title: Detection of micrometastatic breast cancer by means of real time quantitative RT-PCR and immunostaining in perioperative blood samples and sentinel nodes.
abstract: The aim of our study was to detect micrometastatic breast cancer by epithelial glycoprotein-2 (EGP-2) and cytokeratin 19 (CK19), using immunostaining and real time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Fifty-eight breast cancer patients, 52 primary tumors, 75 sentinel nodes (SN) and 149 peripheral blood (PB) samples (from before, during and 4 days after operation) were examined. Immunostaining was performed with antibodies directed against EGP-2 and CK19. Detection limits were one Michigan Cancer Foundation-7 (MCF-7) breast cancer cell line cell/2.10(6) leukocytes (immunostaining) and one MCF-7 cell/10(6) leukocytes qRT-PCR. Control noncancer lymph nodes (n = 10) showed nonspecific CK19 staining, but were qRT-PCR negative; control healthy volunteer PB (n = 11) was always negative. Primary tumor samples, all positive with immunostaining, showed a wide variation of EGP-2 (>10(4) fold) and CK19 mRNA expression (>10(3) fold). SN (n = 19) from 16 patients were tumor-positive with routine haematoxylin-eosin (H&E) and/or immunostaining. SN tumor presence was positively correlated to qRT-PCR expression, but 3 tumor-positive SN were false negative with qRT-PCR. Three SN were qRT-PCR positive, while tumor negative with H&E and/or immunostaining. No immunostaining positive PB was observed, but 19 patients (33%) had one or more qRT-PCR positive PB samples. We concluded that primary tumors have varying expressions of EGP-2 and CK19 mRNA. Both markers can be used in qRT-PCR to obtain adequate sensitivity for single tumor cell detection. In SN, immunostaining appears more sensitive/specific than H&E or qRT-PCR for tumor detection. No immunostaining positivity was found in PB, while 33% of patients had qRT-PCR positive PB. The clinical value of these findings will have to be clarified.
authors.affiliation: Department of Medical Oncology, University Hospital Groningen, The Netherlands.
authors.forename: Carolien P
authors.initials: CP
authors.lastname: Schröder
authors.affiliation: Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands.
authors.forename: Marcel H J
authors.initials: MHJ
authors.lastname: Ruiters
authors.affiliation: Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands.
authors.forename: Steven
authors.initials: S
authors.lastname: de Jong
authors.affiliation: Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands.
authors.forename: Anton T M G
authors.initials: ATMG
authors.lastname: Tiebosch
authors.affiliation: Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands.
authors.forename: Jelle
authors.initials: J
authors.lastname: Wesseling
authors.affiliation: Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands.
authors.forename: Rense
authors.initials: R
authors.lastname: Veenstra
authors.affiliation: Department of Surgical Oncology, University Hospital Groningen, The Netherlands.
authors.forename: Jaap
authors.initials: J
authors.lastname: de Vries
authors.affiliation: Department of Surgical Oncology, University Hospital Groningen, The Netherlands.
authors.forename: Harold J
authors.initials: HJ
authors.lastname: Hoekstra
authors.affiliation: Department of Pathology and Laboratory Medicine, University Hospital Groningen, The Netherlands.
authors.forename: Lou F M H
authors.initials: LFMH
authors.lastname: de Leij
authors.affiliation: Department of Medical Oncology, University Hospital Groningen, The Netherlands.
authors.forename: Elisabeth G E
authors.initials: EGE
authors.lastname: de Vries
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000951:Antigens, Neoplasm / Q000235:genetics* / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000378:metabolism; D001943:Breast Neoplasms / Q000097:blood / Q000175:diagnosis* / Q000473:pathology; D018270:Carcinoma, Ductal, Breast / Q000097:blood / Q000175:diagnosis / Q000556:secondary; D002285:Carcinoma, Intraductal, Noninfiltrating / Q000097:blood / Q000175:diagnosis / Q000556:secondary; D018275:Carcinoma, Lobular / Q000097:blood / Q000175:diagnosis / Q000556:secondary; D016022:Case-Control Studies; D015815:Cell Adhesion Molecules / Q000235:genetics* / Q000378:metabolism; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D000071858:Epithelial Cell Adhesion Molecule; D005260:Female; D006801:Humans; D007633:Keratins / Q000235:genetics* / Q000378:metabolism; D008207:Lymphatic Metastasis; D008875:Middle Aged; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D012333:RNA, Messenger / Q000032:analysis; D012334:RNA, Neoplasm / Q000097:blood / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D021701:Sentinel Lymph Node Biopsy
chemical_list: D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015815:Cell Adhesion Molecules; D017931:DNA Primers; D000071858:Epithelial Cell Adhesion Molecule; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D007633:Keratins