Extended Data Fig. 5: Modes of dissemination.

a. Sample level definitions of dissemination patterns relative to the primary tumour phylogeny. b. Sample level dissemination patterns with overall case level defined beneath c. Proportion of cases defined as polyclonal or monoclonal divided by whether a single or multiple metastatic samples were available (n single = 77, n multiple = 49). There is increased power to detect polyclonal seeding when multiple metastatic samples were sequenced (in dark red, we see approximately 22.4% of polyclonal cases result from multiple monoclonal seeding patterns). d. Proportion of observed polyclonal metastases when simulating differing numbers of disseminating primary tumour cells (y-axis) and varying the number of primary regions from which this occurs (top and bottom panel). The primary tumour was always simulated with 1% selection while the selection coefficients were varied in the metastasis (x-axis). Increasing selection pressure in the metastasis is associated with the appearance of monoclonal dissemination even if the dissemination from the primary tumour is polyclonal. The fewer the number of disseminating cells, the stronger the effect. e. Kaplan-Meier analyses demonstrate no significant difference in lung-cancer specific disease-free survival across the different dissemination patterns (Log rank test, p = 0.5). f. Proportion of dissemination type on a case level, as seen in the main histologic subtypes (LUAD, n = 65; LUSC, n = 39; Fisher’s exact test, p > 0.05). g. Tumours with polyclonal dissemination and extrathoracic metastases have more metastatic samples acquired (Wilcoxon rank-sum test). h. Comparison of the TRACERx dissemination definitions with MACHINA¹⁸ shows that the majority of dissemination patterns are consistent across the two methods, with only 12/126 cases differing; with the TRACERx definitions being more conservative by classifying these cases as monoclonal whereas MACHINA defines these as polyclonal. i. Summary of MACHINA analysis of a metastasis seeding other sites of disease in 46 cases with multiple metastatic samples. ‘Other’ represents cases where the primary tumour seeds the recurrence and additional metastasis seeding patterns are concurrently observed (e.g., recurrence/progression sample seeding the primary LN, primary LN to primary LN seeding, recurrence seeding a progression sample). The box plots represent the upper and lower quartiles (box limits), the median (centre line) and the vertical bars span the 5th to 95th percentiles. All tests were two-sided unless otherwise specified; LN, lymph node.