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Periodic Report Summary 4 - RATHER (Rational Therapy for Breast Cancer: Individualized Treatment for Difficult-to-Treat Breast Cancer Subtypes)

Project Context and Objectives:
Cancer genotyping has identified a number of correlations between mutations in specific genes and responses to targeted anti-cancer drugs, with many mutations occurring in kinases or downstream signalling components. The RATHER consortium are identifying novel kinase targets for therapy in two poor-prognosis subtypes of breast cancer. These are triple negative (TN) breast tumours which lack the estrogen, progesterone and HER2 receptors, constituting 15% of breast cancers, and invasive lobular carcinomas (ILC) of the breast, which represent 10% of breast tumours. RATHER seeks to address the lack of effective targeted therapies for these cohorts, which constitute a quarter of all breast cancer patients.
The RATHER programme is providing one of the most comprehensive investigations of the contribution of protein kinases and kinase mutations to these distinct biological subtypes of breast cancer. In RATHER, we are identifying and validating novel kinase targets that have potential as prognostic or predictive biomarkers of disease progression, or as potential drug targets. We have applied state-of-the-art techniques to analyse the kinome by DNA sequencing, copy number variation analysis, proteomics and gene expression profiling technologies. Our focus is the identification of activated/altered kinases in these poor prognosis subtypes of breast cancer.
In detail, the RATHER consortium has identified kinase targets via a 5-pronged approach: i) direct re-sequencing of the kinome of 150 TN and 150 ILC tumours, ii) determination of abundance and activation status of kinases in these tumours by reverse phase protein array (RRPA) and tissue microarray (TMA) technologies, iii) determination of copy number variation by SNP arrays, iv) mRNA quantitation of the kinome using DNA microarrays and v) RNA sequencing of 80 ILC breast cancer tumours/cell lines. Data from these five independent genome-scale technologies have been integrated (along with publicly available data), yielding a priority list of potential kinase targets for therapy in ILC and TN breast cancer. These candidate targets for therapy are being validated in preclinical models using RNA interference and/or small molecule inhibitors, whilst prognostic and predictive clinical assays for breast cancer are being developed by the two SMEs involved.
Finally, we have initiated a phase I/II pan-European clinical trial to study the therapeutic potential of a promising isoform-specific inhibitor of phosphatidylinositol 3-kinase (PI3K) in combination with endocrine therapy in breast cancer patients. The drug is being studied in patients with and without mutations in the PI3K gene. The RATHER project will deliver proof-of-concept for the use of this drug in patients who do not respond to current therapies. The RATHER project will also deliver prognostic clinical assays for patient stratification, for up to 25% of breast cancer patients where current treatment options are unsatisfactory.

Project Results:
The foundations of the RATHER programme are the ILC and TN patient tissues (WP1). NKI and CAM successfully reviewed and selected over 300 frozen primary tumour samples (176 TN + 145 ILC), for RNA, DNA and protein extraction, and FFPE tissue collation (for tissue microarrays [TMA]). Matched normal DNA was extracted where possible. Clinical and survival data have enabled us to correlate kinome pathway alterations with clinical outcome.
The discovery WPs (WP2-6) have been completed. NKI completed kinome sequencing for 143 ILC and 150 TN samples and associated controls. The analyses accomplished so far have revealed numerous mutations in ILC (most frequently CDH1 and PI3KCA) and TN (TP53). Similarly, CAM completed 445 hybridizations for copy number analysis, and demonstrated more alterations in TN than ILC. RNA was used for expression arrays and RNA sequencing (WP6). AG has analysed the ability of the MammaPrint assay to classify at least 50% of patients as low risk with a 5-year DMFS/BCSS of at least 90% in lobular cancer patients. NKI have identified a ‘BRCAness’ (BRCA1-like) signature in TN (WP6), that displays a similarity to that seen in tumours with BRCA1 mutations, even though the gene is not mutated or methylated, and consistently predicts benefit of treatments that are effective for BRCA1 mutations. The BRCAness signature has been patented, it is currently being studied in an independent clinical trial, and AG is developing a companion diagnostic assay for TN (WP8). In addition, UCD has delivered RNA-seq data and corresponding bioinformatics analysis for 77 ILC tumours and cell lines where the expression levels of 41 kinase or kinase-related genes were shown to be correlated with ILC patient outcome (WP6).
Kinase (phospho-)protein expression was analysed in WP3&4. IC has generated RPPA data for 168 (phospho-) proteins, in 124 ILC and 153 TN tumour samples and controls. Subgroups of ILC tumours show activation of signalling pathways, with proteins identified that might have a prognostic value in ILC. TMAs have been constructed (NKI/CAM), and TN or ILC status has been confirmed by immunohistochemistry (LU/OM, WP4). TMAs were used to validate key markers from publicly available data (WP10), including AKT3 and CDK7. Data indicate that AKT3 amplification in a subset of TN tumours could be a potential therapeutic target. CDK7 expression was found to specifically predict outcome in TNBC. Additional biomarker candidates were identified (IC), with initial analyses by OM suggesting that GATA3 protein expression is correlated with ILC prognosis (WP4).
In WP8, AG successfully translated their suite of patient assays from microarrays to a next generation sequencing platform. AG also validated the use of MammaPrint as a prognostic assay for ILC, with study results emphasising the independent value of MammaPrint specifically in lymph node-negative invasive lobular breast cancers.
Additionally, OM are validating a novel prognostic signature for early stage breast cancer, termed the OncoMasTR panel; a subset of the OncoMasTR panel will be validated by IHC, initially in an independent cohort of patients.
Comprehensive data integration (WP10) has revealed two subtypes of ILC, termed hormone receptor signalling and immune related subtypes. Survival analysis indicates several molecular markers associated with clinical outcome. AG is developing a companion diagnostic assay utilising the immune related subtype for ILC (WP8). A clinical trial is currently being initiated to validate the IR response prediction in the clinical setting.

