Genomic mapping of metastatic organotropism in lung adenocarcinoma

Abstract

We analyzed 2,532 lung adenocarcinomas (LUAD) to identify the clinicopathological and genomic features associated with metastasis, metastatic burden, organotropism, and metastasis-free survival. Patients who develop metastasis are younger and male, with primary tumors enriched in micropapillary or solid histological subtypes and with a higher mutational burden, chromosomal instability, and fraction of genome doublings. Inactivation of TP53, SMARCA4, and CDKN2A are correlated with a site-specific shorter time to metastasis. The APOBEC mutational signature is more prevalent among metastases, particularly liver lesions. Analyses of matched specimens show that oncogenic and actionable alterations are frequently shared between primary tumors and metastases, whereas copy number alterations of unknown significance are more often private to metastases. Only 4% of metastases harbor therapeutically actionable alterations undetected in their matched primaries. Key clinicopathological and genomic alterations in our cohort were externally validated. In summary, our analysis highlights the complexity of clinicopathological features and tumor genomics in LUAD organotropism.

Figure 1.. Cohort overview.

(A) Number of sequenced specimens in unmatched sample analyses, cohorts 1–4. (B) Clinical outcomes of patients with sequenced primary tumors, cohorts 1–3. Top: Kaplan-Meier curve showing overall survival. Bottom: Metastatic burden per patient (left). Number of patients with metastasis to specific anatomic sites (right). (C) Overview of patients with matched primary and metastatic samples, cohort 5. Overlap of samples included in cohorts 2–4 and matched cohort. (D) Primary and metastatic lesions with whole-exome sequencing (WES). Overlap of samples included in the IMPACT sequencing and WES cohorts. CNS, central nervous system. See also Figure S1 and Table S1.

Figure 2.. Comparison of nonmetastatic (NM) and ever-metastatic (EM) primary tumors.

(A) Oncoprint displaying clinical attributes and genes altered at significantly different frequencies between the two groups. (B) Comparisons of clinicopathologic features. (C) Tumor mutational burden (TMB), fraction of genome altered (FGA), and whole-genome duplication (WGD). Boxplots display median values, interquartile range (IQR) boxes, and whiskers demonstrating 1.5 x IQR. (D) Percentage of samples with the APOBEC mutational signature present. (E) Percentage of samples with alterations for ten canonical oncogenic pathways. (F) Co-occurrence and mutual exclusivity of genes with significant interaction. (G) Genes with significant associations with metastasis-free survival among patients with clinical stage I-III disease (cohorts 1–2, n=576) on univariable analysis. Statistical analyses: (A, B, D-F) Fisher’s exact test. *p<0.05 unless otherwise indicated; q-values correct for multiple comparisons using false-discovery rate (FDR). (C) Wilcoxon rank-sum test for TMB and FGA. Fisher’s exact test for WGD. (G) Cox proportional hazard model. All p-values as indicated, log-rank test. Squares represent hazard ratio (HR) and whiskers display 95% confidence interval (CI). +, genes that remained significant in multivariable model with clinicopathologic factors. Hx, history; pStage, pathologic stage. See also Figure S2 and Table S2.

Figure 3.. Clinicopathologic and genomic features of primary tumors associated with site-specific metastasis and metastatic burden.

