support-information-section).

Background Ovarian cancers are hallmarked by chromosomal instability. New therapies deliver improved patient outcomes in relevant phenotypes, however therapy resistance and poor long-term survival signal requirements for better patient preselection. An impaired DNA damage response (DDR) is a major chemosensitivity determinant. Comprising five pathways, DDR redundancy is complex and rarely studied alongside chemoresistance influence from mitochondrial dysfunction. We developed functional assays to monitor DDR and mitochondrial states and trialled this suite on patient explants. Methods We profiled DDR and mitochondrial signatures in cultures from 16 primary-setting ovarian cancer patients receiving platinum chemotherapy. Explant signature relationships to patient progression-free (PFS) and overall survival (OS) were assessed by multiple statistical and machine-learning methods. Results DR dysregulation was wide-ranging. Defective HR (HRD) and NHEJ were near-mutually exclusive. HRD patients (44%) had increased SSB abrogation. HR competence was associated with perturbed mitochondria (78% vs 57% HRD) while every relapse patient harboured dysfunctional mitochondria. DDR signatures classified explant platinum cytotoxicity and mitochondrial dysregulation. Importantly, explant signatures classified patient PFS and OS. Conclusions Whilst individual pathway scores are mechanistically insufficient to describe resistance, holistic DDR and mitochondrial states accurately predict patient survival. Our assay suite demonstrates promise for translational chemosensitivity prediction.

Background: Ovarian cancer remains the most lethal gynaecological malignancy and there is an urgent need for the development of further therapies to improve patient survival. Tumour infiltrating lymphocyte (TIL) therapy has shown clear efficacy in immunogenic cancers, and TIL can be readily expanded ex vivo from samples of high grade serous ovarian cancer (HGSOC). Antibodies against checkpoint proteins are effective in increasing TIL yield and functional response from ovarian cancer TIL cultures, potentially increasing TIL product efficacy. However, it is unknown whether blockade of other key checkpoints, including programmed death ligand-1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3) and lymphocyte activation gene-3 (LAG-3) increase TIL yield in ex vivo cultures from HGSOC samples.Materials and Methods: TIL cultures were generated from fresh tissue samples and were incubated with CD3/CD28 Dynabeads® and IL-2. TIL were grown with or without the addition of 10µg/mL αPD-1, αTIM-3 or αLAG-3 antibodies as either single or repeated applications for 19 days. The phenotype of cultures was analysed by multiparameter flow cytometry. Interferon gamma (IFN-ᵧ) release in response to TIL co-culture with autologous tumour cultures or digests was measured by ELISA. Results: In keeping with previous findings the majority of TIL expressed at least one checkpoint protein. Specifically, TIL expressing PD-1 and TIM-3 retained a functional phenotype with co expression of OX40 and CD28. Expansion of TIL was increased following a single exposure to αPD-1 (1.20 fold) and αLAG-3 (1.31 fold) which was most marked in cultures with otherwise low proliferation. There was no additional increase in proliferation following repeated dosing. In co-culture with autologous tumour, TIL grown in the presence of checkpoint inhibitors showed increased IFN-ᵧ release suggesting increased functional activity in individual cultures. However, this response was not uniform across all cultures. Conclusions: These data suggests that initial blockade of checkpoint proteins is effective in increasing the yield of TIL expanded from HGSOC tumours, suggesting that checkpoint inhibition during TIL manufacture may be a useful method of increasing expansion efficiency.

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