Eileen Parkes

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.

Clinical Application of Poly(ADP-Ribose) Polymerase Inhibitors in High-Grade Serous Ovarian Cancer

UNLABELLED : High-grade serous ovarian cancer is characterized by genomic instability, with one half of all tumors displaying defects in the important DNA repair pathway of homologous recombination. Given the action of poly(ADP-ribose) polymerase (PARP) inhibitors in targeting tumors with deficiencies in this repair pathway by loss of BRCA1/2, ovarian tumors could be an attractive population for clinical application of this therapy. PARP inhibitors have moved into clinical practice in the past few years, with approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA) within the past 2 years. The U.S. FDA approval of olaparib applies to fourth line treatment in germline BRCA-mutant ovarian cancer, and European EMA approval to olaparib maintenance in both germline and somatic BRCA-mutant platinum-sensitive ovarian cancer. In order to widen the ovarian cancer patient population that would benefit from PARP inhibitors, predictive biomarkers based on a clear understanding of the mechanism of action are required. Additionally, a better understanding of the toxicity profile is needed if PARP inhibitors are to be used in the curative, rather than the palliative, setting. We reviewed the development of PARP inhibitors in phase I-III clinical trials, including combination trials of PARP inhibitors and chemotherapy/antiangiogenics, the approval for these agents, the mechanisms of resistance, and the outstanding issues, including the development of biomarkers and the rate of long-term hematologic toxicities with these agents. IMPLICATIONS FOR PRACTICE The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib has recently received approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA), with a second agent (rucaparib) likely to be approved in the near future. However, the patient population with potential benefit from PARP inhibitors is likely wider than that of germline BRCA mutation-associated disease, and biomarkers are in development to enable the selection of patients with the potential for clinical benefit from these agents. Questions remain regarding the toxicities of PARP inhibitors, limiting the use of these agents in the prophylactic or adjuvant setting until more information is available. The indications for olaparib as indicated by the FDA and EMA are reviewed.

A BRCA1 deficient, NFκB driven immune signal predicts good outcome in Triple Negative breast cancer

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or ‘BRCAness’, is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies. We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways.

Clinical Tumor Staging of Adenocarcinoma of the Esophagus and Esophagogastric Junction

TO THEEDITOR: We congratulate Davies et al 1 on the retrospective analysis of 400 patients treated with neoadjuvant chemotherapy followed by surgery at two high-volume London centers over the course of a decade. Rather than examining the prognostic effect of tumor regression, the authors focused on the downstaging effect of neoadjuvant chemotherapy as a predictor of recurrence-free and overall survival. Following adjustment for known clinicopathologic variables, tumor downstaging was associated with a reduction in the risk of recurrence and death, suggesting that major clinical decisions should be based on postchemotherapy staging. As mentioned by the authors, advanced diagnostic staging procedures, such as positron emission tomography-computed tomography (PET-CT),areimportantfordetectingnodaldiseaseanddistantmetastases.Toclassifyatumorasdownstaged,itisessentialtonotonlyaccurately define the pathologic stage following surgical resection but also to define the clinical stage before chemotherapy. Although much work has been carried out to standardize the reporting of pathologic stage, the use of clinical staging methodologies has varied over time and between centers. 2,3 StagingthatusesPET-CTcandetectoccultmetastaticdiseaseinup

Abstract OT3-02-07: Neo-DDRD: A biomarker-driven neoadjuvant feasibility study in breast cancer

