Laura A. Knight

Activation of STING-Dependent Innate Immune Signaling By S-Phase-Specific DNA Damage in Breast Cancer.

Background: Previously we identified a DNA damage response–deficient (DDRD) molecular subtype within breast cancer. A 44-gene assay identifying this subtype was validated as predicting benefit from DNA-damaging chemotherapy. This subtype was defined by interferon signaling. In this study, we address the mechanism of this immune response and its possible clinical significance. Methods: We used immunohistochemistry (IHC) to characterize immune infiltration in 184 breast cancer samples, of which 65 were within the DDRD subtype. Isogenic cell lines, which represent DDRD-positive and -negative, were used to study the effects of chemokine release on peripheral blood mononuclear cell (PBMC) migration and the mechanism of immune signaling activation. Finally, we studied the association between the DDRD subtype and expression of the immune-checkpoint protein PD-L1 as detected by IHC. All statistical tests were two-sided. Results: We found that DDRD breast tumors were associated with CD4+ and CD8+ lymphocytic infiltration (Fisher’s exact test P < .001) and that DDRD cells expressed the chemokines CXCL10 and CCL5 3.5- to 11.9-fold more than DNA damage response–proficient cells (P < .01). Conditioned medium from DDRD cells statistically significantly attracted PBMCs when compared with medium from DNA damage response–proficient cells (P < .05), and this was dependent on CXCL10 and CCL5. DDRD cells demonstrated increased cytosolic DNA and constitutive activation of the viral response cGAS/STING/TBK1/IRF3 pathway. Importantly, this pathway was activated in a cell cycle–specific manner. Finally, we demonstrated that S-phase DNA damage activated expression of PD-L1 in a STING-dependent manner. Conclusions: We propose a novel mechanism of immune infiltration in DDRD tumors, independent of neoantigen production. Activation of this pathway and associated PD-L1 expression may explain the paradoxical lack of T-cell-mediated cytotoxicity observed in DDRD tumors. We provide a rationale for exploration of DDRD in the stratification of patients for immune checkpoint–based therapies.

Molecular Subgroup of Primary Prostate Cancer Presenting with Metastatic Biology

BACKGROUND Approximately 4-25% of patients with early prostate cancer develop disease recurrence following radical prostatectomy. OBJECTIVE To identify a molecular subgroup of prostate cancers with metastatic potential at presentation resulting in a high risk of recurrence following radical prostatectomy. DESIGN, SETTING, AND PARTICIPANTS Unsupervised hierarchical clustering was performed using gene expression data from 70 primary resections, 31 metastatic lymph nodes, and 25 normal prostate samples. Independent assay validation was performed using 322 radical prostatectomy samples from four sites with a mean follow-up of 50.3 months. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Molecular subgroups were identified using unsupervised hierarchical clustering. A partial least squares approach was used to generate a gene expression assay. Relationships with outcome (time to biochemical and metastatic recurrence) were analysed using multivariable Cox regression and log-rank analysis. RESULTS AND LIMITATIONS A molecular subgroup of primary prostate cancer with biology similar to metastatic disease was identified. A 70-transcript signature (metastatic assay) was developed and independently validated in the radical prostatectomy samples. Metastatic assay positive patients had increased risk of biochemical recurrence (multivariable hazard ratio [HR] 1.62 [1.13-2.33]; p=0.0092) and metastatic recurrence (multivariable HR=3.20 [1.76-5.80]; p=0.0001). A combined model with Cancer of the Prostate Risk Assessment post surgical (CAPRA-S) identified patients at an increased risk of biochemical and metastatic recurrence superior to either model alone (HR=2.67 [1.90-3.75]; p<0.0001 and HR=7.53 [4.13-13.73]; p<0.0001, respectively). The retrospective nature of the study is acknowledged as a potential limitation. CONCLUSIONS The metastatic assay may identify a molecular subgroup of primary prostate cancers with metastatic potential. PATIENT SUMMARY The metastatic assay may improve the ability to detect patients at risk of metastatic recurrence following radical prostatectomy. The impact of adjuvant therapies should be assessed in this higher-risk population.

