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Bone Marrow Transplantation, (1997) 19, 553–556 1997 Stockton Press All rights reserved 0268–3369/97 $12.00 Expression of the hematopoietic stem cell antigen CD34 on blood and bone marrow monoclonal plasma cells from patients with multiple myeloma T Kimlinger1 and TE Witzig2 1Department of Laboratory Medicine and 2Division of Internal Medicine and Hematology, Mayo Clinic and Mayo Foundation, Rochester, MN, USA Summary: led to strategies to deplete the tumor cells from the harvest product prior to reinfusion of the stem cells. Monoclonal plasma cells (CD38+CD45−/dim) are typi- One of the current attempts at purifying the harvest pro- cally present in the blood of patients with active mye- duct uses antibody to the CD34 antigen to positively select loma and can contaminate stem cell harvests. This has and enrich hematopoietic stem cells and in the process led to strategies that select CD34+ cells for use in auto- purge the stem cell product of tumor cells and T cells.11–13 logous stem cell transplantation with the goal of The CD34 antigen identifies a lymphohematopoietic stem decreasing tumor cell contamination. The aim of this cell, is present on 1–5% of adult bone marrow cells, and study was to learn if the CD34 antigen is expressed on is expressed on early B cells. The characteristics of this monoclonal plasma cells in the blood or marrow of important antigen and its clinical relevance have recently patients with multiple myeloma. We used three-color been reviewed.14 CD34+ hematopoietic cells from blood or flow cytometry (surface CD38;CD45 and cytoplasmic marrow can reconstitute hematopoiesis after high-dose kappa or lambda) to identify monoclonal plasma cells therapy programs.15 The number of CD34+ cells reinfused in the blood (n = 24) and marrow (n = 37) from patients predicts the time to engraftment.16,17 with plasma cell proliferative disorders. In each case the If stem cells are to be selected on the basis of their CD38+CD45− and CD38+CD45dim+ monoclonal popu- expression of CD34 for use in transplants for multiple lations were then analyzed for CD34 expression. In all myeloma, it is important to demonstrate that the CD34 anti- 24 blood and 37 marrow samples, the CD38+CD45− gen is not on the myeloma cells. There are two methods to monoclonal plasma cells were negative for CD34 approach this question. The first involves isolating cells expression. CD38+CD45dim+ monoclonal cells were docu- with CD34 antibody followed by analysis of this cell popu- mented in the blood of 11 patients and in the marrow lation for myeloma cells by use of molecular tech- of 33 patients and this cell population was also CD34- niques.9,13,18,19 The aim of this study was to address the negative in all cases. These results indicate that CD34 question with a second approach that identified the known is usually not expressed on the CD38+CD45−CD45dim+ myeloma cells by three-color flow cytometry to learn if the monoclonal plasma cells in the blood or marrow of CD38+CD45−CD45dim+ monoclonal plasma cell population patients with plasma cell proliferative disorders. CD34 in the blood or marrow from patients with plasma cell pro- selection methods should therefore decrease the chance liferative diseases expressed the CD34 antigen. of tumor cell contamination of the stem cell product. Keywords: CD34; multiple myeloma; stem cell trans- plants Materials and methods Patient selection Multiple myeloma is a malignancy of monoclonal plasma All patients had the diagnosis of a plasma cell proliferative cells that are found in highest number in the bone marrow. disorder and were seen in the Dysproteinemia Clinic, Mayo Malignant cells can also circulate in the blood of patients Clinic, Rochester in 1995. Since the sole purpose of the 1–3 especially those with active disease. Current therapeutic study was to examine the expression of CD34 on the blood approaches utilize autologous stem cells to replenish the and marrow tumor cells from patients with a plasma cell marrow of patients treated with high-dose therapy proliferative disorder, the only eligibility criteria were a programs. Several groups have documented that clonal documented plasma cell proliferative disorder and the pres- myeloma cells can contaminate the blood stem cell harvest ence of monoclonal plasma cells detected in a sample of products from patients with multiple myeloma.4–10 This has the bone marrow or blood by three-color (CD38, CD45 and cytoplasmic kappa or lambda) flow cytometry. Patients without detectable myeloma cells on this initial panel were Correspondence: Dr TE Witzig, 920E Hilton Bldg, Mayo Clinic, Roches- not studied further for CD34. Patients gave written infor- ter, MN 55905, USA med consent to donate additional blood and bone marrow Received 26 March 1996; accepted 5 November 1996 at the time that these procedures were being performed for CD34 expression on myeloma cells T Kimlinger and TE Witzig 554 routine