Abstract Tumor mutational burden (TMB) is emerging as a promising predictive biomarker to select cancer patients who will benefit from immunotherapy. However, accurate TMB estimation from sequencing data remains hampered by the ability to correctly distinguish somatic variants from both germline variants and spurious base changes introduced by sample preparation and sequencing processes. The latter is particularly prevalent in poor quality FFPE material where degradation and chemical modifications of the DNA manifest as variant artefacts leading to over-estimation of TMB. One method of removing germline variants and artefacts is to compare tumor variant calls to those obtained from matched normal tissue, however inclusion of a matched normal adds significant logistical and financial challenges to clinical trial assays. Therefore, there remains a critical need to establish alternative approaches enabling accurate TMB estimation from FFPE tumor-only samples. To address these challenges, we have developed a tumor-only somatic variant calling pipeline for accurate determination of TMB from FFPE material profiled by whole exome sequencing (WES). The process is underpinned by two key features (a) enhanced germline variant filtering that uses a systematic rule-based strategy, (b) FFPE artefact removal using an ensemble machine learning model incorporating multiple variant call QC parameters. The germline filtering approach was developed on a matched fresh frozen (FF)/FFPE/blood dataset of 23 cancer patients. The FFPE artefact filtering model was built on a dataset of 59 FF and FFPE matched pairs, where FFPE artefacts were defined as variants present in FFPE but absent in the FF sample. Benchmarked against internal and TCGA datasets of somatic variant calls from >30 FF samples with a matched normal, our germline and FFPE filtering steps decreased false discovery rates by over 20%. This translated into improved TMB scoring performance with tumor-only pipeline estimates from three SeraCare reference standards closely matching expected TMB scores. Critically, we establish the clinical relevance of our approach on a published cohort of 98 patients treated with an immune checkpoint inhibitor. Here we observed increased association between patient response and TMB estimated from our pipeline (p=0.042) compared to a standard pipeline with no enhanced germline or FFPE artefact filters (p=0.56). Overall, our combined germline and FFPE artefact filtering pipeline results in significantly improved somatic variant detection and TMB scoring performance in FFPE samples where a matched normal is unavailable. It is applicable across multiple disease types and adaptable to other platforms. Therefore, the pipeline has utility in clinical laboratories where accurate somatic variant detection is crucial to successful deployment of predictive biomarkers such as TMB. Citation Format: Hui Sun Leong, Emma O'Connor, Nuala McCabe, Sinead Donegan, Steven M. Walker, Jayna Mistry, Karen Keating, Denis P. Harkin, Richard D. Kennedy, Laura A. Knight, James R. Bradford. A machine learning approach to detect FFPE artefacts leads to accurate estimation of tumor mutational burden from sequencing data without a matched normal [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 152.

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.

Abstract Introduction: 40-45% of patients with High Grade Serous Ovarian Cancer (HGSOC) will eventually relapse with platinum resistant disease. Tothill et al and TCGA are two independent gene expression datasets which have demonstrated the presence of a mesenchymal molecular subgroup, characterised by upregulation of angiogenesis regulating genes. Angiogenesis is known to be an integral pathological feature of HGSOC and anti-angiogenics have dominated the field of drug development in EOC. However, despite this, anti-angiogenic agents have failed to demonstrate a significant impact on overall survival (OS) benefit. In this study, we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents and what the underlying biological rationale for this could be. Methods: A meta-analysis of 14 phase II and III clinical trials in EOC were used to investigate the association between platinum resistance and response to anti-angiogenic agents. In addition, we analysed gene expression in 12 matched pre- and post-chemotherapy EOC samples. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the development of an angiogenic phenotype. Further studies were performed in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy. Result: In the clinical trial meta-analysis, an OS benefit for antiangiogenics was observed in platinum-resistant disease (p=0.029), whilst platinum-sensitive EOC only derived progression free survival (PFS) (p=<0.0001) benefit and not OS (p=0.125). In the 12 matched pairs of patient samples, post-platinum samples had a higher micro-vessel density (MVD) relative to their paired treatment-naïve sample (p= 0.0001). Additionally, an in vivo angiogenesis matrigel plug assay demonstrated that cisplatin-resistant EOC cell lines were associated with an increase in MVD (p=<0.0001). MVD was reduced in the platinum-resistant cells following treatment with bevacizumab (p=0.001). Ascites-derived primary cells established from platinum-resistant patients demonstrated overexpression of VEGF-A, consistent with stimulating angiogenesis. Gene expression analysis of pre- and post-platinum paired samples identified that PDGFRα (p=0.007) and PDGFRβ (p=0.005) were differentially expressed in the post-platinum therapy samples. In vitro validation in the platinum-resistant cell lines demonstrated that VEGF-A expression was regulated by PDGFRα. Discussion: We have demonstrated that previous platinum therapy for EOC is associated with an increase in tumor PDGFα and VEGF-A expression, correlating with a response to anti-angiogenic therapies. This data suggests that platinum therapy resistance may inform the selection of EOC patients for novel antiangiogenic therapies in future clinical trials. Citation Format: Aya El Helali, Nuala McCabe, Christopher Steele, Lara Dura Perez, Christina L. O'Neill, Naomi Dickson, Niamh McGivern, Caolan Harkin, Andrena McCavigan, Reinhold J. Medina, Laura A. Knight, Stephen McQuaid, Jacqueline A. James, Caroline O. Michie, Charlie Gourley, W Glenn McCluggage, Denis P. Harkin, Richard H. Wilson, Alan W. Stitt, Richard D. Kennedy. Platinum resistance in epithelial ovarian cancer is dependent on a PDGFR alpha-VEGF-A signalling mechanism that activates downstream angiogenesis pathways [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 7.

