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Background: Multifocal breast cancer (MFBC), defined as multiple synchronous unilateral lesions of invasive breast cancer, is relatively frequent and has been associated with more aggressive features than unifocal cancer. Here, we aimed to investigate the genomic heterogeneity between MFBC lesions sharing similar histo-pathological parameters.Material and methods: The characterization of different lesions from 36 patients with ductal MFBC involved the identification of non-silent coding mutations in 360 protein-coding genes (171 tumour and 36 matched normal samples). We selected only patients with lesions presenting the same grade, ER and HER2 status. Mutations were classified as ‘oncogenic’ in case of recurrent substitutions reported in COSMIC or truncating mutations affecting tumour suppressor genes. All mutations identified in a given patient were further interrogated in all samples from that patient through deep re-sequencing using an orthogonal platform. Whole genome rearrangement screen was further conducted in 8/36 patients.Results: Twenty-four patients (67%) had substitutions/indels shared by all their lesions, of which 11 carried the same mutations in all lesions, and 13 had lesions with both common and private mutations. Three-quarters of those 24 patients shared oncogenic variants. The remaining 12 patients (33%) did not share any substitution/indels, with inter-lesion heterogeneity observed for oncogenic mutation(s) in genes such as PIK3CA, TP53, GATA3 and PTEN. Genomically heterogeneous lesions tended to be further apart in the mammary gland than the homogeneous ones. Genome-wide analysis of a limited number of MFBC nevertheless identified a common somatic background in all studied MFBC, including those with no mutation in common between the lesions. Conclusion and perspectives: As the number of molecular targeted therapies increases and trials driven by genomic screening are ongoing, our findings, based on the targeted sequencing of cancer genes in 36 MFBC tumors, highlight the presence of genomic inter-lesion heterogeneity in one-third of the cases despite similar pathological features. This implies that deeper molecular characterization of all MFBC lesions is warranted for the adequate management of those cancers. [CD, DF, and EP contributed equally to this work. PJC and CS contributed equally to this work.] Citation Format: Christine Desmedt, Debora Fumagalli, Elisabetta Pietri, Gabriele Zoppoli, Serena Nik-Zainal, Gunes Gundem, David Brown, Francois Rothe, Samira Majjaj, Anna Garuti, Enrico Carminati, Sherene Loi, Thomas Van Brussel, Marion Maetens, Laura Mudie, Delphine Vincent, Naima Kheddoumi, Luigi Serra, Ilaria Massa, Alberto Ballestrero, Dino Amadori, Roberto Salgado, Alexandre de Wind, Diether Lambrechts, Martine Piccart, Denis Larsimont, Peter J. Campbell, Christos Sotiriou. Uncovering the genomic heterogeneity of multifocal breast cancer. [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 CT135. doi:10.1158/1538-7445.AM2015-CT135

Background: Microarrays have revolutionized breast cancer research by enabling gene expression comparisons on a transcriptome-wide scale. They have provided clinically useful information such as the development of the intrinsic classification as well as several gene prognostic signatures. Recently, with the advent of next generation sequencing technology, RNA-Seq has emerged as a promising technology for digital read-out of the transcriptome. Here, we will present a comparison of microarray and RNA-Seq data on a series of 58 breast cancer samples. Methods: 58 tumors representing the different molecular subtypes [17 triple negative (ER−, PgR−, HER2−); 14 HER2 positive (HER2+); 17 luminal A (ER+, HER2−, histological grade 1), 10 luminal B (ER+, HER2−, histological grade 3)] were obtained from patients recruited between 2007 and 2011 at Jules Bordet Institute. Their RNA was profiled using the Affymetrix HG-U133 Plus 2.0 chips and sequenced with the ILLUMINA platform, producing ∼30 million 2*50 bp paired-end reads per sample. The reads alignment and expression quantification were performed using Tophat 2 and Cufflinks 2, respectively, on the basis of the Ensembl genome annotations hg19. Affymetrix microarray data were normalized using fRMA and probesets were selected using JetSet. A selection of 7 clinically relevant gene signatures was evaluated. Results: About 16000 genes were retained for analysis. Comparison of Affymetrix and RNA-Seq data revealed that 50%, 25% and 5% of these genes had a Spearman correlation superior to 0.7, 0.82 and 0.92, respectively. The genes with the highest correlation coefficients between Affymetrix and RNA-Seq tended to have medium/high expression levels (median Affymetrix log2 expression of 7.5) consistent with the fact that microarrays have a limited dynamic range compared to RNA-Seq. We observed an excellent correlation between Affymetrix and RNA-Seq for the clinically relevant genes ER (0.97), PgR (0.95) and HER2 (0.90) respectively. We observed, however, a few discordances between ER and PgR quantification by immunohistochemistry and RNA-Seq/Affymetrix. We next measured the consistency of two molecular classifiers, SCMGENE (Haibe-Kains JNCI2012) and PAM50 (Parker JCO2009), between Affymetrix arrays and RNA-seq. A kappa coefficient of 0.93 (CI 0.85–1) and 0.79 (CI 0.66–0.91) was observed for SCMGENE and PAM50, respectively, with the majority of the discrepancies for PAM50 concerning the HER2 subtype. The first generation proliferation-based gene expression signatures showed an excellent correlation between Affymetrix and RNA-Seq: Genomic Grade Index (0.98), Mammaprint (0.98) and Oncotype (0.97). The immune and stroma gene signatures (Desmedt et al. 2008) showed a correlation of 0.97 and 0.79 respectively. The lower correlation for the stroma signature could partly be explained by the fact that RNA-Seq and Affymetrix are measuring different splice variants. Conclusions: This is to our knowledge the first study to report a systematic comparison between RNA-Seq and Affymetrix HG-U133 Plus2.0 data in breast cancer for individual genes and several prognostic gene signatures. According to these results, RNA-Seq can reliably be used for the quantification of breast cancer transcriptome. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-04-10.

