Abstract Oncogenic KRAS mutations promote tumorigenesis by constitutive activation of multiple, well-characterised signalling pathways. However, there is significant heterogeneity across mutant KRAS tumours in terms of mutation present, mutant allele abundance and downstream signalling strength. It is unclear whether these variations can impact responses to specific therapies. Here, we demonstrate that ∼20% of lung adenocarcinomas (LUAD) show an increase in mutant KRAS dosage (KRAS mutant allele fraction > KRAS wild-type ). Furthermore, we show that KRAS mutant dosage can directly influence clinical outcome and therapeutic susceptibilities in lung cancer. Our findings show that mutant KRAS copy gains specifically affect platinum lung cancer response, promoting resistance to this standard-of-care therapy. Importantly, increases in KRAS mutant dosage are also associated with an increased vulnerability to pS6K inhibition, due to the unique metabolic rewiring of these cells. Together, we show that mutant KRAS dosage contributes to the phenotypic heterogeneity of mutant KRAS NSCLC and that assessment of mutant KRAS content or signalling strength can help optimise treatments strategies for these patients.

PURPOSE: Triple-negative breast cancer (TNBC) is a heterogeneous disease. We previously showed that homologous recombination deficiency (HRD) and the DNA damage immune response (DDIR) signature are prognostic in TNBC. We hypothesized that these biomarkers reflect related but not completely interdependent biological processes, that their combined use would be prognostic, and that simultaneous assessment of the immunologic microenvironment and susceptibility to DNA damaging therapies might be able to identify subgroups with distinct therapeutic vulnerabilities. METHODS: We analyzed the dual DDIR/HRD classification in 341 patients with TNBC treated with adjuvant anthracycline-based chemotherapy on the SWOG S9313 trial and corroborated our findings in The Cancer Genome Atlas breast cancer data set. RESULTS: DDIR/HRD classification is highly prognostic in TNBC and identifies biologically and immunologically distinct subgroups. Immune-enriched DDIR+/HRD+ TNBCs have the most favorable prognosis, and DDIR+/HRD- and DDIR-/HRD+ TNBCs have favorable intermediate prognosis, despite the latter being immune-depleted. DDIR-/HRD-TNBCs have the worst prognosis and represent an internally heterogeneous group of immune-depleted chemoresistant tumors. CONCLUSION: Our findings propose DDIR/HRD classification as a potentially clinically relevant approach to categorize tumors on the basis of therapeutic vulnerabilities.

Figures S1-S9, Tables S1, S2

Background The DNA-damage immune-response (DDIR) signature is an immune-driven gene expression signature retrospectively validated as predicting response to anthracycline-based therapy. This feasibility study prospectively evaluates the use of this assay to predict neoadjuvant chemotherapy response in early breast cancer. Methods This feasibility study assessed the integration of a novel biomarker into clinical workflows. Tumour samples were collected from patients receiving standard of care neoadjuvant chemotherapy (FEC + /−taxane and anti-HER2 therapy as appropriate) at baseline, mid- and post-chemotherapy. Baseline DDIR signature scores were correlated with pathological treatment response. RNA sequencing was used to assess chemotherapy/response-related changes in biologically linked gene signatures. Results DDIR signature reports were available within 14 days for 97.8% of 46 patients (13 TNBC, 16 HER2 + ve, 27 ER + HER2-ve). Positive scores predicted response to treatment (odds ratio 4.67 for RCB 0-1 disease (95% CI 1.13–15.09, P = 0.032)). DDIR positivity correlated with immune infiltration and upregulated immune-checkpoint gene expression. Conclusions This study validates the DDIR signature as predictive of response to neoadjuvant chemotherapy which can be integrated into clinical workflows, potentially identifying a subgroup with high sensitivity to anthracycline chemotherapy. Transcriptomic data suggest induction with anthracycline-containing regimens in immune restricted, “cold” tumours may be effective for immune priming. Trial registration Not applicable (non-interventional study). CRUK Internal Database Number 14232.

AbstractPurpose: 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.

Abstract 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.

