Extended Data Figure 2. Subclonal events in primary mouse tumours become clonal at recurrence.

a, Naive tumours from Ptch^+/−, Ptch^+/−/Trp53^+/− or Ptch^+/−/Trp53^−/− germline mutant mice were analysed by immunohistochemical staining for nuclear p21 (upper panels), demonstrating decreased nuclear p21 expression due to Trp53 pathway dysfunction. Tumours with Trp53 damaging gCIS insertions at recurrence (03-04-11 and 06-28-11) also show decreased immunohistochemical staining for nuclear p21 staining (lower panels), when compared to a recurrent tumour without gCIS Trp53 insertions (02-23-11w) (scale bars, 25 µm and 50 µm as indicated). b, Relative dominance of driver events is shown in one individual tumour where Tead1 is detectable in both primary tumour sample and at recurrence. c, d, Clonal insertions in the local and metastatic recurrences that were found at a subclonal level in the matching primary tumours are shown by mouse. In each case, the number of insertions with evidence for expansion from a subclone of the primary is shown as a proportion (red bar) of the total number of considered events. Green and blue bars depict the proportion of the total number of considered events that were found in local and metastatic recurrences, respectively. The grey bar indicates the proportion of insertions that are also found in an unrelated Sleeping Beauty library of similar depth. We narrowed the analysis on matching primary recurrences with at least 1 clonal insertion in common. This excluded 3 local recurrence cases and 6 metastatic recurrence cases that had no overlap between clonal insertions in the primary and clonal insertions in the matching local recurrences (black stars). c, Local recurrences display statistical support for subclonal derivation from the primary tumours (P = 0.041; n = 7; Mann–Whitney U-test). d, Metastatic recurrences instead show a limited extent of overlap with the matching primaries that does not reach statistical significance (P = 0.298; n = 5; Mann–Whitney U-test). e, Box plot comparing the extent of overlap between primary/local recurrences versus primary/metastatic recurrences, local recurrences (with at least 1 clonal insertion in common with the primary) show a trend for higher evidence of subclonal derivation from their matched primaries than metastatic recurrences (P = 0.051, Mann–Whitney U-test, centre lines show the medians; box limits indicate the 25th and 75th percentiles; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles, samples are represented by dots. n = 7 and 5 sample points).