Abstract P6-04-02: Defining clinically relevant molecular subtypes in lobular breast cancer within the RATHER consortium
Authors: Linn S , Majewski I , Michaut M , Peeters J , Schouten P , de Koning L , He B , Fan Y , Tarrant F , Oconnor D , Rueda O , Chin S-F , Heijmans J , Snel M , Severson T , Bosma A , Mittempergher L , Bismeijer T , Dubois T , Wessels L , Simon I , Gallagher W , Caldas C , Bernards R 
Published: August 2014

Background: The Rational Therapy for Breast Cancer (RATHER) Consortium aims to identify novel kinase targets for therapy in poor-prognosis subtypes of breast cancer for which there are currently no targeted therapies available. In this project, the focus is on invasive lobular carcinomas (ILC), which represent 10% of breast tumors. The main strength of the RATHER project is the unique combination of comprehensive molecular data together with detailed clinical information, which enables translation of state-of-the art genomics analyses to the clinic. Our main goal is to identify and validate novel kinase targets for breast cancer therapy in a comprehensive way using large-scale and complementary genomics data (DNA and RNA sequence, copy number variation, gene expression and protein expression). Integrated analysis of these molecular data is performed to define molecular subtypes of ILC with differential clinical outcome.

Methods: One hundred and fifty ILC samples (fresh frozen) with &gt;5 years follow-up were collected from two institutes (Cambridge, Netherlands Cancer Institute). All samples were processed following one standard operating protocol to isolate RNA, DNA and protein of high quality. We used a five-pronged approach to identify and validate novel kinase targets for therapy in ILC, namely i) direct re-sequencing of the kinome of 150 ILC breast tumors, ii) determination of abundance and activation status of kinases in these tumors by reverse phase protein lysate array (RPPA) technology, iii) determination of copy number variation (CNV) by genome-wide SNP arrays, iv) mRNA quantitation of both genome and kinome using DNA microarrays and v) RNAseq of a subset of ILC tumors. ILC are compared to triple negative, which allows us to highlight differences and/or similarities between these subtypes and provide clues for therapeutic targets.

Results: Data from these independent genome-scale technologies were integrated, yielding a prioritized list of potential kinase targets for therapy in ILC breast cancer. Deep sequencing of the kinome has revealed somatic mutations characteristic of ILC, which are currently being validated via mass spectrometry-based genotyping technology and their possible effects confirmed with gene expression, protein expression and phosphorylation changes. In addition, on a subset of the ILC samples, RNA sequencing was performed to confirm expression of particular mutants. Known gene mutations in ILC such as loss of CDH1 were confirmed. Moreover, the PI3K pathway is found to be frequently altered (50% of the samples). Gene expression analysis, as well as integrative analysis of CNV and gene expression data revealed subsets of ILCs that significantly regulate alternate biological processes and show differential clinical outcome. Such biological subsets are currently being validated with clinical and follow-up data. Updated results will be presented at the meeting.

Conclusion: The RATHER project aims to deliver proof-of-concept for novel therapeutic interventions, together with companion molecular diagnostic assays for patient stratification, for up to 10% of breast cancer patients, where current treatment options are unsatisfactory. Ongoing validation of a number of potential targets proves to be promising.

Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P6-04-02.