(A) Metastatic burden stratified by anatomic site. Each bar represents all ever-metastatic patients with metastasis to a given site; colors correspond to proportion of patients with 1, 2, or ≥3 distinct metastatic sites. (B) Age of patients, stratified by metastatic burden. (C) Fraction of genome altered (FGA), stratified by metastatic burden. Boxplots display median values, interquartile range (IQR) boxes, and whiskers demonstrating 1.5 x IQR. (D) Metastatic burden for significantly different genes between altered and wild-type (WT) tumors. (E) Clinicopathologic and genomic features across each organ site. The fill color corresponds to features enriched in tumors ever metastatic to a given site or not metastatic to a given site. (F) Frequency of significant clinicopathologic features and genomic alterations in nonmetastatic and ever-metastatic tumors with metastasis to lymph node and bone on multivariable analysis. (G) Hazard ratio (HR) for clinicopathologic and genomic features in relation to time to metastasis across each organ site. HR >1 indicates shorter time to metastasis; HR <1 indicates longer time to metastasis. (H) Kaplan-Meier curves demonstrating site-specific metastasis-free survival (MFS) for Hippo pathway alterations for central nervous system (CNS) metastases and CDKN2A alterations for bone metastases. Statistical Analyses: (B) Pearson’s correlation. (C-D) Wilcoxon rank-sum test. *p<0.05. (E) Univariate logistic regression. *p<0.05 for clinicopathologic variables on univariable analysis; °q<0.05 for genomic features on univariable analysis. q-values correct for multiple comparisons using the false-discovery rate (FDR). (F) Multivariate logistic regression. Features shown are significant by p<0.05. (G) Cox proportional hazards model. p-values calculated from log-rank test. *p<0.05 for clinicopathologic variables on univariate analysis; °q<0.05 for genomic features on univariate analysis. Met, metastatic; N, no; pStage, pathologic stage; Y, yes. See also Figures S3, S4, and Table S3.

Figure 4.. Genomic comparisons between primary tumors and metastases.

(A) Left to right: distributions of tumor mutational burden (TMB), fraction of genome altered (FGA), whole-genome duplication (WGD), gene alteration frequencies, pathway alteration frequencies, and frequency of samples presenting APOBEC signatures across groups. Boxplots display median values, interquartile range (IQR) boxes, and whiskers demonstrating 1.5 x IQR. Heat map lists alteration frequencies for genes and pathways as percentages. Darker colors correspond to higher percentage of altered samples. Statistical analysis: Wilcoxon rank-sum test for TMB and FGA; Fisher’s exact test for WGD, gene, pathway, and APOBEC signature frequencies. Significance indicated with a red or green * for q<0.05, false-discovery rate (FDR) adjusted. (B) Top to bottom: distributions of oncogenic alteration types, number of actionable alterations (levels 1 to 3A), and highest level of actionability across grouped sites. (C) Percentage of samples with actionable alterations. Color in the left column corresponds to level of actionability. (D) Mutational signature profiles of primary and metastatic lesions in WES cohort, with metastases stratified by anatomic site. (E) Frequency of samples with signature present for three mutational signatures of interest (SBS2, SBS4, SBS13). Statistical analysis: Fisher’s exact test. ^ and * indicate q<0.05, FDR adjusted, for comparisons of primary vs. lesion site and metastatic lesion site vs. all other metastatic lesions, respectively. CNS, central nervous system; M, metastasis; P, primary tumor; VUS, variant of unknown significance. See also Figures S5–S7 and Tables S4–S6.

Figure 5.. Genomic comparisons between patient-matched primary tumors and metastases.

(A) Median tumor mutational burden (TMB) and fraction of genome altered (FGA) between matched primary tumors and metastases. Statistical analysis: Wilcoxon rank-sum test. p-values as indicated. Right: Proportion of samples with whole-genome duplication (WGD) private or shared between primary and metastatic samples. (B) Distribution of shared and private alterations stratified by alteration types across sites. (C) Actionability of alterations private or shared between primary and metastatic samples across sites. (D) Cancer cell fraction (CCF) of shared mutations between primary and metastatic samples. CCF <0.8 is considered subclonal. (E) Proportion of shared and private mutations, stratified by clonality status for each gene (left) and specific amino acid changes (right). Clonality status nomenclature describes mutation clonality first in metastasis and then primary sample. CNA, copy number alteration; CNS, central nervous system; Fus, fusion; LN, lymph node; Met, metastasis; Mut, mutation; VUS, variant of unknown significance. See also Figure S8.

Figure 6.. Individual patient comparisons of matched primary and metastatic samples.

(A) Summary of genomic and clinical characteristics of patients with one primary and ≥2 matched sequenced metastases. Each numbered cluster represents a patient. One column represents a sequenced sample with the first column representing the primary sample and subsequent columns representing metastatic samples. For example, Patient 1 has one primary and five metastatic samples. (B) Phylogenetic trees for two patients with multiple matched samples, including timeline of sample acquisition and treatment course. CNS, central nervous system; FGA, fraction of genome altered; LN, lymph node; TMB, tumor mutational burden; Tx, treatment; WGD, whole-genome duplication; XRT, radiation.