title: Capturing intra-tumor genetic heterogeneity by de novo mutation profiling of circulating cell-free tumor DNA: a proof-of-principle.
abstract: Plasma-derived cell-free tumor DNA (ctDNA) constitutes a potential surrogate for tumor DNA obtained from tissue biopsies. We posit that massively parallel sequencing (MPS) analysis of ctDNA may help define the repertoire of mutations in breast cancer and monitor tumor somatic alterations during the course of targeted therapy.
  
  
  
  A 66-year-old patient presented with synchronous estrogen receptor-positive/HER2-negative, highly proliferative, grade 2, mixed invasive ductal-lobular carcinoma with bone and liver metastases at diagnosis. DNA extracted from archival tumor material, plasma and peripheral blood leukocytes was subjected to targeted MPS using a platform comprising 300 cancer genes known to harbor actionable mutations. Multiple plasma samples were collected during the fourth line of treatment with an AKT inhibitor.
  
  
  
  Average read depths of 287x were obtained from the archival primary tumor, 139x from the liver metastasis and between 200x and 900x from ctDNA samples. Sixteen somatic non-synonymous mutations were detected in the liver metastasis, of which 9 (CDKN2A, AKT1, TP53, JAK3, TSC1, NF1, CDH1, MML3 and CTNNB1) were also detected in >5% of the alleles found in the primary tumor sample. Not all mutations identified in the metastasis were reliably identified in the primary tumor (e.g. FLT4). Analysis of ctDNA, nevertheless, captured all mutations present in the primary tumor and/or liver metastasis. In the longitudinal monitoring of the patient, the mutant allele fractions identified in ctDNA samples varied over time and mirrored the pharmacodynamic response to the targeted therapy as assessed by positron emission tomography-computed tomography.
  
  
  
  This proof-of-principle study is one of the first to demonstrate that high-depth targeted MPS of plasma-derived ctDNA constitutes a potential tool for de novo mutation identification and monitoring of somatic genetic alterations during the course of targeted therapy, and may be employed to overcome the challenges posed by intra-tumor genetic heterogeneity.
  
  
  
  www.clinicaltrials.gov, NCT01090960.
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona; Universitat Autònoma de Barcelona, Barcelona, Spain; Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: L
authors.initials: L
authors.lastname: De Mattos-Arruda
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: B
authors.initials: B
authors.lastname: Weigelt
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona.
authors.forename: J
authors.initials: J
authors.lastname: Cortes
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: H H
authors.initials: HH
authors.lastname: Won
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: C K Y
authors.initials: CKY
authors.lastname: Ng
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona.
authors.forename: P
authors.initials: P
authors.lastname: Nuciforo
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA; Department of Medical Oncology, Institut Curie, Paris, France.
authors.forename: F-C
authors.initials: FC
authors.lastname: Bidard
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona.
authors.forename: C
authors.initials: C
authors.lastname: Aura
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona.
authors.forename: C
authors.initials: C
authors.lastname: Saura
authors.affiliation: Department of Pathology, Vall d'Hebron University Hospital, Barcelona, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Peg
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: S
authors.initials: S
authors.lastname: Piscuoglio
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona.
authors.forename: M
authors.initials: M
authors.lastname: Oliveira
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Smolders
authors.affiliation: Genentech, Inc., San Francisco.
authors.forename: P
authors.initials: P
authors.lastname: Patel
authors.affiliation: Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA.
authors.forename: L
authors.initials: L
authors.lastname: Norton
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona; Universitat Autònoma de Barcelona, Barcelona, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Tabernero
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA.
authors.forename: M F
authors.initials: MF
authors.lastname: Berger
authors.affiliation: Vall d'Hebron Institute of Oncology, Vall d'Hebron University Hospital, Barcelona; Universitat Autònoma de Barcelona, Barcelona, Spain; Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Seoane
authors.affiliation: Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, USA. Electronic address: reisfilj@mskcc.org.
authors.forename: J S
authors.initials: JS
authors.lastname: Reis-Filho
mesh_terms: D001483:Base Sequence; D014408:Biomarkers, Tumor / Q000235:genetics; D001859:Bone Neoplasms / Q000235:genetics / Q000556:secondary; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000235:genetics*; D002474:Cell-Free System; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics*; D005260:Female; D018740:Genetic Heterogeneity; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000556:secondary; D008875:Middle Aged; D051057:Proto-Oncogene Proteins c-akt / Q000037:antagonists & inhibitors; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism; D017422:Sequence Analysis, DNA
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D051057:Proto-Oncogene Proteins c-akt; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; C508053:ERBB2 protein, human
keywords: breast cancer; cell-free tumor DNA; disease monitoring; intra-tumor heterogeneity; massively parallel sequencing