title: CD34 selections from myeloma peripheral blood cell autografts contain residual tumour cells due to impurity, not to CD34+ myeloma cells.
abstract: Malignant cells in haemopoietic autografts can contribute to post-transplant relapse. Engraftment of myeloma patients with CD34+ peripheral blood progenitors selected from total autografts reduces the number of tumour cells infused by 2.7-4.5 logs. Residual tumour cells detected in CD34+ selected cells may be due to selection impurity or the existence of malignant CD34+ progenitors. In three patients we evaluated the CD34 purity and tumour load of total autografts, CD34+ progenitors selected with immunomagnetic beads and highly purified CD34+ progenitors obtained in two rounds of selection (combining magnetic with flow cytometry activated cell sorting) to determine the cause of residual tumour cells in CD34 selections. Using allele-specific oligonucleotides (ASO) complementary to the unique Ig heavy chain sequence (CDRIII region) of the malignant clone, semi-quantitative ASO-PCR was capable of detecting one malignant cell in 10(4)-10(5) normal white blood cells. Selection of CD34+ cells from bone marrow (BM) with approximately 20% malignant plasma cells resulted in a 1.4 log reduction of tumour burden. Using two-colour flow-cytometry we observed CD34-, BB4+ malignant plasma cells contaminating this CD34 selection. Prior to sorting, peripheral blood cell autografts (PBCA) contained approximately 0.1% malignant cells. Selection of > 99% pure CD34+ cells using immunomagnetic beads (Dynal) resulted in an approximate 2 log reduction of malignant cells, but residual tumour cells were still detectable. ASO-PCR detected no malignant cells in > 99.9% pure CD34+ peripheral blood progenitors obtained with two rounds of selection (combining magnetic with flow cytometry activated cell sorting). We conclude that CD34+ malignant cells are not detectable in myeloma PBCA and that residual tumour cells in CD34 selections are due to contaminating CD34-negative cells.
authors.affiliation: Department of Internal Medicine, University Hospital Nijmegen, The Netherlands.
authors.forename: P
authors.initials: P
authors.lastname: Willems
authors.forename: A
authors.initials: A
authors.lastname: Croockewit
authors.forename: R
authors.initials: R
authors.lastname: Raymakers
authors.forename: R
authors.initials: R
authors.lastname: Holdrinet
authors.forename: G
authors.initials: G
authors.lastname: van Der Bosch
authors.forename: E
authors.initials: E
authors.lastname: Huys
authors.forename: E
authors.initials: E
authors.lastname: Mensink
mesh_terms: D018952:Antigens, CD34 / Q000032:analysis; D001483:Base Sequence; D001853:Bone Marrow / Q000473:pathology; D005434:Flow Cytometry; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000473:pathology*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D018189:Immunomagnetic Separation; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000097:blood* / Q000473:pathology; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D018952:Antigens, CD34; D007143:Immunoglobulin Heavy Chains