Clinical Implications of Inter- and Intratumor Heterogeneity of Immune Cell Markers in Lung Cancer

Abstract

Background: Immune cell transcriptome signatures have been widely used to study the lung tumor microenvironment (TME). However, it is unclear to what extent the immune cell composition in the lung TME varies across histological and molecular subtypes (intertumor heterogeneity [inter-TH]) and within tumors (intratumor heterogeneity [ITH]) and whether ITH has any prognostic relevance.

Methods: Using RNA sequencing in 269 tumor samples from 160 lung cancer patients, we quantified the inter-TH of immune gene expression and immune cell abundance and evaluated the association of median immune cell abundance with clinical and pathological features and overall survival. In 39 tumors with 132 multiregion samples, we also analyzed the ITH of immune cell abundance in relation to overall survival using a variance-weighted multivariate Cox model not biased by the number of samples per tumor.

Results: Substantial inter-TH of 14 immune cell types was observed even within the same histological and molecular subtypes, but early stage tumors had higher lymphocyte infiltration across all tumor types. In multiregion samples, an unbiased estimate of low ITH of overall immune cell composition (hazard ratio [HR] = 0.40, 95% confidence interval [CI] = 0.21 to 0.78; P = .007), dendritic cells (HR = 0.24, 95% CI = 0.096 to 0.58; P = .002), and macrophages (HR = 0.50, 95% CI = 0.30 to 0.84; P = .009) was strongly associated with poor survival. The ITH of 3 markers, including CD163 and CD68 (macrophages) and CCL13 (dendritic cells), was enough to characterize the ITH of the corresponding immune cell abundances and its association with overall survival.

Conclusion: This study suggests that lack of immune cell diversity may facilitate tumor evasion and progression. ITH inferred from CCL13, CD163, and CD68 could be used as a prognostic tool in clinical practice.

Figure 1.

Summary of inter-tumor heterogeneity (inter-TH) and intra-tumor heterogeneity (ITH) of EAGLE-lung samples. A) Unsupervised clustering of RNA-based abundance of 14 immune cells across 269 tumor samples from 160 tumors. Histological types, molecular subtypes, sex, smoking status, consensus clusters (cluster count K = 3), and immune classes are indicated by different colors of the column sidebars. The P values are based on the χ² test of the immune classes and clinical features. B) Summary of immune classes of samples from the same subjects. 39 subjects with at least 2 samples per subject that passed the purity filtering were analyzed. Histological types of subjects are indicated by different colors of the diamond symbols. C) Inter- and intratumor mRNA heterogeneity quadrant of 18 927 expressed genes in 132 samples from 39 multiregion tumors. Each dot represents a gene. Genes of immune signatures are indicated by colors. 2D density contour is indicated by blue lines. The plot is split into 4 quadrants by average inter-TH and ITH, defined as Q1 to Q4, respectively (ie, x- and y-axes indicate mean values of ITH and inter-TH scores, respectively). The numbers of all genes and immune genes are indicated by circles on the left and right sides of the figure. All statistical tests were 2-sided. DC = dendritic cells; NK = natural killer; Treg = regulatory T cells.

Figure 2.

Association of immune cell abundances with clinical and pathological features. A) Comparison of 8 immune cell abundances in former (n = 73) and current smokers (n = 80). The immune cell abundances were determined by the average expression level across all marker genes for each immune cell type and were plotted in the same scale across all figures in the panel. The nominal P value is based on a 2-sided Student t-test. B) Distribution of the natural kill (NK) cell and CD8 T-cell abundances in patients with different smoke duration periods. P values are based on ordinal regression adjusted for smoking status. C) Comparison of 5 immune cell abundance in subjects with different tumor stages. P values are based on a 2-sided Student t-test comparing the stage I patients vs stage II-IV patients. D) Comparison of NK cell abundance in different histological types. The P value is based on a 2-sided Student t-test of adenocarcinoma (n = 100) and squamous and/or epidermoid carcinoma (n = 45). DC = dendritic cell.

Figure 3.

Association between overall survival and intratumor heterogeneity (ITH) of immune cells in subjects with multiregion tumor samples. A-C) Kaplan-Meier curves for overall survival stratified by the median APITH of (A) overall immune cells, (B) dendritic cells, and (C) macrophages. P values and hazard ratios indicated are based on the multivariate model. D-F) Distribution of log₁₀ (P values) of the multivariate Cox proportional-hazards model for 500 random signatures of identical sizes of (D) overall immune cells, (E) dendritic cells, and (F) macrophages. P values of corresponding immune ITH are indicated by arrows. All statistical tests were 2-sided. CI = confidence interval; HR = hazard ratio.

Figure 4.

Association between overall survival and intratumor heterogeneity (ITH) of single immune gene markers. A-C) Scatter plots of the gene expression levels of immune markers and the corresponding immune cell abundances for (A) CCL13 (DC marker), (B) CD163 (macrophage marker), and (C) CD68 (macrophage marker). D-F) Scatter plots of the single gene-based APITH and APITH of the corresponding immune cells for (D) CCL13, (E) CD163, and (F) CD68. G-I) Kaplan-Meier curves for overall survival stratified by the median APITH of (G) CCL13, (H) CD163, and (I) CD68. P values and hazard ratios indicated are based on the multivariate model. J-L) Distribution of log₁₀ (P values) of the multivariate Cox proportional hazards model for all expressed genes (n = 18 927). P values of (J) CCL13, (K) CD163, and (L) CD68 are indicated by arrows. CI = confidence interval; DC = dendritic cell; HR = hazard ratio; TPM = transcripts per million .