*2.3. Primary Prostate Samples with Relapse Post Radical Prostatectomy: 12543, 5545, 5684, and 13179*

At the time of surgery, none of these cases had evidence of metastatic disease on staging scans. All men subsequently relapsed with incurable disease, including bone metastases (5545, 5684 and 13179) and persistent BCR with eventual CRPC (12543). While TMBs were similar (range 1.4–1.9), there was more marked variability in their PGAs (range 3.1–26%). Genomic alterations with patient-specific relevance are summarized in Figure 5.

#### 2.3.1. Case 12543: Left Prostate Tumour Core Biopsy

This patient's tumour is characterized by *KMT2C* mutation, copy number losses (supported by large deletion) in *PTEN* and *FOXP1* and an *ETV1-ACSL3* fusion. *ETV1-ACSL3* fusion may account for this patient's prolonged ADT sensitivity (no evidence of metastatic disease following 10 years on ADT for BCR). *ACSL3* is an androgen responsive gene and thus, this fusion may lead to a strong reliance on androgen signalling [72]. Despite *PTEN* loss, loss of *FOXP1* may restore androgen receptor signalling, further enhancing this patient's response to ADT despite the *PTEN* loss [73]. At development of CRPC, this reliance on AR signalling may be exploited further with the addition of a novel ASI to his ADT, rather than docetaxel.

## 2.3.2. Case 5545: Left Prostate Tumour Core Biopsy

This case is characterized by a deleterious somatic *SPOP* missense variant (rs193921065, c.399C > G; p.Phe133Leu; VAF 44%) [7] and a large hemizygous deletion encompassing *CHD1*. We also predict the *LRB1B* mutation (c.3178A > G; p.Cys1060Arg) to be deleterious. Notable copy number losses include *TP53BP1* and the TSG *RB1* and the DDR genes *FANCA* and *PPP2R2A*, while a deletion overlapped *LRP1B.* Unique to WGM, we identified a large deletion involving *FILIP1L*, a gene commonly hypermethylated in PCa [74].

Point mutations in *SPOP* occur in approximately 11% of primary PCas [7] and are commonly associated with *CHD1* loss [75]. This combination of alterations is associated with increased abiraterone sensitivity in CRPC [76]. These tumours are also characterized by increased genomic instability due to error-prone double-strand DNA break repair, which results in more SVs, as seen in this case, and potential vulnerability to DNA damaging treatment such as irradiation, PARP inhibition and platinum chemotherapy [77]. Loss of *FANCA,* a gene involved in homologous recombination, may also sensitise this cancer to PARP inhibition. A recent retrospective study found that *LRP1B* alterations may predict for sensitivity to pembrolizumab [78].

*SPOP*/*CHD1* co-altered clones persist across the disease spectrum in studies of serial patient samples [76]. Therefore, knowledge of this case's genomic data from radical prostatectomy would lead to a preference for abiraterone over docetaxel at development of CRPC. These alterations may also increase his responsiveness to PARP inhibition, though evidence is limited and preclinical models have shown that this vulnerability is reliant on elevated 53BP1 protein levels [77] and so the copy number loss in *TP53BP1* may counteract this vulnerability. This combination of alterations highlights the importance of understanding the entire genomic landscape in an individual.

#### 2.3.3. Case 5684: Right Prostate Tumour Core Biopsy

This case harbours a small frameshift deletion in *TP53* between exons 11 and 12, in addition to heterozygous copy number loss and a deletion on SV analysis. He also presented with SCNA in *CDH1* and alterations in other DDR genes including: an SNV in *CDK12* and SCNAs in *PPP2R2A* and *FANCA.* Losses in genes affecting proliferative pathways include those in *PIK3R1,* the loss of which activates the PI3K pathway [79] and *MAP3K1,* which is associated with MEK signalling [80]. The inclusion of WGM for 5684 revealed a large insertion overlapping *SPOCK1*, which encodes a protein found to promote tumorigenesis and metastases in PCa [81]. WGM also identified an insertion in *CREBBP*, a coactivator of AR that is usually overexpressed in CRPC and the upregulation of which is associated with ADT resistance [82].

*TP53* loss confers a worse prognosis and improved outcomes with chemotherapy compared to novel ASI agents [62]. Knowledge of his primary tumour *TP53* status may have guided ordering of therapies with a preference for chemotherapy, particularly upon progression to CRPC. A study of co-targeting PARP and Wee1 kinase with olaparib and AZD1775 is currently underway for *TP53* mutated solid tumours (NCT02576444). The losses in *PIK3R1* and *MAP3K1* may confer sensitivity to PI3K and MEK inhibitors respectively though these agents would only be used on a suitable clinical trial.