Abstract 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 DDRD' had significantly higher PCM scores and DDRD immune scores compared to the other subgroups (p < 2E-12). This subgroup of patients showed elevated leukocyte fraction and expression of immune checkpoint genes: CD274 (PDL1), CTLA4, ICOS, IDO1, HAVCR2 (TIM3) & LAG3 (p < 2E-6). Genomic instability with amplification of 8q and a larger prevalence of somatic mutations including that of TP53 was also detected in this subgroup. The ‘Metastatic-like DDRD' subgroup was found to have a significant association with poor survival outcome in TCGA (multivariable: p < 0.008), a result that has also been replicated in an independent dataset in both univariate (p < 0.001) and multivariable analysis (p < 0.01). Conclusions We have identified and validated a poor prognostic subgroup, representing 10-20% of early prostate cancer patients that are at increased risk of developing metastatic disease and present with targetable immune biology. These patients may represent a viable target population for immune checkpoint and DNA damaging therapies in prostate cancer. 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.

5578 Background: Patients with High Grade Serous Ovarian Cancer (HGSOC) initially respond to SOC platinum based treatment but most will eventually relapse with platinum resistant disease. Angiogenesis is known to be an integral pathological feature of HGSOC and anti-angiogenic agents have been trialed in this population, but have failed to demonstrate a significant impact on overall survival (OS). Here, we asked if platinum resistance could be associated with an improved response to anti-angiogenic therapies. Methods: A meta-analysis of 14 phase II and III clinical trials in EOC were used to investigate the association between platinum resistance and response to anti-angiogenic agents. In addition we analysed gene expression in 12 matched pre-and post-chemotherapy samples. Novel cisplatin-resistant HGSOC cell lines and novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy were developed to investigate the relationship between angiogenesis and platinum resistance. Results: The meta-analysis revealed an OS benefit for anti-angiogenics in platinum-resistant disease (p = 0.029), whilst platinum-sensitive disease derived only PFS benefit (p = < 0.0001). In the matched pairs of patient samples, post-platinum samples had a higher micro-vessel density (MVD) relative to their paired treatment-naïve sample (p = 0.0001). Additionally, an in vivo angiogenesis matrigel plug assay demonstrated that cisplatin-resistant EOC cell lines were associated with an increase in MVD (p = < 0.0001). MVD was reduced in the platinum-resistant cells following treatment with bevacizumab (p = 0.001). Ascites-derived cells established from platinum-resistant patients demonstrated overexpression of VEGF-A through increased PDGFRa and PDGFRb expression. Conclusions: We have demonstrated that previous platinum therapy for EOC is associated with an increase in tumour PDGFα and VEGF-A expression, correlating with a response to anti-angiogenic therapies. This data suggests that platinum therapy resistance may inform the selection of EOC patients for novel anti-angiogenic therapies in future clinical trials.

12111 Background: 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 a mesenchymal phenotype, poor prognosis and resistance to platinum.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. Methods: Using TCGA data we show the existence of the mesenchymal subgroup across a range of solid tumours including stomach, bladder colon, lung, melanoma and prostate cancer. Further to this, a common gene list was generated to include only transcripts with high variability and expression across diseases, and used as a starting list for the development of a 15 transcript assay which can be used to prospectively identify the mesenchymal subgroup from archived tissue. The 15 gene expression assay was tested in preclinical model systems to assess its utility at predicting response to MEK inhibitors. Results: The 15 gene expression mesenchymal assay was a poor prognostic marker in 13 different solid tumours: overall HR = 1.78 [95% CI:1.65-1.92]) p < 0.0001. Additionally the assay was associated with a mesenchymal phenotype (migration, invasion) and activated MAPK (phospho-MAPK) signalling in preclinical cell line models. The assay also predicted phospho-MEK expression in clinical samples (p < 0.05). The assay score was reduced by MEK inhibition (p < 0.05) and elevated by KRAS, NRAS and MEK1 overexpression (p < 0.05). The assay predicted response to the MEK inhibitors Trametinib and Selumetinib across cell line models from multiple diseases (p < 0.001) and to Trametinib in mouse xenograft studies of lung cancer cell lines. Conclusions: A 15 gene expression assay has been developed from FFPE samples across multiple diseases to detect a mesenchymal molecular subgroup associated with MAPK signalling. The assay predicted sensitivity to MEK inhibitors in pre-clinical cell line and mouse model systems. Further work aims to validate the assay as a predictive biomarker in clinical samples from patients treated with MEK targeted therapies.