Background Anthracycline-based chemotherapy reduces the risk of early breast cancer recurrence but to date it has not been possible to predict which patients specifically benefit from this treatment. The Almac DDRD assay, a 44 gene signature, has been developed to detect DNA damage response deficiency (DDRD) within breast and other cancer types. The DDRD assay has been shown to predict benefit from DNA damaging chemotherapy in retrospective analyses in neoadjuvant and adjuvant settings (Mulligan et al, JNCI 2014). This pilot study aims to prospectively evaluate the feasibility and utility of this signature in the neoadjuvant setting in breast cancer. It will inform the design of a phase II randomised study where the DDRD assay will be utilised to guide neoadjuvant chemotherapy selection. Study design A single-centre, non-randomised feasibility study of 30 women with a histologically confirmed diagnosis of breast cancer, where neoadjuvant chemotherapy is the treatment modality of choice. Inclusion criteria: Age >18 years Early (T1-2, N0-1), locally advanced or inflammatory breast cancer Normal left ventricular function, haematological and biochemical parameters ECOG PS 0-1 Exclusion criteria: Metastatic disease Bilateral breast cancer Pregnancy/breastfeeding Inability to give informed consent At diagnosis, patients consented to providing two core needle biopsies in addition to a diagnostic biopsy. Axillary nodal status was determined by axillary ultrasound, fine needle aspiration cytology, and pre-treatment sentinel node biopsy if pre-operative axillary staging was negative. Chemotherapy was administered as per standard institutional practice: 6 cycles fluourouracil, epirubicin and cyclophosphamide (FEC) in node-negative patients; 3 cycles of FEC followed by 3 cycles of docetaxel in node positive patients. Neoadjuvant trastuzumab was given in Her2 positive patients. Imaging (mammography and ultrasound) was repeated after three cycles, and repeat FFPE and snap frozen core biopsies undertaken. At conclusion of treatment, patients underwent surgery as appropriate (mastectomy or breast conservation at the discretion of the operating surgeon), with axillary node clearance for patients who were node positive at diagnosis. Further biopsies (FFPE and fresh frozen) were taken intra-operatively. Plasma samples were obtained at diagnosis, mid-treatment and surgery. Pathological reporting of the surgical specimen was standardised using the residual cancer burden (RCB) reporting system. Aims · To assess the feasibility of carrying out the DDRD assay on diagnostic core biopsy specimens · To evaluate the turn-around time for the assay to assess feasibility of integration into the breast cancer treatment pathway · To assess the correlation of DDRD assay scores and pathological tumour response · Exploratory biomarker analysis will be carried out within Queen9s University, Belfast, including: o correlation of DDRD score with changes in Ki67 protein level after three cycles of chemotherapy o exploratory analysis of chemokine expression in peripheral plasma samples, and correlation with pathological response to treatment Accrual The study opened in April 2014. Accrual to date is 14 patients. Citation Format: McIntosh SA, Parkes EE, James CR, Lioe T, Lowry K, Keating KE, Khambata-Ford S, Kennedy RD. Neo-DDRD: A biomarker-driven neoadjuvant feasibility study in breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT3-02-07.

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.

Review of applications of high-throughput sequencing in personalized medicine: barriers and facilitators of future progress in research and clinical application

Abstract There has been an exponential growth in the performance and output of sequencing technologies (omics data) with full genome sequencing now producing gigabases of reads on a daily basis. These data may hold the promise of personalized medicine, leading to routinely available sequencing tests that can guide patient treatment decisions. In the era of high-throughput sequencing (HTS), computational considerations, data governance and clinical translation are the greatest rate-limiting steps. To ensure that the analysis, management and interpretation of such extensive omics data is exploited to its full potential, key factors, including sample sourcing, technology selection and computational expertise and resources, need to be considered, leading to an integrated set of high-performance tools and systems. This article provides an up-to-date overview of the evolution of HTS and the accompanying tools, infrastructure and data management approaches that are emerging in this space, which, if used within in a multidisciplinary context, may ultimately facilitate the development of personalized medicine.

Abstract 4000: A DNA damage response deficiency (DDRD) group in breast cancer is associated with activation of the STING innate immune pathway and PD-L1 expression

We have previously identified a DNA damage response deficient (DDRD) subgroup in breast cancer, associated with loss of the Fanconi Anemia/BRCA DNA repair pathway. The 44-gene DDRD signature developed to prospectively identify this subgroup has been validated as predictive of response to DNA damaging chemotherapy in the treatment of breast cancer. This subgroup is defined by immune signalling, with immune genes, such as the chemokines CXCL10 and CCL5, and immune checkpoints IDO1 and PD-L1 (CD274) overexpressed within the signature and subgroup. Here we report innate immune pathway activation in this subgroup, and association with PD-L1 expression. Methods: We used isogenic cell line models to identify and model pathways constitutively active in DDRD cancer cells. CD4+, CD8+ T-cell infiltration and PD-L1 expression was identified by immunohistochemistry (IHC) on a previously described breast TMA of 191 breast tumor samples, of which 65 were in the DDRD subgroup. DNA microarray data from FFPE samples was used to identify other immune checkpoints associated with this subgroup. Results: CD4+ and CD8+ T-cell infiltration, and PD-L1 expression were significantly associated with DDRD breast tumors on IHC analysis. In addition, expression of immune checkpoints such as IDO1, TIM-3 and LAG-3 were associated with the DDRD subgroup. Increased migration of peripheral blood mononuclear cells was identified into media conditioned by DDRD cells, this was dependent on chemokines secreted by DDRD cells. Endogenous activation of the innate immune pathway STING/TBK1/IRF3 was identified in DDRD cancer cells, and required for expression of chemokines CXCL10 and CCL5. This chemokine expression was associated with cytosolic DNA detected by cGAS and activation of the innate immune pathway STING/TBK1/IRF3. Importantly, this pathway was cell cycle related with upregulation of chemokine expression in S-phase of the cell cycle. Chemokine expression could be induced by S-phase DNA damaging chemotherapy but not by taxanes. Similarly, PD-L1 was induced by treatment with DNA damaging agents in cancer cells dependent on STING. Conclusions: We have identified constitutive activation of the innate immune STING pathway in a DNA damage response deficient subgroup of breast cancer, which is associated with CD4+, CD8+ infiltration and PD-L1 expression. We propose activation of the STING pathway as an important signal for lymphocytic infiltration, independent of tumor-associated neoantigens. The DDRD signature could identify patients with innate immune activation in response to endogenous DNA damage, which may allow stratification for immune checkpoint targeted treatments. Moreover, S-phase DNA damaging agents activate PD-L1 expression, and a combination approach of immune checkpoint inhibitors and S-phase specific chemotherapy may be synergistic in the clinic. Citation Format: Eileen E. Parkes, Steven M. Walker, Nuala McCabe, Laura E. Taggart, Laura Hill, Niamh E. Buckley, Kienan I. Savage, Manuel Salto-Tellez, Stephen McQuaid, Mary T. Harte, Paul B. Mullan, D Paul Harkin, Richard D. Kennedy. A DNA damage response deficiency (DDRD) group in breast cancer is associated with activation of the STING innate immune pathway and PD-L1 expression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4000.

