Hematopathology / CD5 Expression on Normal B Cells Significant CD5 Expression on Normal Stage 3 Hematogones and Mature B Lymphocytes in Bone Marrow Franklin S. Fuda, DO, Nitin J. Karandikar, MD, PhD, and Weina Chen, MD, PhD Key Words: Hematogones; Mature B cells; CD5; Multiparameter flow cytometry DOI: 10.1309/AJCPU5E3NXEKLFIY Abstract B-cell maturation from hematogones to mature B cells in bone marrow exhibits a consistent, complex spectrum of sequential antigen expression. CD5 expression, however, has not been characterized. We studied the dynamics of CD5 expression on developing B cells by 4-color flow cytometry in 32 patients, aged 9 months to 63 years, with hematogone hyperplasia (>3.5% of total events). The mean percentage of hematogones was 8.1%. We demonstrate consistent CD5 expression on normal, polytypic B cells in a continuum, predominantly at later stages of maturation, specifically on stage 3 hematogones and mature B cells. Awareness of this normal pattern of CD5 expression on B-cell subsets has implications in the analysis of minimal residual disease of CD5+ B-lineage non-hodgkin lymphomas. B-cell maturation from hematogones to mature B cells in bone marrow has been well characterized. 1-6 Hematogones are normal, maturing, B-lineage precursors that exhibit a consistent, reproducible, complex spectrum of sequential antigen expression. This defines hematogones into 3 stages of maturation. Stage 1 hematogones express CD34, high levels of CD10 and CD38, a moderate level of CD22, and absence of CD20. These progress to the intermediate stage 2 as they down-regulate CD34 completely and CD10 partially, while increasing expression of CD22 and CD20. Finally, at stage 3, CD20 expression reaches the intensity of mature B cells and CD10 and CD38 are slightly down-regulated with increasing expression of polytypic surface immunoglobulin (sig) light chains. Subsequently, these cells mature into CD20+/CD10 mature B cells. We had anecdotally observed CD5 expression on a subset of hematogones and mature B cells in bone marrow, particularly in cases with hematogone hyperplasia. The expression pattern of CD5 on B-cell subsets at different stages of maturation and surface light chain expression on these CD5+ B cells had not been systematically studied and became the focus of this study. CD5 is a T cell associated antigen. Its expression on neoplastic B cells is a defining feature of mantle cell lymphoma and chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma. Lack of awareness of CD5 expression on normal B cells may lead to erroneous interpretation, particularly in the analysis of minimal residual disease of these CD5+ lymphomas. Therefore, in this study, we systematically evaluated the pattern of CD5 expression on normal B-cell subsets in bone marrow by 4-color flow cytometry and examined whether the differential antigen expression including CD5 would permit the distinction of normal CD5+ B cells from neoplastic CD5+ B cells. Am J Clin Pathol 2009;132:733-737 733 733 DOI: 10.1309/AJCPU5E3NXEKLFIY 733
Fuda et al / CD5 Expression on Normal B Cells Materials and Methods As CD5 expression on normal B cells was readily appreciable in cases with hematogone hyperplasia, 32 bone marrow cases with hematogone hyperplasia, defined as a percentage of hematogones higher than 3.5% of total events, were prospectively studied. All cases were immunophenotyped using a 4-color FACSCalibur flow cytometry instrument with CellQuest software (Becton Dickinson, San Jose, CA) and analyzed using cluster analysis with Paint-a-Gate Software (Becton Dickinson). Bone marrow processing and antibody staining were performed as previously described. 