title: Isolation by size of epithelial tumor cells in peripheral blood of patients with breast cancer: correlation with real-time reverse transcriptase-polymerase chain reaction results and feasibility of molecular analysis by laser microdissection.
abstract: The aim of this study is the counting and the immunomorphological and molecular characterization of circulating tumor cells (CTCs) by the isolation by size of epithelial tumor cells (ISET) method in the peripheral blood of patients with breast cancer. An evaluation of the method's ability to reveal the presence of occult carcinoma cells in blood of a patient with breast cancer was performed and the results compared with those obtained by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay for the evaluation of cytokeratin-19 (CK-19) mRNA expression. The feasibility of molecular analysis of CTCs after laser microdissection of filters used in ISET was illustrated, referring to HER-2 amplification. Blood samples drawn from 44 patients with breast cancer were preoperatively analyzed by ISET. From the same samples, total RNA was extracted and submitted to quantitative real-time RT-PCR for the detection of CK-19 mRNA-positive cells using TaqMan technology. HER-2 amplification was measured by real-time RT-PCR on DNA extracted from cells recovered by laser microdissection from 7 selected ISET-positive filters. Of 44 samples, 12 (27%) showed the presence of epithelial cells on the filter (mean +/- SE: 8.5 +/- 2.4 cells per milliliter of blood). A statistically significant agreement (P = .001) was observed between real-time RT-PCR results and those obtained by ISET. With regard to HER-2 amplification, a good correspondence was found between the results obtained from microdissected CTCs and those obtained using DNA extracted from the primary tumor (R = 0.918; P < .01), as well as the immunohistochemistry results. The ISET method allows for the collection of breast carcinoma cells by filtration despite their smaller dimension relative to other carcinoma cell types. The sensitivity and specificity of the method is comparable with those obtained using the quantitative real-time RT-PCR assay for the evaluation of CK-19 mRNA expression. Moreover, the laser microdissection technique allows for the recovery of nucleic acids for further molecular analysis and CTC characterization.
authors.affiliation: Clinical Biochemistry Unit, Department of Clinical Physiopathology, University of Florence, 50100 Florence, Italy. p.pinzani@dfc.unifi.it
authors.forename: Pamela
authors.initials: P
authors.lastname: Pinzani
authors.forename: Benedetta
authors.initials: B
authors.lastname: Salvadori
authors.forename: Lisa
authors.initials: L
authors.lastname: Simi
authors.forename: Simonetta
authors.initials: S
authors.lastname: Bianchi
authors.forename: Vito
authors.initials: V
authors.lastname: Distante
authors.forename: Luigi
authors.initials: L
authors.lastname: Cataliotti
authors.forename: Mario
authors.initials: M
authors.lastname: Pazzagli
authors.forename: Claudio
authors.initials: C
authors.lastname: Orlando
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001943:Breast Neoplasms / Q000097:blood* / Q000235:genetics / Q000473:pathology; D045744:Cell Line, Tumor; D048429:Cell Size; D004847:Epithelial Cells / Q000473:pathology; D005240:Feasibility Studies; D005260:Female; D018734:Genes, erbB-2; D006801:Humans; D007633:Keratins / Q000032:analysis; D007834:Lasers; D042282:Microdissection; D008875:Middle Aged; D025202:Molecular Diagnostic Techniques; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D012333:RNA, Messenger; D012334:RNA, Neoplasm; D007633:Keratins