Validation of a Metastatic Assay using biopsies to improve risk stratification in patients with prostate cancer treated with radical radiation therapy

Abstract Background Radiotherapy is an effective treatment of intermediate/high-risk locally advanced prostate cancer, however, >30% of patients relapse within 5 years. Clinicopathological parameters currently fail to identify patients prone to systemic relapse and those whom treatment intensification may be beneficial. The purpose of this study was to independently validate the performance of a 70-gene Metastatic Assay in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Patients and methods A bridging cohort of prostate cancer diagnostic biopsy specimens was profiled to enable optimization of the Metastatic Assay threshold before further independent clinical validation in a cohort of diagnostic biopsies from patients treated with radical radiotherapy and androgen deprivation therapy. Multivariable Cox proportional hazard regression analysis was used to assess assay performance in predicting biochemical failure-free survival (BFFS) and metastasis-free survival (MFS). Results Gene expression analysis was carried out in 248 patients from the independent validation cohort and the Metastatic Assay applied. Ten-year MFS was 72% for Metastatic Assay positive patients and 94% for Metastatic Assay negative patients [HR = 3.21 (1.35–7.67); P = 0.003]. On multivariable analysis the Metastatic Assay remained predictive for development of distant metastases [HR = 2.71 (1.11–6.63); P = 0.030]. The assay retained independent prognostic performance for MFS when assessed with the Cancer of the Prostate Assessment Score (CAPRA) [HR = 3.23 (1.22–8.59); P = 0.019] whilst CAPRA itself was not significant [HR = 1.88, (0.52–6.77); P = 0.332]. A high concordance [100% (61.5–100)] for the assay result was noted between two separate foci taken from 11 tumours, whilst Gleason score had low concordance. Conclusions The Metastatic Assay demonstrated significant prognostic performance in patients treated with radical radiotherapy both alone and independent of standard clinical and pathological variables. The Metastatic Assay could have clinical utility when deciding upon treatment intensification in high-risk patients. Genomic and clinical data are available as a public resource.

Abstract 322: High EMSY expression defines a BRCA-like subgroup of high-grade serous ovarian carcinoma with superior survival and platinum sensitivity

EMSY, encoding a BRCA2-binding protein, is reportedly amplified in 6-18% of high grade serous ovarian carcinomas (HGSOCs). As a negative regulator of BRCA2, HGSOCs with EMSY overexpression may mimic BRCA-mutant patients, who experience superior clinical outcome and hypersensitivity to platinum chemotherapy and PARP inhibition by virtue of homologous recombination deficiency (HRD). Here we investigate the impact of EMSY expression, extracted from local and publicly available transcriptomic data, on the outcome and platinum sensitivity of HGSOCs. Platinum response data were collected from the Edinburgh Ovarian Cancer Database. Overall survival (OS) and progression-free survival (PFS) differences were assessed using multivariate cox regression accounting for age, stage at diagnosis and debulking status. Within the Edinburgh cohort, high EMSY expression was associated with superior OS and PFS (table 1). Datasets from the MRC ICON7 trial control arm, Tothill et al, Mateescu et al, Pils et al, and TCGA cohorts demonstrated similar benefit for high-EMSY patients. Within the Edinburgh cohort, high-EMSY patients displayed significantly superior complete response (CR) rate to platinum at second (radio CR 44.4% vs 12.5%, P = 0.035; CA125 CR 88.0% vs 55.0%, P = 0.002) and third exposure (radio CR 50.0% vs 5.9%, P = 0.080; CA125 CR 53.3% vs 21.3%, P = 0.021), with prolonged time to progression (median 127 vs 83.5 days from second platinum, P = 0.084; median 151.5 vs 60.5 days from third platinum, P = 0.004). Within high-risk (advanced stage suboptimally debulked) HGSOCs, more high-EMSY patients remained recurrence-free 5 and 10 years from diagnosis (17.6% vs 2.7%, P = 0.031; 12.5% vs 0.9%, P = 0.041). Together these data demonstrate a subgroup of HGSOCs defined by high EMSY expression experience superior clinical outcome and platinum sensitivity, consistent with HRD. This subgroup may therefore represent HGSOCs that are also sensitive to PARP inhibition. Citation Format: Robert L. Hollis, Michael Churchman, Tzyvia Rye, Andrena M. McCavigan, Laura A. Knight, Richard Kennedy, C. Simon Herrington, Charlie Gourley. High EMSY expression defines a BRCA-like subgroup of high-grade serous ovarian carcinoma with superior survival and platinum sensitivity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 322.

