Katherine E. Keating

Identification and Validation of an Anthracycline/Cyclophosphamide–Based Chemotherapy Response Assay in Breast Cancer

Background There is no method routinely used to predict response to anthracycline and cyclophosphamide–based chemotherapy in the clinic; therefore patients often receive treatment for breast cancer with no benefit. Loss of the Fanconi anemia/BRCA (FA/BRCA) DNA damage response (DDR) pathway occurs in approximately 25% of breast cancer patients through several mechanisms and results in sensitization to DNA-damaging agents. The aim of this study was to develop an assay to detect DDR-deficient tumors associated with loss of the FA/BRCA pathway, for the purpose of treatment selection. Methods DNA microarray data from 21 FA patients and 11 control subjects were analyzed to identify genetic processes associated with a deficiency in DDR. Unsupervised hierarchical clustering was then performed using 60 BRCA1/2 mutant and 47 sporadic tumor samples, and a molecular subgroup was identified that was defined by the molecular processes represented within FA patients. A 44-gene microarray-based assay (the DDR deficiency assay) was developed to prospectively identify this subgroup from formalin-fixed, paraffin-embedded samples. All statistical tests were two-sided. Results In a publicly available independent cohort of 203 patients, the assay predicted complete pathologic response vs residual disease after neoadjuvant DNA-damaging chemotherapy (5-fluorouracil, anthracycline, and cyclophosphamide) with an odds ratio of 3.96 (95% confidence interval [Cl] =1.67 to 9.41; P = .002). In a new independent cohort of 191 breast cancer patients treated with adjuvant 5-fluorouracil, epirubicin, and cyclophosphamide, a positive assay result predicted 5-year relapse-free survival with a hazard ratio of 0.37 (95% Cl = 0.15 to 0.88; P = .03) compared with the assay negative population. Conclusions A formalin-fixed, paraffin-embedded tissue-based assay has been developed and independently validated as a predictor of response and prognosis after anthracycline/cyclophosphamide–based chemotherapy in the neoadjuvant and adjuvant settings. These findings warrant further validation in a prospective clinical study.

Cross-laboratory validation of the OncoScan® FFPE Assay, a multiplex tool for whole genome tumour profiling

BackgroundAdoption of new technology in both basic research and clinical settings requires rigorous validation of analytical performance. The OncoScan® FFPE Assay is a multiplexing tool that offers genome-wide copy number and loss of heterozygosity detection, as well as identification of frequently tested somatic mutations.MethodsIn this study, 162 formalin fixed paraffin embedded samples, representing six different tumour types, were profiled in triplicate across three independent laboratories. OncoScan® formalin fixed paraffin embedded assay data was then analysed for reproducibility of genome-wide copy number, loss of heterozygosity and somatic mutations. Where available, somatic mutation data was compared to data from orthogonal technologies (pyro/sanger sequencing).ResultsCross site comparisons of genome-wide copy number and loss of heterozygosity profiles showed greater than 95% average agreement between sites. Somatic mutations pre-validated by orthogonal technologies showed greater than 90% agreement with OncoScan® somatic mutation calls and somatic mutation concordance between sites averaged 97%.ConclusionsReproducibility of whole-genome copy number, loss of heterozygosity and somatic mutation data using the OncoScan® assay has been demonstrated with comparatively low DNA inputs from a range of highly degraded formalin fixed paraffin embedded samples. In addition, our data shows examples of clinically-relevant aberrations that demonstrate the potential utility of the OncoScan® assay as a robust clinical tool for guiding tumour therapy.

Analytical Performance of a 15-Gene Prognostic Assay for Early-Stage Non–Small-Cell Lung Carcinoma Using RNA-Stabilized Tissue

