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Introduction: We previously identified an immune-driven gene expression signature recognizing loss of the Fanconi Anemia/BRCA DNA repair pathway - the DNA damage immune response (DDIR) assay. This 44-gene expression-based assay predicted response to DNA damaging chemotherapy in breast cancer, with a 5-year relapse-free survival hazard ratio of 0.37 post adjuvant treatment in assay positive patients. We demonstrated constitutive activation of the cGAS-STING pathway as the underlying mechanism driving immune gene expression in this subgroup, as a result of aberrant cytoplasmic DNA due to deficient DNA repair. We sought to evaluate efficacy of this immune-based signature in predicting response to neoadjuvant DNA-damaging chemotherapy (NAC) in breast cancer. Methods: Patients with early breast cancer were recruited from 2 Northern Ireland centres. All received 3 cycles of 5FU, epirubicin and cyclophosphamide (FEC) chemotherapy initially, with subsequent taxane/HER2 targeted therapies as indicated. Formalin fixed paraffin embedded tumor samples were obtained at baseline, post 3 cycles of FEC and at resection. Chemotherapy response was assessed by residual cancer burden (RCB) scoring. Gene expression data was obtained from all baseline samples and 18 midpoint samples containing sufficient residual tumor. DDIR assay call was determined using a predetermined cut-off. Tumor infiltrating lymphocytes (TILs) were assessed at each time point on H&E stained sections. In addition, gene expression signatures (n=62) encompassing 6 hallmarks of cancer (Immune response, Genome instability, Angiogenesis, EMT, Cell death and Proliferation) were reported using the Almac ClaraT platform. Results: 46 patients were recruited - 26 were assay positive, with high immune gene expression, and 20 assay negative. The DDIR assay predicted NAC response with an odds ratio for RCB 0-1 of 4.67 (95% CI 1.13 - 15.09; p = 0.03). In patients with DDIR positive tumors, there was a strong association with immune response and immune checkpoint response signatures (p < 0.0001), as well as TILs in baseline biopsies (p =0.0262). Matched baseline and mid-point (after 3 cycles of FEC chemotherapy) gene expression analysis was available for 17 non-responders and 4 responders. In responding tumors a trend to reduced immune gene expression was seen, with an associated reduction in TILs (p = 0.0573), probably reflecting eradication of DDIR positive tumor cells (decreased tumor content on midpoint specimens in responders was noted compared to non-responders (p = 0.0257)). Conversely, increased DDIR signature score was observed in non-responders post FEC chemotherapy (p = 0.0332), with a trend to increased TIL infiltration (p = 0.0792). Immune checkpoint gene expression also increased following NAC in these patients. DDIR negative non-responding tumors demonstrated upregulation of angiogenesis signatures at baseline (p < 0.01), along with higher scores for TGFβ driven immune resistance signatures (p < 0.05), suggesting other potential therapeutic approaches for these patients. Conclusions: The cGAS-STING driven DDIR gene expression assay is predictive of response to neoadjuvant DNA damaging chemotherapy and is associated with high baseline immune infiltration. In keeping with the recent TONIC trial, we note anthracycline-based treatment as a potent activator of innate immunity. In non-responders, although TIL infiltration and immune gene expression is induced by DNA damaging chemotherapy, tumors do not respond, presumably due to intact DNA repair pathways and immune checkpoint gene induction. Therefore, there is a rationale to offer this population induction anthracycline-based chemotherapy followed by immune checkpoint targeting treatment to improve outcome. Citation Format: Stuart A McIntosh, Kienan I Savage, Colin James, Tong Lioe, Steven Walker, Keith Lowry, Laura Knight, Andrena McGavigan, Gemma Logan, Jane Hurwitz, Stephen J Kirk, Paul Harkin, Richard D Kennedy, Eileen E Parkes. cGAS-STING driven immune activation predicts response to neoadjuvant chemotherapy and suggests rational IO combination therapies: Results of the Neo-DDIR study [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-10-17.

