title: Non-invasive detection of genomic imbalances in Hodgkin/Reed-Sternberg cells in early and advanced stage Hodgkin's lymphoma by sequencing of circulating cell-free DNA: a technical proof-of-principle study.
abstract: BACKGROUND
  Hodgkin's lymphoma is one of the most common lymphoid neoplasms in young adults, but the low abundance of neoplastic Hodgkin/Reed-Sternberg cells in the tumour hampers the elucidation of its pathogenesis, biology, and diversity. After an incidental observation that genomic aberrations known to occur in Hodgkin's lymphoma were detectable in circulating cell-free DNA, this study was undertaken to investigate whether circulating cell-free DNA can be informative about genomic imbalances in Hodgkin's lymphoma.
  
  
  METHODS
  We applied massive parallel sequencing to circulating cell-free DNA in a prospective study of patients with biopsy proven nodular sclerosis Hodgkin's lymphoma. Genomic imbalances in Hodgkin/Reed-Sternberg cells were investigated by fluorescence in-situ hybridisation (FISH) on tumour specimens.
  
  
  RESULTS
  By non-invasive prenatal testing, we observed several genomic imbalances in circulating cell-free DNA of a pregnant woman, who was subsequently diagnosed with early-stage nodular sclerosis Hodgkin's lymphoma stage IIA during gestation. FISH on tumour tissue confirmed corresponding genomic imbalances in Hodgkin/Reed-Sternberg cells. We prospectively studied circulating cell-free DNA of nine nodular sclerosis Hodgkin's lymphoma cases: eight at first diagnosis and one at first relapse. Seven patients had stage IIA disease and two had stage IVB disease. In eight, genomic imbalances were detected, including, among others, gain of chromosomes 2p and 9p, known to occur in Hodgkin's lymphoma. These gains and losses in circulating cell-free DNA were extensively validated by FISH on Hodgkin/Reed-Sternberg cells in biopsy samples. Initiation of chemotherapy induced normalisation of circulating cell-free DNA profiles within 2-6 weeks. The cell cycle indicator Ki67 and cleaved caspase-3 were detected in Hodgkin/Reed-Sternberg cells by immunohistochemistry, suggesting high turnover of Hodgkin/Reed-Sternberg cells.
  
  
  CONCLUSIONS
  In early and advanced stage nodular sclerosis Hodgkin's lymphoma, genomic imbalances in Hodgkin/Reed-Sternberg cells can be identified by massive parallel sequencing of circulating cell-free DNA at diagnosis. The rapid normalisation of circulating cell-free DNA profiles on therapy initiation suggests a potential role for circulating cell-free DNA profiling in early response monitoring. This finding creates several new possibilities for exploring the diversity of Hodgkin's lymphoma, and has potential implications for the future clinical development of biomarkers and precision therapy for this malignancy.
  
  
  BACKGROUND
  KU Leuven-University of Leuven and University Hospitals Leuven.
authors.affiliation: Department of Human Genetics, Center for Human Genetics, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium; Department of Haematology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium. Electronic address: peter.vandenberghe@uzleuven.be.
authors.forename: Peter
authors.initials: P
authors.lastname: Vandenberghe
authors.affiliation: Department of Human Genetics, Center for Human Genetics, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Iwona
authors.initials: I
authors.lastname: Wlodarska
authors.affiliation: Department of Pathology, Translational Cell and Tissue Research, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Thomas
authors.initials: T
authors.lastname: Tousseyn
authors.affiliation: Department of Human Genetics, Center for Human Genetics, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Luc
authors.initials: L
authors.lastname: Dehaspe
authors.affiliation: Department of Haematology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Daan
authors.initials: D
authors.lastname: Dierickx
authors.affiliation: Department of Obstetrics and Gynaecology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Magali
authors.initials: M
authors.lastname: Verheecke
authors.affiliation: Department of Paediatrics, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Anne
authors.initials: A
authors.lastname: Uyttebroeck
authors.affiliation: Department of General Medical Oncology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Oliver
authors.initials: O
authors.lastname: Bechter
authors.affiliation: Department of Haematology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Michel
authors.initials: M
authors.lastname: Delforge
authors.affiliation: Department of Radiology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Vincent
authors.initials: V
authors.lastname: Vandecaveye
authors.affiliation: Department of Human Genetics, Center for Human Genetics, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Brison
authors.affiliation: Department of Haematology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Gregor E G
authors.initials: GE
authors.lastname: Verhoef
authors.affiliation: Department of Human Genetics, Center for Human Genetics, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Eric
authors.initials: E
authors.lastname: Legius
authors.affiliation: Department of Obstetrics and Gynaecology, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Frederic
authors.initials: F
authors.lastname: Amant
authors.affiliation: Department of Human Genetics, Center for Human Genetics, KU Leuven - University of Leuven, University Hospitals Leuven, Leuven, Belgium.
authors.forename: Joris R
authors.initials: JR
authors.lastname: Vermeesch
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D002648:Child; D002869:Chromosome Aberrations; D055028:Comparative Genomic Hybridization; D004247:DNA / Q000097:blood*; D005260:Female; D023281:Genomics; D006689:Hodgkin Disease / Q000235:genetics*; D006801:Humans; D008297:Male; D008875:Middle Aged; D011247:Pregnancy; D011446:Prospective Studies; D016539:Reed-Sternberg Cells / Q000473:pathology*; D017422:Sequence Analysis, DNA; D055815:Young Adult
chemical_list: D004247:DNA