination and subtype were statistically significant (P<0.01).

Figure 6. Patient outcomes compared among molecular subtypes.

Overall survival is displayed for patients from cohorts with survival follow-up (Bhattacharjee et al., Chitale et al., Shedden et al., Tomida et al., UNC, and Zhu et al.; n = 807) (A). Overall survival among the three subtypes was compared by a log-rank test (two-sided P′). Patient disease-specific-survival from the Zhu et al. JBR.10 trial 25] is displayed by treatment type, for each subtype (B). Hazard ratios (HR) compare treatment to observation. Gefitinib sensitivity scores, derived from a cell line expression signature, are displayed for all patients with EGFR mutation status (Bhattacharjee et al., Ding et al., Chitale et al., Tomida et al., UNC; n = 561) (C). Predicted gefitinib sensitivities were compared by Kruskal-Wallis tests among the three subtypes in the EGFR mutation group and separately for the EGFR wild type group (two-sided P″). Wilcoxon rank-sum tests evaluated the hypothesis that Bronchioid tumors have not greater scores than Squamoid and Magnoid tumors within each EGFR mutation group separately (one-sided P″′).