Background: The aim of this study was to interrogate primary/metastatic breast cancer (BC) genetic heterogeneity using next generation whole exome sequencing and copy number analysis from an autopsy series of BC patients (pts). Methods:To reconstruct the trajectories of BC progression, we performed exome sequencing (Illumina HiSeq2000, Truseq DNA sample preparation kit v2 and Exome Enrichment Kit v2; alignement done with BWA, substitutions and indels called with GATK and DINDEL respectively) coupled to validation by Sequenom and deep re-sequencing (ongoing), as well as copy number (CN) analyses (Oncoscan, Affymetrix) on DNA from matched primary (n=1-6/pt), axillary lymph node (ALN, n=1-6 for 4 pts), local recurrence (for 1 pt) and distant metastatic (n=1-5/pt) FFPE samples obtained from an autopsy series of 10 BC pts. Results:1/ The degree of primary/metastatic genetic heterogeneity is proportional to the time elapsed between the diagnosis of the primary tumor and the emergence of the metastases (mutations: corr=0.76, CN: corr=0.63); metastases from pts with a longer cancer history being genetically more different from their corresponding primary tumor than those from pts with a shorter cancer history. 2/ The phylogenetic analyses revealed that in all but 1 pt, the various distant metastases are present on the same branch of the phylogenetic tree. This suggests that the majority of the metastases arise from a single metastasizing event, with one or more distant sites further re-seeding additional organs. ALN metastases are genetically less evolved compared to distant metastases. 3/ Distant metastases from a same patient only share between 11 and 50% metastasis-specific mutations. This implies that metastasis-specific potentially targetable genetic alterations might either be present in all metastases from a given patient (such as EGFR, PDGFRA amplification for pt 3, AR amplification for pt 8) or only in some metastases (such as NOTCH2 mutation in the liver metastasis of pt 6). 4/ Primary tumor samples and metastases from one ER+/HER2- pt were hypermutated (696 unique mutations in total). The substitutions were enriched for the APOBEC-related mutational signature, possibly linked to the somatic APOBEC3B D316N mutation observed in all samples from this pt. Conclusions: Here we report for the first time that primary/metastatic BC genetic heterogeneity is proportional to the time elapsed between the diagnosis of the primary tumor and the emergence of the metastases. These results therefore suggest that it is extremely relevant to interrogate distant metastatic lesions, multiple if possible to account for inter-metastatic heterogeneity, to guide treatment, especially but not only, in those pts relapsing a few to many years after initial diagnosis. Citation Format: Christine Desmedt, David Brown, Borbala Szekely, Dominiek Smeets, Marcell A. Szasz, Pierre-Yves Adnet, Françoise Rothé, Zsofia I. Nagy, Zsofia Farago, Anna-Maria Tokes, Dimitrios Zardavas, Gabriele Zoppoli, Michail Ignatiadis, Lajos Pusztai, Martine Piccart, Denis Larsimont, Diether Lambrechts, Janina Kulka, Christos Sotiriou. Unraveling breast cancer progression through geographical and temporal sequencing. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 986. doi:10.1158/1538-7445.AM2014-986

522 Background: HER2 overexpression predicts response to anti-HER2 agents. Molecular profiling often suggests that ER+/HER2+ (double positive-DP) BC is a distinct entity from ER-/HER2+ BC. We inves...

In recent years there has been considerable interest in lighting deep within building interiors with guided sunlight. In this paper a new device known as the prism light guide is described and its potential application to daylighting is analyzed. The prism light guide is a hollow acrylic plastic pipe which has optically precise corrugations on the external surface. This shape causes light rays which hit the pipe exterior to reflect back into the pipe, by means of the highly efficient process of total internal reflection. A system has been designed to direct concentrated sunlight into a distribution network based on the prism light guide. An economic analysis of this proposed system is very encouraging. Depending on energy costs and sunlight availability, payback times will range from 3 to 30 years. With mass production of the components, the comparison to all-electric lighting will become even more favourable.