Abstract
Homologous recombination deficiency (HRD)-targeting agents such as platinums (Pt) and small molecule Poly-ADP Ribose Polymerase inhibitors (PARPi) are effective in treating patients with defects in BRCA1, BRCA2 or PALB2 genes, key players in homologous recombination (HR). Understanding the prevalence of distinct mechanisms of resistance to PARPi and cross resistance with Pt in breast cancer may inform therapy choices. Prior pre-clinical work has identified multiple mechanisms of HRD-targeted treatment resistance, but their existence and relative frequency in clinical disease is unclear. The aim of this project was to use integrated genomics and functional analyses to; (a) explore resistance to HRD-targeting agents in patients with metastatic BRCA1, BRCA2 and PALB2 deficient breast cancer using patient derived material collected in a longitudinal manner along the patient’s treatment journey; (b) determine whether functional analyses of HR and replication fork (RF) dynamics can be used to predict response to HRD-targeted treatment in patients with metastatic BRCA1/2 or PALB2 deficient breast cancer.Longitudinal mutation profiling of circulating tumour (ct)DNA was carried out in 47 patients with germline BRCA1/2 and PALB2 mutations who exhibited resistance to HRD-targeted treatment, using a novel >750 gene intron/exon targeted sequencing panel. Where available, matched tumour biopsies were whole exome and RNA-whole transcriptome sequenced and developed into patient derived cancer models for subsequent analysis. RAD51 and BRCA1 nuclear foci analyses were performed in breast cancer patient tumours as a functional readout of restoration of HR proficiency before and after HRD-targeted treatment resistance. In addition, DNA RF dynamics, using DNA fibre combing assays were carried out in breast cancer patient derived organoids (PDOs). This RF data was compared to clinical responses in the same patients in order to assess how such assays might predict therapy resistance. ctDNA analysis revealed BRCA1/2 reversion mutations, including novel types of reversion, are the most common mechanism of HRD-targeted treatment resistance occurring in advanced breast cancer. Non-reversion mechanisms of resistance were also detected (mutations in TP53BP1, RIF1 or PAXIP1), and in some cases occurred in parallel with reversion mutations, challenging the notion that resistance mechanisms in individual patients are singular. Importantly, the presence of a BRCA1/2 reversion mutation in ctDNA prior to starting HRD-targeted treatment was associated with a shorter time to progression, which may impact treatment decisions.
The majority of patients, whether they had de novo or acquired resistance, exhibited high RAD51 scores in post-resistance tumour FFPE samples, suggesting restoration of HR function is a dominant mechanism of PARPi resistance in this cohort. RAD51 nuclear foci correlated with multiple mechanisms of HR restoration and showed potential as a biomarker of clinical PARPi resistance. RF dynamics were assessed in breast cancer PDOs for the first time, and an association between RF stability and PARPi resistance was observed.
In conclusion, this work reports the prevalence of HRD-targeted treatment resistance mechanisms occurring in a real-world cohort of patients with HRD metastatic breast cancer. I show how the RAD51 nuclear foci assay can be used to characterise such resistance mechanisms, and has the potential to predict HRD-targeted treatment resistance in longitudinally collected tumour FFPE samples. In addition, I also demonstrate that analysis of DNA RF dynamics in relation to PARPi response can be carried out in breast cancer PDOs and could be further explored to better understand PARPi resistance.
Date of Award | 1 Aug 2024 |
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Original language | English |
Awarding Institution |
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Supervisor | Andrew Tutt (Supervisor) & Natasha Lukashchuk (Supervisor) |