LMS⁺ primary tumors are larger and can be experimentally selected for during primary tumor growth. (A) A box-and-whisker plot comparing the size distributions for LMS⁻ and LMS⁺ primary tumors in the NKI-295/EMC-344 cohort. Shown are the medians, 25th and 75th percent quartiles, and 1.5 times the interquartile range. P values for the box plots were calculated by using a Wilcoxon rank sum test. (B) Transcriptomic microarray profiling was performed on parental MDA-MB-231 cells, the LMS⁺ in vivo selected lung metastatic LM2 subpopulation (LM2–4175) (15), and cells derived from xenografted parental MDA-MB-231 mouse mammary fat pad tumors. Mammary tumor denotes a sample from which in vivo mRNA expression was assessed directly from a fresh frozen mouse mammary tumor. The heatmap corresponds to relative gene expression levels for 95 previously identified lung metastasis genes (113 probe sets), with red being high and blue indicating low expression. Gene labels are provided for gene clusters of interest due to partial selection during outgrowth of a parental MDA-MB-231 mammary tumor. Genes highlighted in bold are functionally validated mediators of lung metastasis or are included in the 18-gene LMS. (C) Confirmation of microarray-based gene expression levels by using quantitative RT-PCR analysis. Expression values for representative lung metastasis genes were normalized to parental MDA-MB-231 expression levels and displayed graphically as a heatmap. Tumor-derived A and B represent in vitro analyses of independent isolates of cells purified from dissociated MDA-MB-231 mammary tumors.

LMS⁺ primary tumors show a marked rise in metastatic risk after reaching ≥2 cm. (A) Factors that influence the risk of metastasis for patients from the NKI-295/EMC-344 cohort were determined by a random survival forest analysis. Clinical and pathological variables that include tumor size, patient age, histological grade, estrogen receptor status, and the number of positive lymph nodes were simultaneously entered into the model. This method is virtually free of model assumptions and involves constructing survival trees from bootstrap samples by using randomly selected covariates for tree splitting to deliver an ensemble cumulative hazard estimate for metastasis-free survival. The expected frequency of patients developing metastasis from the 128 patients with LMS⁺ tumors (Left) and the 511 patients with LMS⁻ tumors (Right) is obtained from the ensemble estimate and plotted for each covariate. Shown are the results for tumor size. Results for other covariates are shown in SI Fig. 9. Patients that actually developed metastasis are indicated in red along with a lowess regression line through these points shown in magenta. (B) A concordance index from a random survival forest analysis modeling the influence of the LMS, tumor size, and other breast cancer prognostic gene expression signatures on the risk for lung metastasis was calculated (indicated by “All”) by using the NKI-295 cohort. This was then repeated with each of the indicated gene signatures or tumor size omitted from the full model (indicated above the blue bracket “Variable Removed”). The results from 50 runs are shown as a box-and-whisker plot. Nonoverlapping notches are considered significant. Both lung metastasis (Left) and overall survival (Right) were separately analyzed.

Expression of the LMS predicts for increased risk of distant failure selectively in the lung and is associated with other markers of poor prognosis. (A) Kaplan–Meier curves representing the probability of cumulative lung metastasis-free survival for the MSK (Left) and combined NKI-295/EMC-344 (Right) cohorts. (B) Distribution of site(s) of first distant failure (simultaneous metastasis sites included) in the NKI-295/EMC-344 cohort according to LMS status. The P value for the difference in distribution for lung metastasis is shown and was calculated by a χ² test. Patients with LMS⁺ primary tumors are shown in red, and LMS⁻ tumors are in blue. (C) Hierarchical clustering was performed on the NKI-295 cohort with the indicated pathological and genomic markers consisting of the 70-gene prognosis signature (9, 10) (NKI 70), 16-gene recurrence score (18) (Rec score), molecular subtype (19) (Subtype), and wound response signature (17) (Wound). The legend for the color codes for each marker is shown. The luminal A, luminal B, and normal-like subtypes were grouped together as “Other” and consist of 5.8, 1.9, and 7.7% of LMS⁺ tumors, respectively.

LMS⁺ primary tumors intravasate upon reaching larger tumor sizes. (A) Mice bearing parental or LM2 tumors grown in the mammary gland to various sizes were killed and bled via cardiac puncture. Mouse blood was lysed and plated in culture. Tumor cell colonies were scored and plotted as a function of tumor volume. (B) A suspension of 200 LM2 or parental MDA-MB-231 cells expressing luciferase were injected into the tail vein of mice. The development of lung metastasis was monitored over the indicated time course by using noninvasive luciferase bioluminescence imaging and quantitated by measuring photon flux. Values are the mean of four animals ± SEM. Representative mice from each group are shown.

Tumor size influences the probability of lung metastasis for LMS⁺ tumors in a manner that is independent of discernable uniform changes in gene expression. (A) Box-and-whisker plot comparing tumor size distributions between the MSK and NKI-295/EMC-344 cohorts. The P value was calculated by using a Wilcoxon rank sum test. (B) Lung metastasis-free survival for patients with LMS⁺ tumors (red) compared with LMS⁻ tumors (blue) in the NKI-295/EMC-344 cohort after excluding patients with a tumor size <3 cm (the approximate median size for the MSK cohort). P value was calculated by using the log-rank test. (C) Gene expression changes in tumors <2 cm in size and tumors ≥2 cm in size among LMS⁺ primaries from the NKI-295 cohort were compared by using SAM. Shown is a Q–Q plot with the Δ value corresponding to a median FDR of 10% marked by the dashed line. Genes that lie outside of this dashed line are deemed significant. In this case, no genes are determined to be significant at an FDR of 10%. The inset shows the number of significant genes as a function of increasing FDR.