11616 Background: Epithelial–mesenchymal transition (EMT) is the conversion of epithelial cells to mesenchymal cells and involves loss of cell–cell adhesion and cell polarity and increased motility, invasiveness and metastases. The EMT process has therefore been under investigation as a new target for anticancer drug discovery. The aim of this work was to develop an EMT biomarker suitable for formalin-fixed paraffin embedded (FFPE) tissue that could be used for patient treatment selection. Methods: Unsupervised hierarchical clustering of ovarian cancer gene expression data (TCGA. 2011) previously identified an EMT subgroup. We confirmed this EMT subgroup in FFPE tissue using 265 high grade serous ovarian cancer (HGSOC) FFPE samples. The analysis was extended to show existence of an EMT subgroup across a range of solid tumours including colon, lung, melanoma and prostate cancer. Further to this, a common gene list was generated to include only transcripts with high variability and expression across diseases, and used as a starting list for development of a 15 transcript assay which can be used to prospectively identify the EMT subgroup from archived tissue. Results: The 15 gene expression assay was a poor prognostic marker in 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 was associated with activated MAPK (phospho-MAPK) in preclinical models and clinical samples (p < 0.05). The assay score was reduced by MEK inhibition (p < 0.05) and elevated by KRAS, NRAS and MEK1 overexpression (p < 0.05). The assay predicted response to the MEK inhibitors Trametinib and Selumetinib in cell line models (p < 0.001). Conclusions: A 15 gene expression assay has been developed from FFPE samples across multiple diseases to detect an EMT molecular subgroup associated with MAPK signalling. The assay predicted sensitivity to MEK inhibitors in pre-clinical model systems. Further work aims to validate the assay as a predictive biomarker in clinical samples from patients treated with EMT or MEK targeted therapies.

Abstract Introduction: Platinum resistant High Grade Serous Ovarian Cancer (HGSOC) has a poor outcome with limited treatment options. Angiogenesis is a key pathological feature of ovarian cancer and anti-angiogenics have dominated the field of drug development in EOC, particularly in the second-line setting (Marachini et al 2013). In this study we asked if platinum resistance could be associated with an improved response to anti-angiogenic agents. Method: A review of phase III anti-angiogenic clinical trials was used to investigate the association between platinum resistance and response to anti-angiogenic agents. To investigate the effect of chemotherapy on predefined ovarian cancer molecular subgroups (Gourley, et al. J Clin Oncol 32:5s, 2014), we analysed 35 matched pre- and post-chemotherapy samples by gene expression. Novel isogenic cisplatin-resistant HGSOC cell lines were established to study the mechanisms of cisplatin section pressure and shift to an angiogenic phenotype. This was further validated in novel ascites-derived primary cell lines from HGSOC patients with known outcomes following platinum-based chemotherapy. Results: Critical review of 22 phase III anti-angiogenic trials suggested that there was a better response to anti-angiogenics following previous platinum-based chemotherapy. Our analysis demonstrated that 67% of treatment naïve tumours that were initially classified as non-angiogenic shifted to an angiogenic biology, which was associated with platinum resistance. Additionally we found that cisplatin resistant cancer cell lines demonstrated hallmarks of vascular mimicry and an associated increase in vessel density using an angiogenesis Matrigel plug assay in Athymic nude mice (p-value=<0.0001). In addition, cell lines established from platinum-resistant patients as well as cell lines made platinum resistant in-vitro, demonstrated overexpression of VEGFa which would be expected to stimulate angiogenesis. Conclusion: We have demonstrated that platinum-resistance in HGSOC is associated with angiogenic biology supporting the use of anti-angiogenic agents in this setting. Citation Format: Aya El Helali, Nuala McCabe, Naomi Dickson, Lara Dura Perez, Denis P Harkin, Richard Wilson, Richard Kennedy. Acquired platinum resistance enhances tumour angiogenesis through activation of vascular mimicry [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 776. doi:10.1158/1538-7445.AM2017-776

