title: Enrichment and Molecular Analysis of Breast Cancer Disseminated Tumor Cells from Bone Marrow Using Microfiltration.
abstract: OBJECTIVE
  Molecular characterization of disseminated tumor cells (DTCs) in the bone marrow (BM) of breast cancer (BC) patients has been hindered by their rarity. To enrich for these cells using an antigen-independent methodology, we have evaluated a size-based microfiltration device in combination with several downstream biomarker assays.
  
  
  METHODS
  BM aspirates were collected from healthy volunteers or BC patients. Healthy BM was mixed with a specified number of BC cells to calculate recovery and fold enrichment by microfiltration. Specimens were pre-filtered using a 70 μm mesh sieve and the effluent filtered through CellSieve microfilters. Captured cells were analyzed by immunocytochemistry (ICC), FISH for HER-2/neu gene amplification status, and RNA in situ hybridization (RISH). Cells eluted from the filter were used for RNA isolation and subsequent qRT-PCR analysis for DTC biomarker gene expression.
  
  
  RESULTS
  Filtering an average of 14×106 nucleated BM cells yielded approximately 17-21×103 residual BM cells. In the BC cell spiking experiments, an average of 87% (range 84-92%) of tumor cells were recovered with approximately 170- to 400-fold enrichment. Captured BC cells from patients co-stained for cytokeratin and EpCAM, but not CD45 by ICC. RNA yields from 4 ml of patient BM after filtration averaged 135ng per 10 million BM cells filtered with an average RNA Integrity Number (RIN) of 5.3. DTC-associated gene expression was detected by both qRT-PCR and RISH in filtered spiked or BC patient specimens but, not in control filtered normal BM.
  
  
  CONCLUSIONS
  We have tested a microfiltration technique for enrichment of BM DTCs. DTC capture efficiency was shown to range from 84.3% to 92.1% with up to 400-fold enrichment using model BC cell lines. In patients, recovered DTCs can be identified and distinguished from normal BM cells using multiple antibody-, DNA-, and RNA-based biomarker assays.
authors.affiliation: Washington University School of Medicine, Dept. of Surgery, St. Louis, Missouri, United States of America.
authors.forename: Sreeraj G
authors.identifier: http://orcid.org/0000-0002-0285-0813
authors.initials: SG
authors.lastname: Pillai
authors.affiliation: Creatv MicroTech, Inc., Rockville, Maryland, United States of America.
authors.forename: Peixuan
authors.initials: P
authors.lastname: Zhu
authors.affiliation: Washington University School of Medicine, Dept. of Surgery, St. Louis, Missouri, United States of America.
authors.forename: Chidananda M
authors.initials: CM
authors.lastname: Siddappa
authors.affiliation: Creatv MicroTech, Inc., Monmouth Junction, New Jersey, United States of America.
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Adams
authors.affiliation: Creatv MicroTech, Inc., Rockville, Maryland, United States of America.
authors.forename: Shuhong
authors.initials: S
authors.lastname: Li
authors.affiliation: Creatv MicroTech, Inc., Chicago, Illinois, United States of America.
authors.forename: Olga V
authors.initials: OV
authors.lastname: Makarova
authors.affiliation: Creatv MicroTech, Inc., Potomac, Maryland, United States of America.
authors.forename: Pete
authors.initials: P
authors.lastname: Amstutz
authors.affiliation: Washington University School of Medicine, Dept. of Orthopedic Surgery, St. Louis, Missouri, United States of America.
authors.forename: Ryan
authors.initials: R
authors.lastname: Nunley
authors.affiliation: Creatv MicroTech, Inc., Potomac, Maryland, United States of America.
authors.forename: Cha-Mei
authors.initials: CM
authors.lastname: Tang
authors.affiliation: Washington University School of Medicine, Dept. of Pathology and Immunology, St. Louis, Missouri, United States of America.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Watson
authors.affiliation: Washington University School of Medicine, Dept. of Surgery, St. Louis, Missouri, United States of America.|John Cochran Veterans Administration Hospital, St. Louis, Missouri, United States of America.
authors.forename: Rebecca L
authors.initials: RL
authors.lastname: Aft
mesh_terms: D000328:Adult; D000368:Aged; D001854:Bone Marrow Cells / Q000378:metabolism / Q000473:pathology; D001943:Breast Neoplasms / Q000235:genetics / Q000473:pathology*; D002469:Cell Separation / Q000379:methods*; D048429:Cell Size; D005260:Female; D005374:Filtration / Q000379:methods; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D017403:In Situ Hybridization; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics*; D009360:Neoplastic Cells, Circulating / Q000378:metabolism / Q000473:pathology*; D012333:RNA, Messenger / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics
chemical_list: D009363:Neoplasm Proteins; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human