Abstract 2270: A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer

Triple negative (TNBCs) and the closely related Basal-like (BLBCs) breast cancers are a loosely defined collection of cancers with poor clinical outcomes. Both show strong similarities with BRCA1-mutant breast cancers and BRCA1 dysfunction, or ‘BRCAness’, is observed in a large proportion of sporadic BLBCs. BRCA1 expression and function has been shown in vitro to modulate responses to radiation and chemotherapy. Exploitation of this knowledge in the treatment of BRCA1-mutant patients has had varying degrees of success. This reflects the significant problem of accurately detecting those patients with BRCA1 dysfunction. Moreover, not all BRCA1 mutations/loss of function result in the same histology/pathology or indeed have similar effects in modulating therapeutic responses. Given the poor clinical outcomes and lack of targeted therapy for these subtypes, a better understanding of the biology underlying these diseases is required in order to develop novel therapeutic strategies. We have discovered a consistent NFκB hyperactivity associated with BRCA1 dysfunction as a consequence of increased Reactive Oxygen Species (ROS). This biology is found in a subset of BRCA1-mutant and triple negative breast cancer cases and confers good outcome. The increased NFκB signalling results in an anti-tumour microenvironment which may allow CD8+ cytotoxic T cells to suppress tumour progression. However, tumours lacking this NFκB-driven biology have a more tumour-promoting environment and so are associated with poorer prognosis. Tumour-derived gene expression data and cell line models imply that these tumours may benefit from alternative treatment strategies such as reprogramming the microenvironment and targeting the IGF and AR signalling pathways. Citation Format: Niamh E. Buckley, Paula Haddock, Ricardo De Matos Simoes, Eileen Parkes, Gareth Irwin, Frank Emmert-Streib, Stephen McQuaid, Richard D. Kennedy, Paul B. Mullan. A BRCA1 deficient, NFκB driven immune signal predicts good outcome in triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2270.

Abstract C105: DNA damage response deficiency (DDRD) in breast cancer is associated with a STING-dependent innate immune response

Background In breast cancer the presence of an immune infiltration has been recognised as a prognostic factor, however the mechanisms underpinning this response are not clearly defined. Previously we identified a gene expression signature that defines a DNA damage response defective (DDRD) molecular subgroup in breast cancer, that is enriched for BRCA1 and 2 mutations, and has a good outcome following DNA-damaging chemotherapy. Importantly, this DDRD subgroup is associated with up-regulation of type I Interferon-related genes. We therefore investigated the mechanism activating this immune response in the context of abnormal DNA repair. Results IHC analysis demonstrated that both intra-tumoral and stromal CD8+ and CD4+ T cell infiltration were associated with DDRD positive breast tumors. The CXCL10 and CCL5 cytokines were shown to be significantly up-regulated in DDRD positive breast tumours and in tissue culture models for the DDRD molecular subgroup. Furthermore, conditioned media from DDRD positive cell lines stimulated inward migration of peripheral blood mononuclear cells, when compared to media from DDRD negative cells, indicating the presence of active cytokines. We identified constitutive activation of the innate immune pathway STING/TBK1/IRF3 specifically in DDRD positive cells when compared to DDRD negative cells and found that binding of the DNA sensor cGAS to cytosolic Histone H3 was required for this immune response. Treatment of HeLa cells with DNA damaging chemotherapies but not taxanes resulted in STING-dependent expression of CXCL10 and CCL5 cytokines. Conclusion We have identified that the STING/TBK1/IRF3 immune pathway is activated by endogenous DNA damage in DDRD breast cancers and may explain lymphocytic infiltration observed in this subtype of breast tumours. These data suggest that the DDRD signature can identify an innate immune response to DNA damage in breast cancer, which may have therapeutic applications for immune targeted therapies. Citation Format: Eileen E. Parkes, Steven M. Walker, Nuala McCabe, Laura E. Taggart, Laura Hill, Karen D. McCloskey, Niamh E. Buckley, Manuel Salto-Tellez, Paul B. Mullan, D. P. Harkin, Richard D. Kennedy. DNA damage response deficiency (DDRD) in breast cancer is associated with a STING-dependent innate immune response. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C105.