RATHER is structured into a number of work packages – units of work with clearly defined goals and deliverables. This page provides an overview of the 11 work packages of the project.
- Work Package 1 - Clinical Samples
- Work Package 2 - Kinome Re-sequencing
- Work Package 3 - Reverse Phase Protein Lysate Arrays
- Work Package 4 - Tissue Microarray Analysis
- Work Package 5 - SNP Arrays
- Work Package 6 - DNA Microarrays
- Work Package 7 - Validation of Kinase Targets
- Work Package 8 - Development of Biomarker Assays for Clinical Trail
- Work Package 9 - Initiation of Phase I/II Clinical Trial
- Work Package 10 - Data Integration
- Work Package 11 - Project and Database Management
Work Package 1 – Clinical Samples
Researchers in RATHER are investigating more than 300 clinical samples from breast cancer patients (150 invasive lobular carcinomas [ILC], and 150 triple negative [TN] breast cancers). These samples were sourced from biobanks at the University of Cambridge and the Netherlands Cancer Institute (NKI). DNA and RNA extractions, and the preparation of protein lysates and cell pellet arrays, were performed in succeeding work packages.
Work Package 2 – Kinome Re-sequencing
RATHER is particularly interested in the human kinome – the complete set of 500+ kinases. In this work package, led by NKI, we have sequenced the kinome DNA of each clinical sample. Exome capture technology from Agilent was be used to isolate kinome DNA, and researchers identified several kinase mutations specific to ILC or TN samples.
Work Package 3 – Reverse Phase Protein Lysate Arrays
This work package, led by the Institut Curie, investigated the protein expression levels of human kinases, as well as the phosphorylation status of their targets. This was accomplished using reverse phase protein lysate arrays (RPPA). The RPPA technique consists of printing very small amounts (1ng) of cell or tissue lysates in a microarray format to detect proteins of interest using highly specific antibodies. The activation status of a particular kinase target can be assessed by using antibodies directed against regular and phosphorylated form(s) of the protein. Institut Curie generated protein expression data for 168 kinases and identified potential biomarkers in ILC and TN samples, as well as finding 4 subgroups of ILC samples with biologically distinct features.
Work Package 4 – Tissue Microarray Analysis
OncoMark Ltd. are leading this work package and have utilised various techniques to detect proteins, as well as DNA/RNA molecules, in the clinical samples. Conventional IHC and tissue immunoblotting approaches are being used to investigate the protein expression levels of the human kinases, as well as the phosphorylation status of their targets. One publication emanating from this work package describes the amplification of the AKT-3 gene and increased expression of the Akt-3 protein in TN, which were associated with poorer prognosis (O'Hurley et al., 2013). Biomarkers of interest, identified by Institut Curie in WP3 and potentially correlated with poor prognosis in ILC, are currently being validated in RATHER tissue microarrays (TMAs). TMAs incorporate multiple cores of tissue from patient biopsies; they allow for high-throughput analysis and reduce wastage of precious clinical material. Fluorescent and chromogenic in situ hybridisation have also been used to analyse kinase gene alterations in the clinical samples.
Work Package 5 – SNP Arrays
In this work package, led by University of Cambridge, we used Affymetrix 6.0 SNP arrays to perform genome-wide investigations of the clinical samples. The aim of this work was to generate comprehensive maps of copy number and allelic state of the protein kinase genes within the samples. Matched normal DNA from each of the patients was also profiled in order to call both copy number aberrations (CNAs) and allelic imbalances (AIs). This comprehensive analysis (identifying of all possible types of somatic kinase aberrations) has been integrated with data from the other platforms in WP10.
Work Package 6 – DNA Microarrays
Led by Agendia, RATHER used DNA microarrays to quantify the expression levels of all kinases in the clinical samples. These data have been integrated with data from the preceding work packages, and gene signatures that are predictive of particular kinase alternations/pathway activations have been identified. Severson et al., 2015, describes the identification of a subtype of TN called BRCA1-like, defined by a genomic pattern. The BRCA1-like subtype of TN appears to have a poorer prognosis, but may respond to targeted drugs. A second theme of this work package involves the development of a prognostic signature for ILC. Agendia has already developed and commercialised the breast cancer prognostic test MammaPrint, which predicts the risk of breast cancer recurrence based on a gene expression signature. Here, we aim to improve the prognostic power of the test by developing a signature that is specific for ILC. Additionally, University College Dublin (UCD) have conducted RNA sequencing on 69 ILC samples to complement the data generated by other partners. This has enabled us to detect 'RNA editing' in the expressed kinome in ILC.
Work Package 7 – Validation of Kinase Targets
Collectively, work packages 2-6, combined with the integration efforts of WP10, have identified a number of candidate mutations/pathway alterations that appear to be specific to ILC and/or TN. Within this work package, led by NKI, researchers are further investigating these and other candidates in an attempt to determine which of them represent true drivers of the oncogenic phenotype. We are using siRNA and shRNA libraries to knock down the relevant genes in cell line models, and then analysing the consequent effects on cell proliferation and survival. In a second approach, candidate drug targets are being inhibited with small molecule drugs/antibodies that are in clinical development (most are available for research use from commercial vendors today), either as mono-therapy or in combination with established breast cancer therapies. The output of this work package is a priority list of functionally validated kinase targets for ILC and TN.
Work Package 8 – Development of Biomarker Assays
In this work package, the two commercial partners, Agendia and OncoMark, are developing biomarker assays that can be used to stratify patients for treatment (companion diagnostic), or to predict risk of recurrence (prognostic assay). We are focusing our efforts on technologies that are already well-validated in the clinical context, namely use of PCR-based methods for mutation detection and quantification of RNA changes, as well as IHC and FISH for assessment of protein expression/activity and CNAs, respectively. Agendia are focusing on the development of a companion diagnostic assay that uses predictive biomarkers identified in work packages 2-6, and 10, as well as a prognostic assay for ILC. OncoMark are currently validating a protein expression-based prognostic assay based on a novel panel of biomarkers, for early stage breast cancer.
Work Package 9 – Initiation of Phase I/II Clinical Trial
RATHER initiated a phase I/II clinical trial in 2014, to evaluate patient drug responses in a clinical setting. The trial is studying single kinase inhibitor (in combination with endocrine therapy) in breast cancer patients, and will examine kinase gene mutations in patients to assess whether these affect the treatment. A range of breast cancer patients will be included in the trial, but one study group will include only ILC patients. Translational studies will be carried out by all partners to closely examine the genetics and proteomics (proteins) of samples from each patient, and how they respond to treatment. These translational studies should help us to identify other biomarkers; these could lead to new drug targets or clinical diagnostic assays. We expect phase II to be completed in 2019.
Work Package 10 – Data Integration
The primary aim of this work package, led by UCD, was to develop and apply a computational framework that would enable the integration of data from all the project partners. Specifically, it has focused on analysing and integrating data from other work packages; these analyses serve as the basis for functional validation of the candidate kinase targets for therapy in WP7. This work package is linked to WP11 (project/database management), the latter providing a secure and comprehensive database to store data from WP1-10.
Work Package 11 – Project and Database Management
Due to the complex nature of the RATHER project and the associated data generated, project and data management activities are key to a successful delivery. The specific objectives of this work package, led by OncoMark Ltd., are (1) to ensure governance and co-ordination of the whole project, (2) to create a dedicated consortium webpage to facilitate communication within and without the consortium, and (3) to design, create and populate a secure database to hold data generated across the entire project.