Abstract 283: Identification of a high-risk subgroup in primary prostate cancers presenting with targetable immune biology

Background Recent studies have demonstrated limited success of immune checkpoint therapies in unselected prostate cancer. We therefore assessed an immune based DNA Damage Repair Deficiency (DDRD) assay, that we have previously reported represents activation of the cGAS STING pathway in the TCGA dataset of primary prostate cancers, to investigate the presence of targetable immune biology in prostate cancer. In addition we applied a second assay (the prostate cancer metastatic signature-PCM) that predicts risk of metastatic recurrence for early prostate cancer to assess if immune therapy could have a role in treating high risk disease. Methods 498 samples in the TCGA dataset with RNA sequencing data were scored with the PCM and DDRD assays. Integrative analysis was performed on 488 of those samples with matched RNA sequencing, promoter site methylation, somatic mutation and somatic copy number variation. Gene expression of n=6 immune checkpoint targets was investigated with the subgroups identified using T-tests. The prevalence of immune infiltration in each subgroup was tested by applying a cut off to the leukocyte fraction. The viability of reproducing those subgroups with RNA sequencing alone was tested in the TCGA dataset and an independent validation dataset of 321 resected primary prostate cancers. Cox proportional hazards regression analysis was performed for biochemical recurrence and metastatic events in both datasets. Results Integrative analysis of the TCGA dataset identified four patient subgroups characterised primarily by variances in copy number and genomic mutation. One of these subgroups ‘Metastatic-like DDRD9 had significantly higher PCM scores and DDRD immune scores compared to the other subgroups (p Citation Format: Emma Reilly, Andrena McCavigan, Steven M. Walker, Nuala McCabe, Eileen Parkes, Denis P. Harkin, Richard D. Kennedy, Laura A. Knight. Identification of a high-risk subgroup in primary prostate cancers presenting with targetable immune biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 283.

Abstract 1777: Development of a pan-cancer 15 gene expression signature to detect a subgroup driven by MAPK signalling

INTRODUCTION Unsupervised hierarchical clustering of gene expression data from 265 High Grade Serous Ovarian Cancer (HGSOC) patients identified 3 major molecular subgroups. One subgroup is driven by activation of the MAPK pathway and is associated with poor prognosis and resistance to platinum chemotherapy. The MAPK pathway is currently being targeted by novel therapeutics and hence an assay to detect activation of the pathway across cancers would be highly valuable as a clinical trial enrichment tool. Using internal and publicly available gene expression datasets we have demonstrated that the MAPK subgroup also exists in other cancer types and is associated with poor prognosis. The aim of this study was to develop a gene expression signature to predict the MAPK subgroup across multiple cancer types. METHODS Evaluation of gene expression data in a range of tumours (ovarian, colon, lung, melanoma and prostate) identified a common gene list (CGL) of 7802 genes showing high variability and high expression across diseases. The CGL was input to different machine learning algorithms developing signatures under 10x5-fold cross-validation (CV), trained against the MAPK HGSOC subgroup. Filter-Feature-Selection removed 10% of genes under CV based upon ranked correlation adjusted t-scores and the final model selected to satisfy a number of key criteria: AUC for predicting the endpoint; association with survival (C-Index); and functional relevance of signature content. RESULTS A 15 gene signature was selected, yielding an AUC=0.87 [95% CI:0.84-0.89] with respect to the MAPK subgroup. This model has validated as a poor prognostic marker in several other cancer types (Colorectal, Relapse free survival: HR=1.46 [95% CI:1.07-1.98]; Lung, Relapse free survival: HR=2.18 [95% CI:1.33-3.56]; Prostate cancer, Biochemical recurrence: HR 2.49 CI: 1.43-4.34), and is suppressed by MEK inhibition (p=0.0023) and elevated by KRAS, NRAS and MEK1 overexpression in cell line models (p=0.0443, CONCLUSION A 15 gene signature has been developed from formalin fixed paraffin embedded samples across multiple diseases to detect a molecular subgroup driven by MAPK signalling. This assay predicts sensitivity to MEK inhibitors in pre-clinical model systems and in primary cells derived from patients. Further work aims to validate the signature in clinical samples from patients treated with a MEK inhibitor. This assay may be helpful for clinical trial enrichment to select patients that are likely to benefit from MAPK targeted therapies. Citation Format: Laura A. Knight, Bethanie Price, Andrena McCavigan, Aya El-Helali, Charlie Gourley, Denis P. Harkin, Richard Kennedy, Nuala McCabe. Development of a pan-cancer 15 gene expression signature to detect a subgroup driven by MAPK signalling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1777. doi:10.1158/1538-7445.AM2017-1777