Comprehensive multiregional analysis of molecular heterogeneity in bladder cancer

Abstract

Genetic alterations identified in adjacent normal appearing tissue in bladder cancer patients are indicative of a field disease. Here we assessed normal urothelium transformation and intra-tumour heterogeneity (ITH) in four patients with bladder cancer. Exome sequencing identified private acquired mutations in a lymph node metastasis and local recurrences. Deep re-sequencing revealed presence of at least three and four subclones in two patients with multifocal disease, while no demarcation of subclones was identified in the two patients with unifocal disease. Analysis of adjacent normal urothelium showed low frequency mutations in patients with multifocal disease. Expression profiling showed intra-tumour and intra-patient co-existence of basal- and luminal-like tumour regions, and patients with multifocal disease had a greater degree of genomic and transcriptomic ITH, as well as transformation of adjacent normal cells, compared to patients with unifocal disease. Analysis of the adjacent urothelium may pave the way for therapies targeting the field disease.

Figure 1

Detailed analysis of patient 1. (a) Upper heat map: Deep targeted sequencing was applied to 28 LMD regions from four tumour samples (one from each of the four tumours), a lymph node metastasis, as well as seven LMD adjacent normal samples. Tumour sample S1 was procured from the muscle invasive tumour (clinical stage T3b) and tumour samples S2–S4 were procured from three non-muscle invasive tumours (clinical stage Ta). Presented are all validated mutations. Furthermore, only mutations present in at least 5% of the tumour regions were included. LMD tumour regions were grouped using unsupervised hierarchical cluster analysis and clusters are indicated above the heat map. Data from normal samples were clustered independently (sample wise). Variants were classified as shared if present in more than 90% of the tumour samples. Oncogenes (red), tumour suppressor genes (blue), and IntOGen bladder cancer drivers (green) are annotated to the right of the heat map. Further, the Qiagen Clinical Insight Software was used to identify variants, for which therapeutics are available (T), in clinical trials (C_T), or if the variant is pathogenic (P). Allele frequencies are presented ranging from >0 to 1 (an error rate of 1% was applied, hence if present, the variant is at least present at 1% in the sample). Grey indicates less than 4 alternate reads but an allele frequency greater than or equal to 1%, and could hence potentially be present if read depth was higher. Light blue indicates a failed amplicon. Dark blue equals no mutation. Lower heat map: transcriptomic profiling of corresponding regions presented in the upper heat map. Presented are normalized Ct values of genes of the following gene classes: luminal, differentiation, basal, high progression risk, and low progression risk. ND (grey): not determined. Missing value (white): sample has not been profiled. (b) Normal sample analysis: targeted sequencing of normal samples with a less stringent error correction applied (0.5%). The allele frequencies are presented on a different scale from present (>0) to 5%. (c) Illustration of sampling from cystectomy: see Supplementary Figure S2 for detailed overview of LMD regions.

Figure 2

Detailed analysis of patient 2. Deep targeted sequencing was applied to 63 LMD tumour regions procured from multifocal tumour samples (4 samples from tumour 1 and one sample from each of tumour 2–4) as well as a sample from a local recurrence. Further, deep sequencing was applied to seven normal samples. The figure is annotated as Fig. 1. See Supplementary Figure S3 for detailed overview of LMD regions.

Figure 3

Detailed analysis of patient 3 and patient 4. (a) Patient 3. Deep targeted sequencing was applied to 28 LMD regions from four tumour samples taken from one large unifocal tumour, a sample from a local recurrence, as well as five normal samples. (b) Patient 4. Deep targeted sequencing was applied to 10 LMD regions from two tumour samples from a unifocal tumour as well as nine normal samples. (c) Illustration of sampling from cystectomy for patient 3: see Supplementary Figure S4 for detailed overview of LMD regions. (d) Illustration of sampling from cystectomy for patient 4: see Supplementary Figure S5 for detailed overview of LMD regions. The figure is annotated as Fig. 1.

Figure 4

Models of field disease development in bladder cancer. (a) Intra-epithelial migration of multiple subclones gives rise to tumours of a clonal origin and presence of variants throughout the urothelium. (b) Luminal seeding followed by implantation gives rise to tumours of clonal origin. (c) Acquisition genetic alterations such as loss of 9p or 9q or mutation in TP53 in an urothelial stem cell generates a pool of cancer stem cells of a clonal origin. Due to the increased proliferation, numerous mutations are acquired in an initial burst followed by intermixing and parallel expansion throughout the urothelium. This model gives rise to multiple fields.