Expression of the Hematopoietic Stem Cell Antigen CD34 on Blood and Bone Marrow Monoclonal Plasma Cells from Patients with Multiple Myeloma

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 transplants 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 ␮l PBS/BSA and 100 ␮l 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 chemotherapy; 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 population 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 у5; monoclonal lambda as a kappa:lambda ratio р0.5. 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. These stra- patients with a documented plasma cell proliferative dis- tegies include collection of blood stem cells during the order. Both blood and marrow were utilized from 23 response phase of disease when circulating tumor cells are patients; marrow alone in 14; and blood only in one. Six lower than at the time of diagnosis or relapse29 and prior to patients had a monoclonal gammopathy of undetermined significant exposure to alkylating agents that may otherwise significance (MGUS); four had indolent or smoldering mul- impair engraftment of the stem cells. Purging by positive tiple myeloma (SMM); 15 had new multiple myeloma; two selection of CD34+ stem cells has been shown to enrich the had stable disease after chemotherapy; and 11 had relapsed stem cell product for CD34+ cells12 and reduce the contami- multiple myeloma. nating tumor cells9,13,18 without affecting engraftment. CD34 expression on myeloma cells T Kimlinger and TE Witzig 555 Gate 1 Gate 2 five patients no myeloma clonal sequences were detected. 11% 3% This same group used this technique to analyze myeloma cell contamination of the blood stem cell product in 14 patients before and after CD34 selection. Tumor was detected in eight of 14 before and in only three after CD34 selection.13 Gazitt et al9 studied mobilized blood stem cell CD38PE harvests before and after a purification procedure that used counterflow elutriation centrifugation, treatment with phe-

CD45 nylalanine methylester and flow sorting. They demonstrated Gate 1 Gate 2 that this procedure led to an approximately 50-fold enrich- 6% 94% 1% 99% ment in stem cells and a depletion of tumor cells in the CD34+ Lin− Thy+ population. Takashita et al19 used flow cytometry to sort blood lymphocytes from three patients – + + 45 45 into a CD34 population. Clonal sequences were found in + + this CD34+ cell population by use of clone-specific probes + CD38 CD38 made from the myeloma cells. CD34 cells from the bone marrow were not tested. These results are at odds with the results of the other published investigations9,13,18 and need Kappa Kappa to be confirmed on a larger number of patients. One poten- 99.6% 0.4% 96% 4% tial explanation is that because a large number of cells (up to 106) were sorted to obtain the CD34+ population, it is possible that some CD34− cells that were part of the mye- – +

45 45 loma clone may have been included and subsequently + + amplified by PCR thereby influencing the final results.

CD38 CD38 Our study investigated whether myeloma cells identified by three-color flow cytometry express the CD34 antigen. We identified these cells by their characteristic Lambda Lambda CD38+CD45− CD45dim+ monoclonal cytoplasmic light 99.95% 0.05% 98.89% 1.11% chain immunophenotype. By this method we were able to study samples of blood and marrow that contained myeloma cells from a large number of patients and demonstrate – +

45 45 that the CD34 antigen is not expressed on these cells. It is + + possible that there are cells that are part of the myeloma + CD38 CD38 clone that could have been CD34 that were not identified by gating on CD38+CD45−CD45dim+ cells. However, our results are consistent with the studies cited above that show + CD34 CD34 that the clonal myeloma cells are not in the CD34 popu- 9,13,18 Figure 1 Results of three-color flow cytometric study of a sample of lation. bone marrow from a patient with multiple myeloma. Row 1: display of The literature cited and the results from our study indi- CD38 and CD45 showing both CD38+CD45− and CD38+CD45+ popu- cate that the CD34 stem cell selection strategies currently lations of myeloma cells. Rows 2 and 3: cytoplasmic light chain + − + + utilized for patients with multiple myeloma should decrease expression on the CD38 CD45 and CD38 CD45 gates showing the cells the likelihood that significant numbers of tumor cells will to be positive for kappa and negative for lambda. Row 4: lack of CD34 antigen expression on both CD38+CD45− and CD38+CD45+ populations be reinfused. At this time it is questionable whether CD34 of myeloma cells. selection strategies will lead to an improvement in overall survival after autologous transplant. Patients with high numbers of monoclonal plasma cells in the blood stem cell Because very early, immature B cells can express the harvest product have been shown to have a shorter time to CD34 antigen14 there has been concern that procedures that progression after autologous blood stem cell transplants and select CD34+ cells for use in transplantation of patients with a shorter overall survival.30 Studies of genetically marked multiple myeloma may, in addition to enriching for stem stem cells harvested from the blood have documented that cells, also include myeloma cells. Three studies using mol- they can restore hematopoietic function when reinfused; ecular techniques have shown that the enriched CD34+ cell however, it has not yet been shown that the infused mye- population is devoid or depleted of myeloma tumor cells. loma cells are responsible for relapse after blood stem cell Vescio et al18 obtained marrow myeloma cells from five transplants.31 As the preparative regimens and post-trans- patients and developed a unique, patient-specific oligonu- plant therapy improve, it will become more important to cleotide primer based on the complementarity determining provide a stem cell product free of myeloma cells. region of each myeloma immunoglobulin sequence. The CD34+ cells were isolated from the same marrow sample Acknowledgements by an immunoadsorption column and flow cytometry sorting. The patient-specific oligonucleotide was then used to This work was supported in part by grant number CA62242 from amplify the DNA from the CD34-selected cells and in all the National Cancer Institute (National Institutes of Health). CD34 expression on myeloma cells T Kimlinger and TE Witzig 556 References stem cells (PBSCs) collected after recombinant granulocyte colony-stimulating factor (rhG-CSF): an analysis of factors 1 Berenson J, Wong R, Kim K et al. Evidence for peripheral correlating with the tempo of engraftment after transplan- blood B lymphocyte but not T lymphocyte involvement in tation. Br J Haematol 1994; 87: 825–831. multiple myeloma. Blood 1987; 70: 1550–1553. 17 Tricot G, Jagannath S, Vesole D et al. Peripheral blood stem 2 Witzig T, Dhodapkar M, Kyle R, Greipp P. Quantitation of cell transplants for multiple myeloma: identification of favor- circulating peripheral blood plasma cells and their relationship able variables for rapid engraftment in 225 patients. 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