The architecture of clonal expansions in morphologically normal tissue from cancerous and non-cancerous prostates

Abstract

Background: Up to 80% of cases of prostate cancer present with multifocal independent tumour lesions leading to the concept of a field effect present in the normal prostate predisposing to cancer development. In the present study we applied Whole Genome DNA Sequencing (WGS) to a group of morphologically normal tissue (n = 51), including benign prostatic hyperplasia (BPH) and non-BPH samples, from men with and men without prostate cancer. We assess whether the observed genetic changes in morphologically normal tissue are linked to the development of cancer in the prostate.

Results: Single nucleotide variants (P = 7.0 × 10^-03, Wilcoxon rank sum test) and small insertions and deletions (indels, P = 8.7 × 10^-06) were significantly higher in morphologically normal samples, including BPH, from men with prostate cancer compared to those without. The presence of subclonal expansions under selective pressure, supported by a high level of mutations, were significantly associated with samples from men with prostate cancer (P = 0.035, Fisher exact test). The clonal cell fraction of normal clones was always higher than the proportion of the prostate estimated as epithelial (P = 5.94 × 10^-05, paired Wilcoxon signed rank test) which, along with analysis of primary fibroblasts prepared from BPH specimens, suggests a stromal origin. Constructed phylogenies revealed lineages associated with benign tissue that were completely distinct from adjacent tumour clones, but a common lineage between BPH and non-BPH morphologically normal tissues was often observed. Compared to tumours, normal samples have significantly less single nucleotide variants (P = 3.72 × 10^-09, paired Wilcoxon signed rank test), have very few rearrangements and a complete lack of copy number alterations.

Conclusions: Cells within regions of morphologically normal tissue (both BPH and non-BPH) can expand under selective pressure by mechanisms that are distinct from those occurring in adjacent cancer, but that are allied to the presence of cancer. Expansions, which are probably stromal in origin, are characterised by lack of recurrent driver mutations, by almost complete absence of structural variants/copy number alterations, and mutational processes similar to malignant tissue. Our findings have implications for treatment (focal therapy) and early detection approaches.

Keywords: Benign prostatic hyperplasia; Clonal expansions; Field effect; Genomics; Mutational signatures; Normal tissue; Prostate cancer.

Fig. 1

Mutations in morphologically normal tissue: A From top to bottom: whether clonal expansions under positive selection were detected; sample type (morphologically normal tissue in prostate cancer patients, BPH tissue in prostate cancer patients, tissue from non-prostate cancer patients, BPH fibroblast cell culture); number of single nucleotide variants (SNVs) detected per sample; number of indels (insertions, deletions and complex insertions/deletions) per sample. Each column represents a sample and they are ordered according to sample type and decreasing number of SNVs. Eight rearrangements (not represented in figure) were detected across all patients (sample 0063_N (n = 1), 0127 (n = 3), 0073_N (n = 1), 0074_N (n = 1), 0006_N1 (n = 1) and sample 0006_N3 (n = 1)). A BRCA2 SNP (chr13:32,945,095) was detected in the blood of donor 0063. No copy number alterations were detected. B Plot showing the distribution of the number of SNVs found in BPH samples and non-BPH normal samples in prostate cancer patients; C the number of SNVs between normal samples from people with or without prostate cancer; D the number of indels between normal samples from people with or without prostate cancer

Fig. 2

Relationship between clonal cell fraction (CCF) of clones in morphologically normal sample and estimated cellular composition. A Scatter plot of average stromal content estimated by histopathological review and the CCF for each morphologically normal sample from men with prostate cancer. Line is the best fit linear line. Colour is whether the sample is BPH or not. B Comparison between the CCF and the percentage epithelial content for each morphologically normal sample from men with prostate cancer

Fig. 3

Phylogenies of patients with multiple samples. Phylogenies revealing the relationships between clones for each case. A patients where we have collected multiple tumours and normal. B patients where there was data from a tumour, non-BPH normal tissue, and BPH normal tissue. Each coloured line represents a clone/subclone detected in a particular sample. When two or more coloured lines are together, they represent a clone that is found in all the samples represented. The length of the line is proportional to the weighted number of single nucleotide variants present in each clone; the thickness represents the clonal cell fraction associated with that clone (more detail in Additional file 3). For example, case 0077 contains a shared subclone with 8% N, 33% BPH and 2% T (Tb) supported by 113 SNVs and 4 indels. Dotted lines are associated with samples that have no evidence of a unique sample specific clone. The very low fraction tumour subclone (< 4%) shared with normal and BPH tissue in case 0077 and between normal and tumour in case 0072 suggests cancer targeted tissue contained some of the N/BPH cells. Additional phylogenies can be found in Supplementary Fig. 3

Fig. 4

Mutational spectra. Mutational signatures detected in tumour and matched morphologically normal tissue from prostate cancer patients and normal tissue from men without prostate cancer. The mutational spectra of each sample, as defined by the triplets of nucleotides around each SNV, were deconvoluted into mutational signatures (SigProfiler [41]) using the set of signatures defined by Alexandrov et al. [47]. The colour of the first row indicates patient when there is more than a normal-tumour (N-T) pair analysed. Six patients had more than two samples analysed and one patient had only a morphologically normal sample without a matched tumour

Fig. 5

Tumours show a distinct mutation profile to normal tissue. A The difference between the number of single nucleotide variants (SNVs) detected in normal tissue compared to tumour tissue. Where multiple samples of either type were present the median number was used. B The distribution of the number of SNVs detected in morphologically normal tissue, tumour tissue with low CNAs (percentage genome altered (PGA) < 6%) and tumour tissue with high CNAs (PGA > 6%). Data from these last two categories came from Wedge et al. [52]