Intratumoural evolutionary landscape of high-risk prostate cancer: the PROGENY study of genomic and immune parameters

M Linch, G Goh, C Hiley, Y Shanmugabavan, N McGranahan, A Rowan, Y N S Wong, H King, A Furness, A Freeman, J Linares, A Akarca, J Herrero, R Rosenthal, N Harder, G Schmidt, G A Wilson, N J Birkbak, R Mitter, S Dentro, P Cathcart, M Arya, E Johnston, R Scott, M Hung, M Emberton, G Attard, Z Szallasi, S Punwani, S A Quezada, T Marafioti, M Gerlinger, H U Ahmed, C Swanton, M Linch, G Goh, C Hiley, Y Shanmugabavan, N McGranahan, A Rowan, Y N S Wong, H King, A Furness, A Freeman, J Linares, A Akarca, J Herrero, R Rosenthal, N Harder, G Schmidt, G A Wilson, N J Birkbak, R Mitter, S Dentro, P Cathcart, M Arya, E Johnston, R Scott, M Hung, M Emberton, G Attard, Z Szallasi, S Punwani, S A Quezada, T Marafioti, M Gerlinger, H U Ahmed, C Swanton

Abstract

Background: Intratumoural heterogeneity (ITH) is well recognised in prostate cancer (PC), but its role in high-risk disease is uncertain. A prospective, single-arm, translational study using targeted multiregion prostate biopsies was carried out to study genomic and T-cell ITH in clinically high-risk PC aiming to identify drivers and potential therapeutic strategies.

Patients and methods: Forty-nine men with elevated prostate-specific antigen and multiparametric-magnetic resonance imaging detected PC underwent image-guided multiregion transperineal biopsy. Seventy-nine tumour regions from 25 patients with PC underwent sequencing, analysis of mutations, copy number and neoepitopes combined with tumour infiltrating T-cell subset quantification.

Results: We demonstrated extensive somatic nucleotide variation and somatic copy number alteration heterogeneity in high-risk PC. Overall, the mutational burden was low (0.93/Megabase), but two patients had hypermutation, with loss of mismatch repair (MMR) proteins, MSH2 and MSH6. Somatic copy number alteration burden was higher in patients with metastatic hormone-naive PC (mHNPC) than in those with high-risk localised PC (hrlPC), independent of Gleason grade. Mutations were rarely ubiquitous and mutational frequencies were similar for mHNPC and hrlPC patients. Enrichment of focal 3q26.2 and 3q21.3, regions containing putative metastasis drivers, was seen in mHNPC patients. We found evidence of parallel evolution with three separate clones containing activating mutations of β-catenin in a single patient. We demonstrated extensive intratumoural and intertumoural T-cell heterogeneity and high inflammatory infiltrate in the MMR-deficient (MMRD) patients and the patient with parallel evolution of β-catenin. Analysis of all patients with activating Wnt/β-catenin mutations demonstrated a low CD8+/FOXP3+ ratio, a potential surrogate marker of immune evasion.

Conclusions: The PROGENY (PROstate cancer GENomic heterogeneitY) study provides a diagnostic platform suitable for studying tumour ITH. Genetic aberrations in clinically high-risk PC are associated with altered patterns of immune infiltrate in tumours. Activating mutations of Wnt/β-catenin signalling pathway or MMRD could be considered as potential biomarkers for immunomodulation therapies.

Clinical trials.gov identifier: NCT02022371.

Keywords: intratumoural heterogeneity; mismatch repair; neoepitopes; prostate cancer; tumour infiltrating lymphocytes; wnt signalling.

© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology.

Figures

Figure 1.
Figure 1.
Intratumoural heterogeneity in prostate cancer at the somatic nucleotide variation (SNV) and somatic copy number alteration (SCNA) levels. (A) Number of somatic exonic mutations identified in each tumour region, fraction of SNVs and SCNAs that were ubiquitous (present in every tumour region of a given patient) (blue), shared (present in more than one tumour region, but not all) (light orange) or private (present in only one tumour region) (dark orange). Data tracks below indicate if patient was metastatic on presentation (red), Gleason grade (shades of green), level of tumoural inflammatory infiltrate (shades of brown), and if the tumour had undergone whole-genome doubling (purple, triangle indicating heterogeneous genome doubling). (B) Scatterplot showing correlation between degree of SNV and SCNA heterogeneity. (C and D) Boxplots comparing the fraction of genome affected by SCNA and SNV mutational burden in metastatic hormone naive prostate cancer (mHNPC) versus high-risk localised prostate cancer (hrlPC).
Figure 2.
Figure 2.
Clonal and subclonal driver events in prostate cancer. List of driver genes previously reported as significantly mutated in primary prostate cancer (blue), metastatic castrate-resistant prostate cancer (mCRPC) (grey), or both (black). Ubiquitous ETS fusion (purple), homozygous loss (dark blue), heterozygous deletion (blue), amplification (red) in each tumour is depicted by a coloured square, and heterozygous events are indicated with a triangle. Nonsynonymous mutations are depicted as smaller squares, whether missense (green), frameshift (yellow) or nonsense (dark blue). Clonal and subclonal mutations are indicated by a purple and orange outline, respectively. Known recurrent mutations in TP53, PIK3CA, CTNNB1 and BRAF are indicated with a red star. The barplots on the right are an aggregate of clonal/ubiquitous or subclonal/heterogenous events in each gene across all samples. Metastatic hormone naive prostate cancer (mHNPC); high-risk localised prostate cancer (hrlPC).
Figure 3.
Figure 3.
Recurrent somatic copy number alterations (SCNAs) in prostate cancer. (A) An overview of the SCNA landscape across all 25 tumours: fraction of cohort (y-axis) with ubiquitous gains (red), heterogeneous gains (pink), ubiquitous loss (dark blue) and heterogeneous loss (light blue) are shaded across the genome (x-axis). (B) Frequencies of occurrence of previously identified GISTIC focal and arm-level SCNAs across all tumours, metastatic on presentation (mHNPC) and non-metastatic on presentation (hrlPC) tumours. Shades of colours as in A.
Figure 4.
Figure 4.
Parallel evolution in Wnt pathway in PR0139 and association with CD8+/FOXP3+ ratio across 15 tumours. (A) Left, fraction of cancer cells in sequenced tumour regions R1, R2 and R3 harbouring different CTNNB1 mutations, p.S33P (pink), p.T41A (blue) and p.S33C (orange). Right, schematic showing different compositions of subclones in each sequenced tumour region, colours correspond to left panel. (B) Phylogenetic tree showing evolutionary history of PR0139 and acquisition of various driver mutations. Relative sizes of circles correspond to number of SNV mutations in that mutational cluster.
Figure 5.
Figure 5.
T-cell heterogeneity in prostate tumours. (A) Manual quantification of inflammatory infiltrate. Mean is represented by horizontal lines, box and whiskers show the 95% confidence interval and range, respectively. The dotted line marks the threshold for high inflammatory infiltrate. (B) Multiplex immunohistochemistry (IHC) analysis of four different prostate core biopsies (R1–4) from a patient, PR0123, showing heterogeneity in T-cell infiltration. CD8 staining in red, CD4 in brown and FoxP3 in blue. (C) Boxplot comparing CD8+/FOXP3+ ratios between tumours with and without somatic activation of Wnt pathway (gain-of-function mutation in CTNNB1, amplification in RSPO2, loss in APC, RNF43 and ZNRF3) across 15 tumours with digital image analysis.

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