4 The following monoclonal antibodies (clones) were used in the panels to profile B-cell subsets: CD5 (L17F12), CD10 (W8E7), CD19 (SJ25C1), CD20 (L27), CD22 (S-HCL-1), CD22 (4KB128), CD23 (B6), CD38 (HB7), FMC-7, monoclonal κ (TB28-2), and monoclonal λ (I-155-2). Four-color antibody combinations (fluorescein isothiocyanate/phycoerythrin/peridinin chlorophyll protein/allophycocyanin) were used: CD10/CD19/CD20/CD38, CD10/CD5/CD20/ CD22, FMC-7/CD23/CD5/CD19, and κ/λ/cd5/cd22. Hematogones were divided into 3 stages of maturation, as previously described 4,5 : stage 1 hematogone by the immunophenotype of CD10+ bright /CD45+ dim /CD20 /CD22+; stage 2 by CD10+/CD45+ moderate /CD20+ variable /CD22+; and stage 3 by CD10+/CD45+ moderately bright /CD20+ bright /CD22+ bright. The proportion of cells with CD5 expression was determined for total B-lineage cells, mature B cells, and stage 3 hematogones. Statistical Analysis Statistical analysis was performed using Prism software (version 5.0a, GraphPad Software, La Jolla, CA). The difference of mean hematogones among 3 stages was tested by 1-way analysis of variance (Kruskal-Wallis test) followed by pairwise comparisons (Dunn multiple comparison test). The difference between 2 groups was tested by unpaired t test. A P value of less than.05 was considered statistically significant. Results Patient Characteristics There were 21 males and 11 females aged 9 months to 63 years (median, 35 years). Submitted clinical histories included 27 cases of hematolymphoid neoplasia and 5 cases of cytopenias. Hematolymphoid neoplasia included myelodysplastic syndromes or acute myeloid leukemia in 15 cases, plasma cell myeloma in 6, precursor lymphoblastic leukemia in 4, chronic lymphocytic leukemia in 1, and Fanconi anemia in 1. Flow cytometric findings were unremarkable in 20 (63%) of 32 cases. The remaining 12 cases showed persistence of previously diagnosed neoplasia (11 cases) or newly detected acute myeloid leukemia (1 case). CD5 Expression on Normal B Cells Regardless of the underlying disease and the patient s age, hematogones in each case exhibited a typical, consistent, complex spectrum of sequential antigen expression, as described previously. 4,5 Table 1 summarizes the percentage of total B cells, mature B cells, and each stage of hematogones in bone marrow. Of total events, the mean percentage of total B cells that included mature B cells and hematogones was 10.1%. Likely owing to the selection bias for which only cases with hematogone hyperplasia were included, the mean percentage of hematogones was higher than that of mature B cells (8.13% vs 1.95%; P <.01; unpaired t test). Within the hematogone populations, hematogones were present at all stages of maturation with a predominance of stage 2 hematogones in 31 of 32 cases (mean, 77% vs 8.0% in stage 1 and 15.0% in stage 3 hematogones) Image 1A. In 1 remaining case, moderate hematogone hyperplasia (4.1% of total events) was present with a shift toward immaturity and virtual absence of stage 3 hematogones and mature B cells Image 1B. CD5 was expressed on normal subsets of B cells in a continuum, being most prominent on stage 3 hematogones and mature B cells. Table 2 summarizes the percentage of CD5+ B cells over total B cells, mature B cells, and stage 3 Table 1 The Percentage of Mature B Cells and Hematogones in 32 Bone Marrow Samples Mean ± SD Median Range Total B cells/total events (%) 10.08 ± 4.76 9.4 4.10-19.80 Mature B cells/total events (%) 1.95 ± 2.33 0.99 0.00-9.90 Total hematogones/total events (%) 8.13 ± 4.06 7.10 3.70-18.00 Stage of hematogones/total hematogones (%) 1 12.29 ± 13.11 8.00 1.20-70.00 2 71.28 ± 15.80 77.0 22.00-89.00 3 16.35 ± 11.55 15.0 0.00-45.00 734 Am J Clin Pathol 2009;132:733-737 734 DOI: 10.1309/AJCPU5E3NXEKLFIY