clinical care. This study was approved by the Insti- CD34 expression on blood myeloma cells tutional Review Board of the Mayo Clinic/Foundation. Twenty-four patients had a population of monoclonal CD38+CD45− or CD38+CD45dim+ cells detected in the Three-color flow cytometry blood. Twenty-one of these patients had active multiple myeloma (14 new multiple myeloma; seven relapsed) Plasma cells can be identified by their characteristic strong requiring treatment and three had inactive disease (two with CD38 expression and dim or no expression of CD45.20–22 SMM and one with MGUS) and were observed. The addition of a third marker to detect cytoplasmic light All 24 patients had monoclonal CD38+CD45− cells chain restriction further confirms the clonality of the plasma detected and in 46% (11/24) we were able to establish by cells.23 For this study, blood and marrow monoclonal this technique that there were monoclonal cells in the plasma cells were identified by three-color flow cytometry CD45dim+ population. CD34 expression was not detected on as previously described.23 Briefly, blood monoclonal the CD38+CD45− population in all 24 cases nor in the 11 plasma cells were identified in lysed whole blood speci- cases with a CD38+CD45dim+ monoclonal cell population. mens in which red blood cells were lysed with ammonium We also evaluated the expression of CD34 on the chloride or in mononuclear cells isolated on Ficoll– CD38+CD45dim+ cells that were found to be polyclonal in Hypaque from heparinized blood. The cells to be analyzed the analysis of cytoplasmic kappa and lambda in the other for surface CD38/CD45 and cytoplasmic kappa or lambda 13 cases and they were also negative for CD34 expression. were incubated with phycoerythrin (PE)-conjugated anti- human CD38 (Becton Dickinson, Mountain View, CA, CD34 expression on bone marrow myeloma cells USA) and PerCP-conjugated anti-human CD45 for 15 min; then washed, fixed with 4 ml of lysolecithin solution and The bone marrow from 37 patients were studied and 100% + − incubated for 30 min at 4°C in the dark. After washing with had a population of monoclonal CD38 CD45 or + + 3 ml of PBS/3% BSA the pellet was resuspended in 200 CD38 CD45dim cells detected in the marrow. Twenty-five ml PBS/BSA and 100 ml added to each of two 12 × 75 of these patients had active multiple myeloma (15 new mul- tubes. To these tubes was added either FITC conjugated tiple myeloma; 10 relapsed) requiring treatment and 12 had anti-human kappa or lambda light chain antibody (Tago, inactive disease (two with stable disease after chemo- therapy; four with indolent or SMM; and six with MGUS). Amarillo, CA, USA) and the suspension incubated for 30 + − min in the dark at 4°C. After incubation, the cells were All 37 patients had monoclonal CD38 CD45 cells washed with 2 ml PBS, resuspended in 0.5 ml 1% parafor- detected and CD34 was not expressed on this cell popu- lation in any of the cases. In 89% (33/37) of the cases maldehyde, and analyzed on a FACScan flow cytometer. + Gates were set around the CD38+CD45− and the monoclonal cells were documented in the CD45dim popu- CD38+CD45dim+ quadrant and the percentage of the cells lation and, again the CD34 antigen was not found to be expressing kappa and lambda were compared to determine expressed on this cell population in any of theses cases monoclonality. CD45dim+ cells were those that occupied the (Figure 1). We also evaluated the expression of CD34 on + + gated region between the cells in the sample that did not the CD38 CD45dim cells that were found to be polyclonal stain for CD38 or CD45 and the normal lymphocytes with in the analysis of cytoplasmic kappa and lambda and they their characteristic strong CD45 expression. Monoclonal were also negative for CD34 antigen expression. kappa was defined as a kappa:lambda ratio >5; monoclonal lambda as a kappa:lambda ratio <0.5. After documentation of monoclonal CD38+CD45− or Discussion CD38+CD45dim+ cells in the blood or marrow sample we High-dose therapy programs that use autologous stem cells analyzed a tube containing cells stained with FITC-conju- derived from blood or marrow have been shown to be an gated antiCD34 (Becton Dickinson), PE-CD38, and PerCP- effective modality in the overall treatment program of CD45. The CD38+CD45− and the CD38+CD45dim+ cells patients with new or relapsed myeloma.24–28 Because most were gated on to determine if these cells expressed CD34. patients eventually relapse after this therapy, attention needs to be focused on providing a stem cell product free of myeloma cells, improving the preparative regimens, and Results providing therapy in the post-transplant period that can maintain the response. Patient characteristics The finding that the stem cell harvest products are often contaminated with myeloma cells has led to strategies to The blood or marrow used for this study came from 38 improve the quality and purity of the stem cells.

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