title: Proper genomic profiling of (BRCA1-mutated) basal-like breast carcinomas requires prior removal of tumor infiltrating lymphocytes.
abstract: BACKGROUND
  BRCA1-mutated breast carcinomas may have distinct biological features, suggesting the involvement of specific oncogenic pathways in tumor development. The identification of genomic aberrations characteristic for BRCA1-mutated breast carcinomas could lead to a better understanding of BRCA1-associated oncogenic events and could prove valuable in clinical testing for BRCA1-involvement in patients.
  
  
  METHODS
  For this purpose, genomic and gene expression profiles of basal-like BRCA1-mutated breast tumors (n = 27) were compared with basal-like familial BRCAX (non-BRCA1/2/CHEK2*1100delC) tumors (n = 14) in a familial cohort of 120 breast carcinomas.
  
  
  RESULTS
  Genome wide copy number profiles of the BRCA1-mutated breast carcinomas in our data appeared heterogeneous. Gene expression analyses identified varying amounts of tumor infiltrating lymphocytes (TILs) as a major cause for this heterogeneity. Indeed, selecting tumors with relative low amounts of TILs, resulted in the identification of three known but also five previously unrecognized BRCA1-associated copy number aberrations. Moreover, these aberrations occurred with high frequencies in the BRCA1-mutated tumor samples. Using these regions it was possible to discriminate BRCA1-mutated from BRCAX breast carcinomas, and they were validated in two independent cohorts. To further substantiate our findings, we used flow cytometry to isolate cancer cells from formalin-fixed, paraffin-embedded, BRCA1-mutated triple negative breast carcinomas with estimated TIL percentages of 40% and higher. Genomic profiles of sorted and unsorted fractions were compared by shallow whole genome sequencing and confirm our findings.
  
  
  CONCLUSIONS
  This study shows that genomic profiling of in particular basal-like, and thus BRCA1-mutated, breast carcinomas is severely affected by the presence of high numbers of TILs. Previous reports on genomic profiling of BRCA1-mutated breast carcinomas have largely neglected this. Therefore, our findings have direct consequences on the interpretation of published genomic data. Also, these findings could prove valuable in light of currently used genomic tools for assessing BRCA1-involvement in breast cancer patients and pathogenicity assessment of BRCA1 variants of unknown significance. The BRCA1-associated genomic aberrations identified in this study provide possible leads to a better understanding of BRCA1-associated oncogenesis.
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: m.massink@vumc.nl.
authors.forename: Maarten P G
authors.initials: MP
authors.lastname: Massink
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: ei.kooi@vumc.nl.
authors.forename: Irsan E
authors.initials: IE
authors.lastname: Kooi
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: se.vanmil@vumc.nl.
authors.forename: Saskia E
authors.initials: SE
authors.lastname: van Mil
authors.affiliation: Department of Obstetrics and Gynaecology, Center for Gynaecologic Oncology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: e.jordanova@vumc.nl.
authors.forename: Ekaterina S
authors.initials: ES
authors.lastname: Jordanova
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: n.ameziane@vumc.nl.
authors.forename: Najim
authors.initials: N
authors.lastname: Ameziane
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: jc.dorsman@vumc.nl.
authors.forename: Josephine C
authors.initials: JC
authors.lastname: Dorsman
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: d.vanbeek@vumc.nl.
authors.forename: Daphne M
authors.initials: DM
authors.lastname: van Beek
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: jp.vandervoorn@vumc.nl.
authors.forename: J Patrick
authors.initials: JP
authors.lastname: van der Voorn
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: d.sie@vumc.nl.
authors.forename: Daoud
authors.initials: D
authors.lastname: Sie
authors.affiliation: Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: b.ylstra@vumc.nl.
authors.forename: Bauke
authors.initials: B
authors.lastname: Ylstra
authors.affiliation: Department of Pathology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: c.h.m.vandeurzen@erasmusmc.nl.
authors.forename: Carolien H M
authors.initials: CH
authors.lastname: van Deurzen
authors.affiliation: Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: j.martens@erasmusmc.nl.
authors.forename: John W
authors.initials: JW
authors.lastname: Martens
authors.affiliation: Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: m.smid@erasmusmc.nl.
authors.forename: Marcel
authors.initials: M
authors.lastname: Smid
authors.affiliation: Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: a.sieuwerts@erasmusmc.nl.
authors.forename: Anieta M
authors.initials: AM
authors.lastname: Sieuwerts
authors.affiliation: Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: v.deweerd@erasmusmc.nl.
authors.forename: Vanja
authors.initials: V
authors.lastname: de Weerd
authors.affiliation: Department of Medical Oncology, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: j.foekens@erasmusmc.nl.
authors.forename: John A
authors.initials: JA
authors.lastname: Foekens
authors.affiliation: Department of Clinical Genetics, Erasmus MC Cancer Institute, Erasmus University Medical Center, Rotterdam, The Netherlands. Electronic address: a.vandenouweland@erasmusmc.nl.
authors.forename: Ans M W
authors.initials: AM
authors.lastname: van den Ouweland
authors.affiliation: Department of Pathology, Netherlands Cancer Institute, Amsterdam, The Netherlands. Electronic address: e.v.dijk@nki.nl.
authors.forename: Ewald
authors.initials: E
authors.lastname: van Dyk
authors.affiliation: Department of Molecular Carcinogenesis, Netherlands Cancer Institute, Amsterdam, The Netherlands. Electronic address: p.nederlof@nki.nl.
authors.forename: Petra M
authors.initials: PM
authors.lastname: Nederlof
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: q.waisfisz@vumc.nl.
authors.forename: Quinten
authors.initials: Q
authors.lastname: Waisfisz
authors.affiliation: Department of Clinical Genetics, VU University Medical Center, Amsterdam, The Netherlands. Electronic address: h.meijers-heijboer@vumc.nl.
authors.forename: Hanne
authors.initials: H
authors.lastname: Meijers-Heijboer
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics* / Q000276:immunology*; D016000:Cluster Analysis; D056915:DNA Copy Number Variations / Q000235:genetics; D005260:Female; D005434:Flow Cytometry; D018628:Gene Dosage; D015972:Gene Expression Regulation, Neoplastic; D023281:Genomics; D006801:Humans; D016246:Lymphocytes, Tumor-Infiltrating / Q000276:immunology*; D009154:Mutation / Q000235:genetics*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D019313:BRCA1 Protein
keywords: BRCA1; Copy number; FACS analysis; Gene expression; Genomic profiling; Tumor infiltrating lymphocytes