title: Monitoring of tumor cell purging after highly efficient immunomagnetic selection of CD34 cells from leukapheresis products in breast cancer patients: comparison of immunocytochemical tumor cell staining and reverse transcriptase-polymerase chain reaction.
abstract: We studied the efficiency of indirect tumor cell purging via enrichment of CD34+ hematopoietic progenitor cells from leukapheresis products (LP) in breast cancer patients based on immunomagnetic selection of CD34+ cells. Detection of tumor cells was made by immunocytochemical staining. In addition, we evaluated the capacity of cytokeratin 19 (CK19)- and a novel epidermal growth factor receptor (EGF-R)-specific reverse transcriptase-polymerase chain reaction (RT-PCR) for monitoring tumor cell depletion. LP from 13 breast cancer patients were analyzed. Twenty-three CD34 selection procedures were performed. A median of 1.4 x 10(10) total nucleated cells ([TNC] range, 0.88 to 3.5 x 10(10)) with a median CD34 purity of 2.5% (range, 0.4% to 6.3%) were entered into the selection procedure. Immunomagnetic CD34 enrichment resulted in a median purity of 83.3% (range, 45% to 95.4%) and a median recovery of 73.2% (range, 22% to 95%). Retransfusion of CD34-selected cells after high-dose chemotherapy resulted in a rapid and sustained hematologic recovery, reaching an absolute neutrophil count of 500/microL at day +10 and platelet count of 20,000/microL at day +11. Tumor cell depletion was quantified by immunocytochemical detection of CK19-positive cells. By this method, a median tumor cell depletion of 1.9 log (range, 0.7 to > 3 log) could be demonstrated. Immunocytochemical detection of tumor cells was more sensitive than RT-PCR, yielding positive results in 81% of LP (17 to 21) versus 58% positive LP (10 of 17). However, EGF-R-based RT-PCR was much more sensitive than CK19-based RT-PCR (10 of 17 v 1 of 17). Despite highly efficient CD34 selection, tumor cells were still detectable after CD34 enrichment using immunocytochemistry and EGF-R-specific RT-PCR. Thus, this novel EGF-R-specific RT-PCR appears to be of value as an additional method to detect contaminating breast cancer cells within LP.
authors.affiliation: Department of Internal Medicine (Medical Oncology and Tumorimmunology), Humboldt University, Virchow Klinikum-Robert Rössle Klinik, Berlin, Germany.
authors.forename: M Y
authors.initials: MY
authors.lastname: Mapara
authors.forename: I J
authors.initials: IJ
authors.lastname: Körner
authors.forename: M
authors.initials: M
authors.lastname: Hildebrandt
authors.forename: R
authors.initials: R
authors.lastname: Bargou
authors.forename: D
authors.initials: D
authors.lastname: Krahl
authors.forename: P
authors.initials: P
authors.lastname: Reichardt
authors.forename: B
authors.initials: B
authors.lastname: Dörken
mesh_terms: D000328:Adult; D018952:Antigens, CD34 / Q000032:analysis*; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000097:blood* / Q000473:pathology; D002945:Cisplatin / Q000008:administration & dosage; D018076:DNA, Complementary / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis; D015251:Epirubicin / Q000008:administration & dosage; D066246:ErbB Receptors / Q000032:analysis*; D005047:Etoposide / Q000008:administration & dosage; D005069:Evaluation Studies as Topic; D005260:Female; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D018380:Hematopoietic Stem Cell Transplantation; D006801:Humans; D007069:Ifosfamide / Q000008:administration & dosage; D007124:Immunoenzyme Techniques; D018189:Immunomagnetic Separation; D007633:Keratins / Q000032:analysis*; D007937:Leukapheresis; D008558:Melphalan / Q000008:administration & dosage; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000032:analysis*; D009360:Neoplastic Cells, Circulating; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D019172:Transplantation Conditioning
chemical_list: D018952:Antigens, CD34; D014408:Biomarkers, Tumor; D018076:DNA, Complementary; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D016179:Granulocyte Colony-Stimulating Factor; D015251:Epirubicin; D007633:Keratins; D005047:Etoposide; D066246:ErbB Receptors; D002945:Cisplatin; D008558:Melphalan; D007069:Ifosfamide