Background: The DDRD assay was developed to prospectively identify a molecular subtype of cancers defined by DNA damage activated immune signalling. This 44-gene assay has been validated to predict response to anthracycline-cyclophosphamide chemotherapy in both the adjuvant and neoadjuvant settings in breast cancer. The biomarker phase Neo-DDRD trial assessed the clinical utility of this assay prospectively in patients receiving neoadjuvant chemotherapy (NAC). Methods: 48 patients receiving NAC for early (T1-2, N0-1) or locally advanced non-metastatic breast cancer were recruited from March 2014 – Sept 2017. Chemotherapy regimen was selected according to local standard-of-care guidelines (FEC “100” in N0, FEC-D in N1, FEC-DH in HER2 +ve, with the addition of pertuzumab when approved). The DDRD score was applied to RNA expression data obtained from pre-treatment FFPE core biopsies using a custom cDNA microarray (Breast Cancer DSA™). Response to NAC was assessed on resection specimens following definitive surgical treatment by pathologists blinded to the DDRD scores, using the Residual Cancer Burden (RCB) assessment tool. Results: Patients with a positive DDRD assay call had an odds ratio for pathological complete response (pCR) or minimal residual disease (RCB0/RCB1) of 4.64 (95%CI 1.31-16.42; p = 0.0174). Patients with RCB0/RCB1 had significantly higher DDRD assay score than those with class RCB2/RCB3 responses (p =0.0011; unpaired t-test). Tumour subtype and DDRD call are summarised in Table 1. In total, 29.2% of patients had a pathological complete response (pCR) (RCB0). Tumor infiltrating lymphocytes (TILs) were assessed on baseline pre-treatment samples. Increasing DDRD assay score was not significantly associated with increasing stromal TILs (p=0.1403; Pearson correlation). In assessing correlation between stromal TILs and response to chemotherapy, there was no significant difference found across RCB categories (p=0.45; one way ANOVA). Table 1:tumour subtype distribution and DDRD assay resultSubtypeDDRD+DDRD-TotalTriple negative7613HER2+ ER-527HER2+ ER+6612ER+ HER2-8816 Conclusions: Preliminary results from the Neo-DDRD biomarker phase trial support the ability of the DDRD assay to predict response to neoadjuvant chemotherapy. The DDRD assay was confirmed to be a better predictor of neoadjuvant chemotherapy response than the presence of TILs. Translational work from this trial is ongoing: serial tissue and plasma samples collected at treatment mid-point and surgical resection will be used to study changes in RNA expression profiles in both DDRD assay positive and negative during chemotherapy, as well as changes in tumour immune infiltrates, peripheral chemokines and circulating tumour DNA profiles. Based on the results from this pilot study, we propose a biomarker-driven phase II trial in the neoadjuvant setting using the DDRD assay to stratify patients for treatment. Citation Format: Parkes EE, James CR, Lioe T, Walker S, Savage KI, Lowry K, Knight L, McCavigan A, Logan G, Hurwitz J, Kirk SJ, Harkin P, Kennedy RD, McIntosh SA. Biomarker phase neo-DDRD trial: Predicting response to neoadjuvant chemotherapy (NAC) in early breast cancer using the DNA damage repair deficiency (DDRD) assay [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P3-11-12.

Objective: Full-field digital mammography (FFDM) has limited sensitivity for cancer in younger women with denser breasts. Digital breast tomosynthesis (DBT) can reduce the risk of cancer being obscured by overlying tissue. The primary study aim was to compare the sensitivity of FFDM, DBT and FFDM-plus-DBT in women under 60 years old with clinical suspicion of breast cancer. Methods: This multicentre study recruited 446 patients from UK breast clinics. Participants underwent both standard FFDM and DBT. A blinded retrospective multireader study involving 12 readers and 300 mammograms (152 malignant and 148 benign cases) was conducted. Results: Sensitivity for cancer was 86.6% with FFDM [95% CI (85.2–88.0%)], 89.1% with DBT [95% CI (88.2–90%)], and 91.7% with FFDM+DBT [95% CI (90.7–92.6%)]. In the densest breasts, the maximum sensitivity increment with FFDM +DBT over FFDM alone was 10.3%, varying by density measurement method. Overall specificity was 81.4% with FFDM [95% CI (80.5–82.3%)], 84.6% with DBT [95% CI (83.9–85.3%)], and 79.6% with FFDM +DBT [95% CI (79.0–80.2%)]. No differences were detected in accuracy of tumour measurement in unifocal cases. Conclusions: Where available, DBT merits first-line use in the under 60 age group in symptomatic breast clinics, particularly in women known to have very dense breasts. Advances in knowledge: This study is one of very few to address the accuracy of DBT in symptomatic rather than screening patients. It quantifies the diagnostic gains of DBT in direct comparison with standard digital mammography, supporting informed decisions on appropriate use of DBT in this population.

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 Queen's 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.

Invasive lobular cancer has been associated with an increased risk of multifocal and contralateral disease. The literature suggests an incidence of contralateral disease as high as 15%. Current national (NICE) recommendations are that all patients with lobular carcinoma being considered for breast-conserving surgery have a preoperative breast MRI. The objective was to identify the rate of additional MRI-detected multifocal and contralateral disease in patients with a newly diagnosed lobular cancer to determine whether it is as high as the literature suggests. Based on our findings we hope to further explore whether another imaging alternative should be considered.