A 15-gene prognostic signature for early-stage, completely resected, non-small-cell lung carcinoma, (which distinguishes between patients with good and poor prognoses) was clinically validated in prior studies. To achieve operational efficiencies, this study was designed to evaluate the assays performance in RNA-stabilized tissue as an alternative to the fresh-frozen tissue format originally used to develop the assay. The percent concordance between matched tissue formats was 84% (95% Wilson CI, 70%-92%), a level of agreement comparable to the inherent reproducibility of the assay observed within biological replicates of fresh-frozen tissue. Furthermore, the analytical performance of the assay using the RNA-stabilized tissue format was evaluated. When compared to an accredited reference laboratory, the clinical laboratory achieved a concordance of 94% (95% Wilson CI, 81%-98%), and there was no evidence of bias between the laboratories. The lower limit of quantitation for the target RNA concentration was confirmed to be, at most, 12.5 ng/μL. The assay reportable range defined in terms of risk score units was determined to be -4.295 to 4.210. In a large-scale precision study, the assay showed high reproducibility and repeatability. When subjected to a maximal amount of genomic DNA, a potential contaminant, the assay still produced the expected results. The 15-gene signature was confirmed to produce reliable results and, thus, is suitable for its intended use.

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 MIP-055: IDENTIFICATION OF A MOLECULAR SUBTYPE OF HIGH GRADE SEROUS OVARIAN CANCER REPRESENTING MAPK PATHWAY ACTIVATION AND PLATINUM RESISTANCE

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 naive 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. O9Brien, 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.

Abstract 626: Cross-site reproducibility and orthogonal validation of copy number and somatic mutation calls of OncoScan® FFPE Assay Kit in solid tumors

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Objectives Copy number (CN) and somatic mutation (SM) analysis in tumors is rapidly gaining importance in cancer management as a tool for differential diagnosis, determination of prognosis, and selection of therapeutic. Genome-wide copy number and LOH detection as well as a panel of frequently tested somatic mutations can be detected with OncoScan® FFPE Assay Kit. We report on a validation study of OncoScan FFPE Assay Kit CN and SM data by orthogonal technologies (FISH and NGS, respectively) to estimate the sensitivity and specificity parameters of the platform. In addition, we assessed the reproducibility of the platform across three different sites in the UK: Leeds Institute of Cancer and Pathology (LICP), West Midlands Regional Genetics Laboratory (WMRGL), and Almac Diagnostics (Almac). Methods Validation of CN data was performed on a cohort of cancer samples identified as Her2 positive/ambiguous by FISH. The panel of SMs available was validated by a custom targeted amplicon NGS panel on a diverse collection of samples derived from multiple cohorts across several tumor types sourced from both LICP and WMRGL. For the reproducibility study, 162 samples encompassing six different tumor types (breast, colorectal, lung, melanoma, prostate, and ovarian) were collected from LICP and WMRGL. DNA was extracted and plated in triplicate and distributed to the three testing sites: LICP, WMRGL, and Almac. Data from all sites was analyzed for reproducibility of genome-wide CN/LOH calls and SM calls. Results Cross-site comparisons of genome-wide CN and LOH profiles on 162 FFPE solid tumor samples showed greater than 95% average agreement between three sites (LICP, WMGRL, and Almac), while SM classification concordance between the sites averaged 97%. Initial orthogonal validation of Her2 amplification by FISH showed greater than 90% concordance, as did initial test samples used for validating OncoScan SM calls by a targeted amplicon NGS panel. Conclusion In this study we validated both CN and SM calls using OncoScan FFPE Assay Kit and demonstrated a high degree of agreement with orthogonal methods in all aspects. Reproducibility of whole-genome CN, LOH, and SM data using OncoScan FFPE Assay Kit has also been demonstrated for a range of FFPE samples, including highly degraded samples. This study is a step forward in evaluating the potential clinical utility of a platform combining genome-wide copy number and somatic mutation calls within the national health service of the UK. Citation Format: Joseph M. Foster, Assa Oumie, Fiona S. Togneri, Morag Taylor, Sofia Alyas, Paula Wojtowicz, Henry Wood, Emma Tinkler-Hundal, Katie Southward, Dominic McMullan, Phil Quirke, Katherine E. Keating, Mike Griffiths, Karen G. Spink, Fiona Brew, Eric Fung, Jeanette Schmidt. Cross-site reproducibility and orthogonal validation of copy number and somatic mutation calls of OncoScan® FFPE Assay Kit in solid tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 626. doi:10.1158/1538-7445.AM2015-626

Abstract 4820: Identification of a metagene representing SRC activation which is predictive of response to Dasatinib