439 Background: The benefit of checkpoint blockade in advanced gastric cancer is limited and biomarkers related to response are needed. Novel gene expression analysis software was used to identify Hallmarks of Cancer associated with clinical benefit following nivolumab treatment in >2 nd line advanced gastroesophageal adenocarcinoma (GEA). Methods: RNA-sequencing data from baseline GEA patient diagnostic tumor samples (103 from NCT02862535; 5 from NCT02862535) were analyzed using the clara T platform (V2.0.0, Almac Diagnostic Services). 62 gene signatures were quantified representing 6 key Hallmarks of Cancer (Avoiding Immune Destruction, Activating Invasion and Metastases, Sustaining Proliferative Signaling, Inducing Angiogenesis, Resisting Cell Death and Genome Instability and Mutation). Clinical benefit (CB) was defined as tumor response or overall survival (OS) > 1 year. HER2 status was from medical records. Survival analyses used cox proportional hazards models. Results: Gene expression signatures (GES) identified 5 molecular subgroups (C1-C5). Rate of CB in each molecular subtype are outlined in Table. C3 and C4 had significantly improved OS compared to C2, (HR = 0.45; p= 0.02 and HR = 0.42; p= 0.02). Greater proportions of HER2+ subjects were present in C4 and C3 vs. C2, with C3 statistically significant (60% vs. 14%; p= 0.012). Gene expression characterized by chromosomal instability (CIN) and homologous recombination repair deficiency (HRD) were associated with HER2(+) (wilcox p= < 0.05). Patients selected by only using CIN & HRD had significant improvement in OS (HR = 0.63; p= 0.03). Conclusions: Interferon-based GES did not predict benefit from immune checkpoint blockade. GES representing HRD and activation of HER2, EGFR and MAPK (each enriched in CIN) were associated with improved survival upon checkpoint blockade in advanced GEA patients. Clinical trial information: NCT02862535 . [Table: see text]

4539 Background: The Dual Erb B Inhibition in Oesophago-gastric Cancer (DEBIOC) trial reported an acceptable safety profile for neoadjuvant Xelox +/- AZD8931 but limited efficacy. We utilized EAC patient samples from DEBIOC to evaluate the impact of neoadjuvant Xelox +/-AZD8931 on biological pathways using a unique software driven solution. Methods: 24 pre-treatment FFPE EAC biopsies and 17 matched surgical resection specimens were transcriptionally profiled using the Almac Diagnostics Xcel Array. Gene expression data was analyzed using the Almac claraT total mRNA report V3.0.0, reporting on 92 gene expression signatures and 7337 single genes associated with 10 key biologies. Paired Wilcoxon tests (5% significance level) were used to evaluate changes in claraT scores pre- and post-treatment. EGFR and Her2 expression were assessed by IHC and FISH. Results: 15 patients received Xelox+AZD8931 and 9 Xelox alone. Hierarchical clustering of biopsies identified 4 major clusters: Inflammation active, Genomic Instability active, EGFR & MAPK active, and EMT & Angiogenesis active. Comparison of signature scores pre- and post- neoadjuvant treatment demonstrated a significant reduction in scores relating to DNA damage repair (DDR) deficiency (Almac DNA Damage assay, p< 0.0001; BRCAness Profile, p= 0.0025; HRD Gene Signature, p< 0.0001; BRCA1ness Signature, p= 0.0004) and a significant increase in angiogenesis signatures (Almac Angiogenesis Assay, p= 0.0002; Angio Predictive G model, p= 0.0228; Angiogenesis Signature A, p= 0.0034) and EMT signatures (EMT Signature, p= 0.0031, EMT Enrichment Score, p= 0.0013, Pan-Can EMT Signature B, p= 0.0001). Comparing pre- and post-treatment signature scores in patients treated with Xelox +/-AZD8931 revealed a significant reduction in EGFR Sensitivity Signature ( p= 0.0088), ERBB2-specific Gene Expression Signature ( p= 0.0127) and Hallmark PI3K-AKT-MTOR Signaling ( p= 0.0195) in those treated with Xelox + AZD8931 in keeping with the mechanism of action of AZD8931. Downregulation of AKT signaling was confirmed in AZD8931 treated and resistant cell lines. Conclusions: We report the use of a novel software tool to apply 92 gene expression signatures to EAC biopsy and resection specimens from the DEBIOC trial to provide insight into mechanisms of action. Neoadjuvant treatment was associated with a reduction in DDR deficiency and an increase in angiogenesis and EMT signatures whilst a reduction in EGFR, Her2 and AKT pathways was noted with AZD8931 treatment.

PURPOSE To independently validate two biomarkers, a 44-gene DNA damage immune response (DDIR) signature and stromal tumor-infiltrating lymphocytes (sTILs), as prognostic markers in patients with triple-negative breast cancer (TNBC) treated with adjuvant doxorubicin (A) and cyclophosphamide (C) in SWOG 9313. METHODS Four hundred twenty-five centrally determined patient cases with TNBC from S9313 were identified. DDIR signature was performed on RNA isolated from formalin-fixed paraffin-embedded tumor tissue, and samples were classified as DDIR negative or positive using predefined cutoffs. Evaluation of sTILs was performed as described previously. Markers were tested for prognostic value for disease-free survival (DFS) and overall survival (OS) using Cox regression models adjusted for treatment assignment, nodal status, and tumor size. RESULTS Among 425 patients with TNBC, 33% were node positive. DDIR was tested successfully in 90% of patients (381 of 425), 62% of which were DDIR signature positive. DDIR signature positivity was associated with improved DFS (hazard ratio [HR], 0.67; 95% CI, 0.48 to 0.92; P = .015) and OS (HR, 0.61; 95% CI, 0.43 to 0.89; P = .010). sTILs density assessment was available in 99% of patients and was associated with improved DFS (HR, 0.70; 95% CI, 0.51 to 0.96; P = .026 for sTILs density ≥ 20% v < 20%) and OS (HR, 0.59; 95% CI, 0.41 to 0.85; P = .004 for sTILs density ≥ 20% v < 20%). DDIR signature score and sTILs density were moderately correlated ( r = 0.60), which precluded statistical significance for DFS in a joint model. Three-year DFS and OS in a subgroup of patients with DDIR positivity and T1c/T2N0 disease were 88% and 94%, respectively. CONCLUSION The prognostic role of sTILs and DDIR in early-stage TNBC was confirmed. DDIR signature conferred improved prognosis in two thirds of patients with TNBC treated with adjuvant AC. DDIR signature has the potential to stratify outcome and to identify patients with less projected benefit after AC chemotherapy.

