Abstract 986: Unraveling breast cancer progression through geographical and temporal sequencing
Authors: Desmedt Christine , Brown David , Szekely Borbala , Smeets Dominiek , Szasz Marcell A. , Adnet Pierre-Yves , Rothé Françoise , Nagy Zsofia I. , Farago Zsofia , Tokes Anna-Maria , Zardavas Dimitrios , Zoppoli Gabriele , Ignatiadis Michail , Pusztai Lajos , Piccart Martine , Larsimont Denis , Lambrechts Diether , Kulka Janina , Sotiriou Christos 
Published: May 2015

Abstract
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