Fig. 5: Clinical impact of proteomic intra-tumoural heterogeneity in the context of platinum-based neoadjuvant chemotherapy.

a Heatmap showing hierarchical clustering using all 5828 proteins identified across all pre-NAC TUR samples by Ward’s hierarchical agglomerative clustering method based on Euclidean distance. Duplicate samples (N = 36) are displayed with same colour coding and identified by their TUR_ID. Single samples (N = 72) have no colour coding or ´TUR_ID´ shown in the heatmap. b Dot plot shows intra-tumoural heterogeneity (ITH) scores of all patients with duplicate pre-NAC TUR samples. ITH scores were split according to the median (ITH = 21, horizontal dashed lined) into distant (i.e. high ITH) and close (i.e. low ITH). Pre-NAC TUR proteomic cluster calls were made for the combined proteomic signal of both paired samples. c Representative H&E images of two sets of duplicate pre-NAC TUR samples: one with high ITH and one with low ITH as shown in (b). Scale bars represent 100 µm. d Boxplot showing ITH scores between NAC non-responder (≥ypT2N0-3M0) and partial and complete responder (≤ypTa/1/isN0M0) patients. Boxplots include the median (centre line in bar) and the third and first quartiles (upper and lower edges, respectively) of intra-tumour heterogeneity score and the whiskers extend to the most extreme data point which is no more than 1.5 times the IQR from the box. P value from a one-tailed t-test is indicated. Non-responder, N = 18; partial- and complete-responder, N = 18. e Kaplan–Meier plots show RFS and OS stratified between distant and close (split by median) ITH patient tumours in pre-NAC TUR samples. Close, N = 17; distant, N = 18. Note that one CC1-Luminal (classed as ´close´ in this analysis) patient did not have survival data available. f Sankey plot showing consensus clustering of average protein expression from each pair of the pre-NAC TUR samples (N = 72 total samples from N = 36 patients) compared to that of the individual pre-NAC TUR samples consensus centroids used in Fig. 1c. Number of samples in each cluster are indicated in parenthesis. g PCA plot highlighting morphologically similar matched duplicate pre-NAC TUR samples and their corresponding duplicate post-NAC RC counterparts. Only patients with two pre-NAC TUR and two post-NAC RC samples are included (N = 8). Ellipsoids are based on Khachiyan’s algorithm for minimum-volume enclosing. Samples and ellipses representing specimens from the same patient are assigned the same colour. Samples with ID #641 and #689 show distinct proteomic profiles in the TUR or RC specimens, respectively. Abbreviations: H&E hematoxylin and eosin, IQR interquartile range, ITH intra-tumour heterogeneity, NAC neoadjuvant chemotherapy, RC radical cystectomy, TUR transurethral resection. Source data are provided as Source Data file.