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Epithelial ovarian cancer (EOC) is the leading cause of death from gynecological malignancies in the Western World. The aim of this study was to identify ovarian cancer subtypes related to biological pathways which are currently being targeted by novel therapeutics. SRC (sarcoma) is a tyrosine kinase which plays critical roles in mediating many cellular pathways such as proliferation, adhesion, survival, differentiation and cell motility. SRC activity is increased in many human cancers including EOC. Furthermore, up-regulation of the SRC pathway has been shown to be associated with cisplatin resistance and patient overall survival in EOC. We therefore hypothesised that up-regulation of the SRC pathway may represent a subtype of EOC which could be therapeutically exploited. We used a semi-supervised analysis approach to perform hierarchical clustering analysis of 198 serous, stage III and IV EOC using a 917 public gene list generated by the differentially expressed genes, after DNA microarray profiling, in MCF10A cells with normal expression and over-expression of the SRC gene. A number of gene clusters were identified upon hierarchical clustering which segregated the patient samples. To examine which of the genes from these clusters represented SRC activation we used a series of in vitro assays including phospho-SRC expression, sensitivity to SRC siRNA, and sensitivity to the SRC inhibitors Saracatinib and Dasatinib in 16 ovarian cancer cell lines. The ovarian cell lines were scored individually with the probesets representing the genes of interest from each of the individual gene clusters and the cell line in vitro data. Two gene clusters were selected for further investigation. A panel of 15 breast cancer cell lines were next investigated for SRC activation using the same in vitro assays described above. Correlation of SRC activation, by the scoring of the in vitro assays and the scoring of probesets from the individual gene clusters, demonstrated one gene cluster which showed good correlation and which potentially represented a metagene of SRC activation. The gene cluster consists of 16 genes, some of which are already known components of the SRC pathway. Furthermore, using the probesets representing the genes from this gene cluster to score public DNA microarray profiling data we were able to predict response to Dasatinib in two independent datasets of prostate and breast cancer cell lines (p=<0.05). In summary we have used a SRC activation transcriptional profile to cluster ovarian cancer samples and selected a metagene comprised of 16 genes which correlates to SRC activation in vitro using a panel of assays in ovarian and breast cancer cell lines. This has been shown to be predictive of response to Dasatinib in 2 independent datasets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4820. doi:1538-7445.AM2012-4820

Identification of a novel breast cancer molecular subgroup associated with a deficiency in DNA-damage response

10511 Background: Loss of a functional DNA-damage response (DDR) sensitizes tumors to DNA-damaging as well as targeted therapeutics such as PARP-1 inhibitors. However, there is no assay to detect DDR proficiency and guide therapeutic choice. Although abrogation of the DDR can result from multiple mechanisms, including loss of components of the BRCA/Fanconi anemia pathway, the resultant DNA-damage may activate common molecular pathways. We hypothesized that these pathways could define a molecular subgroup and form the basis of a diagnostic test for sensitivity to DNA-damaging and targeted therapies. METHODS Using DNA-microarray technology, we profiled a cohort of BRCA mutant enriched and thus DDR-deficient (DDRD) primary breast tumor samples. Hierarchical agglomerative clustering analysis was performed and identified a molecular subtype in breast cancer characterized by activation of pathways known to respond to DNA damage. Computational classification was performed resulting in the generation of a gene signature that could identify this DDRD molecular subgroup. RESULTS A subset of tumors was identified as displaying biology associated with DDRD. Computational classification was performed based upon expression of this DDRD-related biology resulting in the generation of a 44-gene signature. Retrospective validation in independent breast cancer datasets indicated that the DDRD signature was predictive of response to anthracycline-based chemotherapy with an odds ratio of 15.02 (CI 3.51 - 63.49). In addition, the signature could accurately identify non-responding patients with a negative predictive value of 0.96 (CI 0.88-0.99). CONCLUSIONS We report the identification of a novel molecular subgroup associated with a deficiency in DDR that can be identified in both ER-positive and ER-negative breast cancer using a 44-gene signature. This subgroup is enriched for BRCA1/2 mutant tumors and demonstrates sensitivity to DNA-damaging agents. We propose that the DDRD signature could be used as a patient stratification tool for existing chemotherapy or as a clinical trial enrichment tool for DNA-damaging or repair targeted drugs in development.