In WP7, a list of potential drug targets has been generated by UCD and NKI.
An SFTP repository (the ‘Filestore’) has been created to store RATHER data (WP11). This repository is hosted by UCD, and is available to all members remotely using password-protected encrypted channels.
The POSEIDON clinical trial (WP9) to study the therapeutic potential of a PI3K inhibitor in combination with tamoxifen is progressing well, and phase Ib is complete. Phase II (which recruited its first patient in June 2016) is studying the action of the inhibitor in ductal and lobular breast cancers, with or without PI3KCA mutations.
In summary, the project is progressing very well, with the scientific discovery WPs, data analysis and integration completed, and patient accrual in the POSEIDON clinical trial initiated. The consortium have published 17 scientific papers to date, and filed 4 patents.

Potential Impact:
The RATHER research programme is contributing significantly to our understanding of the mechanisms underlying two difficult-to-treat subtypes of breast cancer (which account for almost a quarter of cases diagnosed), leading to widespread impact at multiple levels, as discussed below.
Scientific impact: The RATHER consortium is addressing key, as yet unanswered, issues relating to a lack of therapeutic (and associated molecular diagnostic) options for ILC and TN breast cancer. Using multiple state-of-the-art approaches, in an integrated manner, we are shedding much needed biological insight into both of these difficult-to-treat subtypes of breast cancer, with the key aim of identifying lead candidate kinase targets for therapeutic intervention, alongside development of tailored molecular diagnostic approaches for prediction of therapeutic response.
Health and societal impact: As discussed, ILC and TN breast subtypes are known to be difficult-to-treat and are typically resistant to standard therapies. By identifying commonly activated kinases and choosing the most appropriate therapeutic agents targeting same, the RATHER consortium predict that patients with these activated kinases will have favourable responses to these therapies. In turn, this will lead to better survival rates and an improved quality-of-life for these patients.
Healthcare economics impact: By identifying activated/mutated kinases in ILC and TN breast cancer, clinical assays for patient stratification can be developed. This will promote the use of personalised therapies for these subgroups, thus avoiding unnecessary treatment of patients who will not benefit from specific treatments.
Providing new commercialisation opportunities and improving the competitiveness of SMEs: The RATHER project has led to novel therapeutic and diagnostic opportunities, primarily for the two associated SMEs. Agendia are developing companion diagnostic tests for TN and ILC breast cancers, while OncoMark are validating an IHC-based prognostic assay for early stage breast cancer patients.
Impact on collaboration: The major advantage of the RATHER consortium is the inclusion of acknowledged world-class experts in the breast cancer and the molecular diagnostics/therapeutics arena. These scientists span 6 EU countries, including Ireland, Netherlands, France, Sweden, Spain, and the UK and have excellent track record for scientific excellence. RATHER has also joined forces with the EurocanPlatform collaborative programme, to lead an exciting pan-European clinical trial. Therefore, the RATHER project provides an exciting platform for integration of this excellence and expertise at a European level.
Impact on publications: We expect the RATHER project will generate several high impact publications. So far, the project has generated 17 publications and had a major high impact publication in Scientific Reports within this period. This publication titled “Integration of genomic and proteomic data identifies two biologically distinct subtypes of invasive lobular breast cancer” describes a multi-omic analysis of a large ILC patient cohort and presents an integrated molecular portrait of ILC. We also expect several high impact publications from the POSEIDON clinical trial. 4 patents have been submitted; (i) Methods and means for subtyping invasive lobular breast cancer, (ii) Methods for molecular classification of BRCA-like breast and / or ovarian cancer, (iii) A method of predicting risk of recurrence of cancer and (iv) Combination therapy - combined MAP2K4/MAP3K1 and MEK/ERK inhibition.
Impact on dissemination: The RATHER project has also had a wide range of dissemination opportunities. There have been numerous invited talks given by the coordinator, Prof. Gallagher, as well as over 20 scientific posters and presentations given by members of the consortium at scientific conferences. Awards have also been given to members of the group including NovaUCD Innovation Award to Prof. William Gallagher (2011) and EU Women Innovator Award (2nd Place) to Prof. Laura van’t Veer (NKI/AG). RATHER hosted a scientific symposium in January 2015, entitled ‘Leveraging the power of systems medicine in personalised oncology’, which was well attended. The agenda featured international leaders in the field of personalised medicine.

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