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 × 1010 total nucleated cells ([TNC] range, 0.88 to 3.5 × 1010) 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/μL at day +10 and platelet count of 20,000/μL 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.