Genetic diversity among metastases is poorly understood but contains important information about disease evolution at secondary sites. Here we investigate inter- and intra-lesion heterogeneity for two types of metastases that associate with different clinical outcomes: lymph node and distant organ metastases in human colorectal cancer. We develop a rigorous mathematical framework for quantifying metastatic phylogenetic diversity. Distant metastases are typically monophyletic and genetically similar to each other. Lymph node metastases, in contrast, display high levels of inter-lesion diversity. We validate these findings by analyzing 317 multi-region biopsies from an independent cohort of 20 patients. We further demonstrate higher levels of intra-lesion heterogeneity in lymph node than in distant metastases. Our results show that fewer primary tumor lineages seed distant metastases than lymph node metastases, indicating that the two sites are subject to different levels of selection. Thus, lymph node and distant metastases develop through fundamentally different evolutionary mechanisms.
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This work was support by grants from the NIH (R37CA225655), AACR (561314) and NHLBI (P01HL142494) to K.N. and from NCI (R00CA22999102) to J.G.R. We thank S. Bian, F. Tang and W. Fu for sharing with us the frequencies of CRC01 subclones across different anatomic sites.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Mean distances between the root normal sample and samples of primary tumors, lymphatic metastases, and distant metastases, respectively. Distance was measured as the number of internal nodes separating a pair of samples and then normalized by the total number of internal nodes in a given phylogeny. Means are 0.51 for N=16 primary tumors, 0.55 for N=16 lymphatic metastases, and 0.68 for N=16 distant metastases. Box plot elements: center line, median; magenta diamond, mean; box limits, lower and upper quartiles; whiskers, lowest and highest value within 1.5 IQR.
Comparison of normalized branch lengths from the normal sample to N=107 primary tumor regions, N=86 lymphatic metastases, and N=34 distant metastases in the validation cohort. Branch lengths were significantly different (p=4.9e-7, Kruskal-Wallis test). Branch lengths for distant metastases were significantly longer than for primary tumor samples (mean 0.9 vs 0.75; p=1.8e-7, Conover’s test) and longer than for lymphatic metastases (mean 0.9 vs 0.77; p=1.9e-6, Conover’s test). Box plot elements: center line, median; magenta diamond, mean; box limits, lower and upper quartiles; whiskers, lowest and highest value within 1.5 IQR.
Spatial classification of primary tumor samples. a, Primary tumor biopsies are classified as luminal or deep by a board-certified pathologist based on established anatomical landmarks. b, Percentages of luminal and deep primary tumor samples in the Naxerova and Reiter/Hung cohorts. c, For each lymphatic and distant metastasis, the closest primary tumor sample is found in the polyguanine marker-based distance matrix. Luminal/deep classifications of closest primary tumor samples are plotted separately for lymphatic and distant metastases. d, As in (c) for the Reiter/Hung cohort. e, as in (c) and (d) for the combined two cohorts. White numbers in panels (b)-(e) denote the number of samples in each group. Two-tailed Fisher’s exact tests were used to calculate the p-values.
Median RDSLN/RDSDM values for simulations of 10 LN-seeding clones and variable numbers of DM-seeding clones. 100 patients were simulated per parameter combination. The experimentally determined fold change (Naxerova & Reiter/Hung & Kim cohorts) is shown as vertical red line.
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Reiter, J.G., Hung, W., Lee, I. et al. Lymph node metastases develop through a wider evolutionary bottleneck than distant metastases. Nat Genet 52, 692–700 (2020). https://doi.org/10.1038/s41588-020-0633-2
Nature Genetics (2020)