e21045 Background: Traditionally gene expression signatures (GES) are used individually to classify patients into subgroups. Signatures targeting the same biology are often developed independently and may not classify identically. We developed the claraT software tool that uses consensus between multiple published GES categorised by the Hallmarks of Cancer (Hanahan & Weinberg, 2011) to classify cancers. As metastatic melanoma represents poor prognostic disease (5-yr survival 15-20%), we applied claraT to the TCGA melanoma dataset to identify targetable biologies, validated in a cohort of melanoma patients treated with Ipilimumab. Methods: TCGA RNA-seq data ( n= 472) was analysed using the claraT platform including GES for immune ( n= 14), angiogenesis ( n= 9) and epithelial-mesenchymal transition (EMT) ( n= 12) Hallmarks. Samples were clustered for the combined and individual Hallmarks. Median progression-free (PFS) and overall-survival (OS) differences were analysed across identified subgroups. Analysis was validated in an Ipilimumab treated melanoma dataset ( n= 42) (Van Allen, 2015). Results: Clustering the combined Hallmarks identified 4 subgroups in the TCGA cohort: 1) Immune active, 2) Immune-EMT active, 3) EMT-Angiogenesis active, 4) All inactive. Groups 1&2 had significantly improved OS compared to Groups 3&4 (HR = 0.50, p< 0.0001). Clustering using single Hallmarks revealed that immune-positive tumours had significantly improved OS (HR = 0.53, p< 0.0001) compared to immune-negative tumours. Angiogenesis-negative tumours displayed improved PFS (HR = 0.73, p= 0.03) and OS (HR = 0.53, p <0.0001) compared to angiogenesis-negative tumours. Interestingly the EMT Hallmark was not found to be individually prognostic. When validated in the Ipilimumab treated dataset, patients classified as immune-positive had improved OS (HR = 0.357, p= 0.010) when compared to immune-negative. Similar trends were also observed for angiogenesis and EMT Hallmarks. Conclusions: This study demonstrates how simultaneous analysis of multiple GES ( n= 35 in this study) can identify robust biologies through consensus expression. This platform may have value in the identification of reliable biomarkers for clinical trials and could inform how combination therapies targeting key biologies may be used in cancer treatment.

ObjectiveCurrent strategies to guide selection of neoadjuvant therapy in oesophageal adenocarcinoma (OAC) are inadequate. We assessed the ability of a DNA damage immune response (DDIR) assay to predict response following neoadjuvant chemotherapy in OAC.DesignTranscriptional profiling of 273 formalin-fixed paraffin-embedded prechemotherapy endoscopic OAC biopsies was performed. All patients were treated with platinum-based neoadjuvant chemotherapy and resection between 2003 and 2014 at four centres in the Oesophageal Cancer Clinical and Molecular Stratification consortium. CD8 and programmed death ligand 1 (PD-L1) immunohistochemical staining was assessed in matched resection specimens from 126 cases. Kaplan-Meier and Cox proportional hazards regression analysis were applied according to DDIR status for recurrence-free survival (RFS) and overall survival (OS).ResultsA total of 66 OAC samples (24%) were DDIR positive with the remaining 207 samples (76%) being DDIR negative. DDIR assay positivity was associated with improved RFS (HR: 0.61; 95% CI 0.38 to 0.98; p=0.042) and OS (HR: 0.52; 95% CI 0.31 to 0.88; p=0.015) following multivariate analysis. DDIR-positive patients had a higher pathological response rate (p=0.033), lower nodal burden (p=0.026) and reduced circumferential margin involvement (p=0.007). No difference in OS was observed according to DDIR status in an independent surgery-alone dataset.DDIR-positive OAC tumours were also associated with the presence of CD8+ lymphocytes (intratumoural: p<0.001; stromal: p=0.026) as well as PD-L1 expression (intratumoural: p=0.047; stromal: p=0.025).ConclusionThe DDIR assay is strongly predictive of benefit from DNA-damaging neoadjuvant chemotherapy followed by surgical resection and is associated with a proinflammatory microenvironment in OAC.

There is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material.

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. (C) 2017 European Association of Urology. Published by Elsevier B.V.