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Prostate cancer

African-specific molecular taxonomy of prostate cancer

Patient cohorts and WGS

Our study included 183 treatment-naive patients with prostate cancer who were recruited under informed consent and appropriate ethics approval (Supplementary Information 2) from Australia (n = 53), Brazil (n = 7) and South Africa (n = 123). While matched for pathological grading, as previously reported, prostate-specific antigen levels are notably elevated within our African patients16 and we cannot exclude on the basis of potential metastasis (as data on metastases in this cohort are unavailable). DNA extracted from fresh tissue and matched blood underwent 2 × 150 bp sequencing on the Illumina NovaSeq instrument (Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research).

WGS processing and variant calling

Each lane of raw sequencing reads was aligned against human reference hg38 + alternative contigs using bwa (v.0.7.15)37. Lane-level BAM files from the same library were merged, and duplicate reads were marked. The Genome Analysis Toolkit (GATK, v.4.1.2.0) was used for base quality recalibration38. Contaminated and duplicate samples (n = 8) were removed. We implemented three main pipelines for the discovery of germline and somatic variants, with the latter including small (SNV and indel) to large genomic variation (CNAs and SVs). The complete pipelines and tools used are available from the Sydney Informatics Hub (SIH), Core Research Facilities, University of Sydney (see the ‘Code availability’ section). Scalable bioinformatic workflows are described in Supplementary Information 4.

Genetic ancestry was estimated using fastSTRUCTURE (v.1.0)39, Bayesian inference for the best approximation of marginal likelihood of a very large variant dataset. Reference panels for African and European ancestry compared in this study were retrieved from previous whole-genome databases19.

Analysis of chromothripsis and chromoplexy

Clustered genomic rearrangements of prostate tumours were identified using ShatterSeek (v.0.4)40 and ChainFinder (v.1.0.1)41. Our somatic SV and somatic CNA call sets were prepared and co-analysed using custom scripts (see the ‘Code availability’ section; Supplementary Information 6).

Analysis of mutational recurrence

We used three approaches to detect recurrently mutated genes or regions based on three mutational types, including small mutations, SVs and CNAs (Supplementary Information 7). In brief, small mutations were tested within a given genomic element as being significantly more mutated than the adjacent background sequences. The genomic elements retrieved from syn5259886, the PCAWG Consortium20, were a group of coding sequences and ten groups of non-coding regions. SV breakpoints were tested in a given gene for their statistical enrichment using gamma–Poisson regression and corrected by genomic covariates12. Focal and arm-level recurrent CNAs were examined using GISTIC (v.2.0.23)42. Known driver mutations in coding and non-coding regions published in PCAWG20,43,44 were also recorded in our 183 tumours, and those specific to prostate cancer genes were also included7,8,12,17,18.

Integrative analysis of prostate cancer subtypes

Integrative clustering of three genomic data types for 183 patients was performed using iClusterplus11,45 in R, with the following inputs: (1) driver genes and elements; (2) somatic CN segments; and (3) significantly recurrent SV breakpoints. We ran iClusterPlus.tune with clusters ranging from 1 to 9. We also performed unsupervised consensus clustering on each of the three data types individually. Association analysis of genomic alteration with different iCluster subtypes was performed in detail (Supplementary Information 8). Differences in driver mutations, recurrent breakpoints and somatic CNAs across different iCluster subtypes were reported.

Comparison of iCluster with Asian and pan-cancer data

To compare molecular subtypes between extant human populations, the Chinese Prostate Cancer Genome and Epigenome Atlas (CPGEA, PRJCA001124)11 was merged and processed with our integrative clustering analysis across the three data types described above, with some modifications. Moreover, we leveraged the PCAWG consortium data13 to define molecular subtypes across different ethnic groups in other cancer types using published data of somatic mutations, SV and GISTIC results by gene. Four cancer types consisting of breast, liver, ovarian and pancreatic cancers were considered due to existing primary ancestries of African, Asian and European with at least 70% contribution. Full details are provided in Supplementary Information 8.4.

PCAWG13 participants with prostate cancer were retrieved to compare with Australian data with clinical follow-up. Only those of European ancestry greater than 90% (n = 139) were analysed for the three genomic data types of iCluster subtyping, as well as individual consensus clustering. Clustering results identical to the larger cohort size mentioned above were chosen for association analyses. Differences in the biochemical relapse and lethal prostate cancer of the participants across the subtypes were assessed using the Kaplan–Meier plot followed by a log-rank test for significance.

Analysis of mutational signatures

Mutational signatures (SBSs, DBSs and indels), as defined by the PCAWG Mutational Signatures Working Group3, were fit to individual tumours with observed signature activities using SigProfiler46. Non-negative matrix factorization was implemented to detect de novo and global signature profiles among 183 patients and their contributions. New mutational genome rearrangement signatures (CN and SV) were also performed using non-negative matrix factorization, with 45 CN and 44 SV features examined across 183 tumours. We followed the PCAWG working classification and annotation scheme for genomic rearrangement26. Two SV callers were used to obtain exact breakpoint coordinates. Replication timing scores influencing on SV detection were set at >75, 20–75 and <20 for early, mid, and late timing, respectively47. Full details of analysis steps, parameters and relevant statistical tests are provided in Supplementary Information 9.

Reconstruction of cancer timelines

Timing of CN gains and driver mutations (SNVs and indels) into four epochs of cancer evolution (early clonal, unspecified clonal, late clonal and subclonal) was conducted using MutationTimeR24. CN gains including 2 + 0, 2 + 1 and 2 + 2 (1 + 1 for a diploid genome) were considered for a clearer boundary between epochs instead of solely information of variant allele frequency. Confidence intervals (tlo – tup) for timing estimates were calculated with 200 bootstraps. Mutation rates for each subtype were calculated according to ref. 24 such that CpG-to-TpG mutations were counted for the analysis because they were attributed to spontaneous deamination of 5-methyl-cytosine to thymine at CpG dinucleotides, therefore acting as a molecular clock.

League model relative ordering was performed to aggregate across all study samples to calculate the overall ranking of driver mutations and recurrent CNAs. The information for the ranking was derived from the timing of each driver mutation and that of clonal and subclonal CN segments, as described above. A full description is provided in Supplementary Information 10.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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