Abstract BACKGROUND: We previously defined 3 molecular subgroups of High Grade Serous Ovarian Cancer (HGSOC), using gene expression data from 265 FFPE samples obtained from treatment naive patients, who received platinum based treatment following surgical resection. The 3 molecular subgroups were Angio: characterised by upregulation of angiogenesis genes; Immune: characterised by upregulation of immune genes and AngioImmune: characterised by upregulation of angiogenesis and immune genes. Patients within these 3 subgroups respond differently to standard of care treatment The Immune subgroup have the best prognosis and the Angio and AngioImmune subgroups have similar worse prognosis. A weighted gene signature to identify each of the molecular subgroups was developed. This dataset was used as a reference to investigate the effect of chemotherapy on molecular subgroup designation. METHODS: To investigate the effect of chemotherapy on predefined molecular subgroups, we analysed 35 matched pre- and post- chemotherapy samples by gene expression. The molecular subgroup assignment for each of the paired samples was determined using the gene expression signatures for each subgroup. Novel cisplatin resistant HGSOC cell lines were generated to study the mechanisms of acquired cisplatin resistance. RESULTS: 40% of the treatment naive samples that were aligned with the AngioImmune subgroup and this increased to 67.5% post-chemotherapy. 10/15 (67%) treatment naïve tumours that were initially assigned to the good prognostic Immune molecular subgroup shifted to the bad prognostic AngioImmune molecular subgroup post chemotherapy. Hence platinum chemotherapy selects for the AngioImmune subgroup, suggesting that this subgroup represents tumours which are innately platinum resistant but also provides a mechanism of acquired resistance. Additionally we demonstrate that the AngioImmune subgroup is driven by activation of the MAPK pathway and shows that cisplatin resistant HGSOC cell lines are specifically sensitive to MEK inhibitors. CONCLUSIONS: The MAPK pathway is a mechanism of innate and acquired platinum resistance in HGSOC. Furthermore the data suggests that original pre-treatment surgical/biopsy samples may fall within a different molecular subgroup to samples taken post-platinum therapy. Citation Format: Aya El-Helali, Nuala McCabe, Charlie Gourley, Andrena McCavigan, Caroline O. Michie, Bethanie Price, Niamh McGivern, Michael Churchman, Aya El-Helai, Eamonn J. O'Brien, Laura Hill, Timothy S Davison, Alistair Williams, W Glenn McCluggage, Katherine E Keating, Denis P Harkin, and Richard Kennedy. IDENTIFICATION OF A MOLECULAR SUBTYPE OF HIGH GRADE SEROUS OVARIAN CANCER REPRESENTING MAPK PATHWAY ACTIVATION AND PLATINUM RESISTANCE [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-055.

Introduction We have previously defined 3 molecular subgroups of High grade serous ovarian cancer (HGSOC), ‘Angio’, ‘Immune’ and ‘Angio_immune’ subgroups using gene expression data from 265 FFPE HGSOC samples obtained from treatment naive patients and who were subsequently treated with platinum-based standard of care (SoC) chemotherapy (carboplatin +/- paclitaxel) (Gourley, et al. J Clin Oncol 32:5s, 2014). Patients within these 3 molecular subgroups respond differently to SOC treatment. The immune subgroup has the best outcome compared to the Angio and Angio_immune subgroups (HR of 0.63 and 0.66 respectively on multivariate analysis). Since it has previously been shown that the MAPK pathway is an important mediator of cisplatin resistance in ovarian cancer, we wanted to investigate if the MAPK pathway was associated with one of the poor prognosis subgroups. Methods A gene signature that could detect each of the subgroups Angio, Immune and Angio_immune was generated from the clinical samples. Data from the Cancer Genome Atlas (TCGA) Project was used to test for correlation between each subgroup and phospho-MEK as measured by Reverse Phase Proteomic Array (RPPA). Sensitivity to the MEK inhibitor trametinib (GSK1120212) and cisplatin was determined by 10-day colony formation assay. Results We found a statistically significant association between the Angio_immune subgroup signature and Phospho-MEK (serine 217/221) expression (p = 0.047) indicating activation of the MAPK pathway in this subgroup. Additionally we have demonstrated that the Angio_immune subgroup signature is suppressed by MEK inhibition (p = 0.0055) and elevated by KRAS, NRAS and MEK1 overexpression in cell line models (0.0072, 0.0004 and Conclusion We have identified a molecular subgroup in HGSOC that is associated with MAPK signalling. A gene signature to detect this subgroup from formalin fixed paraffin embedded samples has been developed and predicts sensitivity to MEK inhibitors in pre-clinical model systems. Further work aims to validate the signature in clinical samples from patients treated with a MEK inhibitor. Citation Format: Nuala McCabe, Charlie Gourley, Andrena McGavigan, Caroline O. Michie, Niamh McGivern, Michael Churchman, Eamonn J. O’Brien, Laura Hill, Timothy S. Davison, Alistair Williams, Glenn McCluggage, Karen E. Keating, Denis P. Harkin, Richard D. Kennedy. MEK activation is associated with a molecular subgroup in high grade serous ovarian 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 453.