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Purpose:The DNA damage immune response (DDIR) assay was developed in breast cancer based on biology associated with deficiencies in homologous recombination and Fanconi anemia pathways. A positive DDIR call identifies patients likely to respond to platinum-based chemotherapies in breast and esophageal cancers. In colorectal cancer, there is currently no biomarker to predict response to oxaliplatin. We tested the ability of the DDIR assay to predict response to oxaliplatin-based chemotherapy in colorectal cancer and characterized the biology in DDIR-positive colorectal cancer.Experimental Design:Samples and clinical data were assessed according to DDIR status from patients who received either 5-fluorouracil (5-FU) or 5FUFA (bolus and infusion 5-FU with folinic acid) plus oxaliplatin (FOLFOX) within the FOCUS trial (n = 361, stage IV), or neoadjuvant FOLFOX in the FOxTROT trial (n = 97, stage II/III). Whole transcriptome, mutation, and IHC data of these samples were used to interrogate the biology of DDIR in colorectal cancer.Results:Contrary to our hypothesis, DDIR-negative patients displayed a trend toward improved outcome for oxaliplatin-based chemotherapy compared with DDIR-positive patients. DDIR positivity was associated with microsatellite instability (MSI) and colorectal molecular subtype 1. Refinement of the DDIR signature, based on overlapping IFN-related chemokine signaling associated with DDIR positivity across colorectal cancer and breast cancer cohorts, further confirmed that the DDIR assay did not have predictive value for oxaliplatin-based chemotherapy in colorectal cancer.Conclusions:DDIR positivity does not predict improved response following oxaliplatin treatment in colorectal cancer. However, data presented here suggest the potential of the DDIR assay in identifying immune-rich tumors that may benefit from immune checkpoint blockade, beyond current use of MSI status.

Supplementary Results

Figures S1-S9, Tables S1, S2

supplementary methods

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.