Hematopathology / Original Article A B C Image 1 Representative flow cytometric plots of hematogones and mature B cells in bone marrow. A, A case with moderate hematogone hyperplasia (6.5% of total events) with all stages of hematogones represented. CD5 expression was evident on stage 3 hematogones and a subset of mature B cells. B, A case with moderate hematogone hyperplasia (4.1% of total events) with a shift toward immaturity (exclusively stage 1 and 2 hematogones detected). Mature B cells were virtually absent. There was no appreciable CD5 expression on stage 1 and 2 hematogones. C, A case with moderate hematogone hyperplasia (4.6% of total events) with all stages of hematogones represented. CD5 was expressed on stage 3 hematogones and a subset of mature B cells. In addition, there was a minute population of clonal B cells (0.22%, in red) with immunophenotype of chronic lymphocytic leukemia (CLL). The differential expression of CD5, CD10, CD20, and surface immunoglobulin light chain (sig) permitted the distinction of CD5+ normal B cells from CD5+ CLL cells. Note that the CD5+ B cells exhibited predominantly polytypic sig expression. APC, allophycocyanin; FITC, fluorescein isothiocyanate; PE, phycoerythrin; PerCP, peridinin chlorophyll protein; green, stage 1 and 2 hematogones; yellow, stage 3 hematogones; blue, mature B cells; red, monotypic B cells. Table 2 CD5 Expression on Mature B Cells and Stage 3 Hematogones in 31 Bone Marrow Samples Mean ± SD Median Range CD5+ B cells/total B cells (%) 22.77 ± 15.1 21.5 0.65-63.00 CD5+ mature B cells/total CD5+ B cells (%) 44.10 ± 30.3 59.3 0-90.49 CD5+ hematogones/total CD5+ B cells (%) 55.82 ± 30.3 50.06 9.51-100 CD5+ mature B cells/mature B cells (%) 59.92 ± 21.7 66.5 16.00-95.00 CD5+ hematogones/stage 3 hematogones (%) 67.07 ± 16.2 67.5 24.00-92.00 Am J Clin Pathol 2009;132:733-737 735 735 DOI: 10.1309/AJCPU5E3NXEKLFIY 735
Fuda et al / CD5 Expression on Normal B Cells hematogones, and their distribution over total CD5+ B cells. The mean percentage of CD5+ B cells over total B cells was 22.8%. CD5+ B cells were approximately equally split between mature B cells (~44%) and stage 3 hematogones (~56%). Within mature B cells, 59.92% were CD5+, whereas 67.07% of the stage 3 hematogones were CD5+. Notably, this subset of CD5+ stage 3 hematogones was also positive for CD10 (a marker used to define all hematogones). Virtually all CD5+ B cells expressed sig, showing a polytypic distribution of light chains. It is interesting that in the 1 case that virtually lacked stage 3 hematogones and mature B cells, CD5 expression on B cells was undetectable (Image 1B). In another case, there was a minute population of CD5+ monotypic B cells (0.22% of total events) with the immunophenotype of CLL. In this case, the differential expression of CD5, CD10, CD20, and sig permitted the distinction of CD5+ normal B cells from CD5+ CLL cells. Compared with normal CD5+ B cells, CLL cells expressed strong CD5, dim CD20, and monotypic sig and lacked CD10 Image 1C. Discussion Hematogones in this study exhibited a typical, consistent, complex spectrum of sequential antigen expression, as described previously. 4,5 The novel finding in this study is CD5 expression on normal subsets of polytypic B cells in a continuum, predominantly at later stages of maturation, specifically on stage 3 hematogones and mature B cells. The differential antigen expression, including CD5 and sig light chains, permits the distinction of CD5+ normal B cells from CD5+ lymphoma cells. In this study, we characterized the pattern of CD5 expression in bone marrow cases with hematogone hyperplasia in which CD5 expression was readily appreciable compared with cases with low numbers of hematogones. The majority of patients had a history of hematolymphoid malignancies and were in remission or had minimal residual disease. Therefore, it is conceivable that hematogone hyperplasia was likely associated with postchemotherapy bone marrow regeneration. Another condition associated with hematogone hyperplasia is copper deficiency, which was illustrated in one of our cases that exhibited exuberant hematogone hyperplasia and no evidence of myelodysplastic syndrome. 7 It has been 2 decades since CD5, first described as a T-cell marker, was identified in malignant human B cells. 8 CD5 was later shown to mark a minority of normal B cells in peripheral blood. 9 These CD5+ B cells likely represent so-called transitional B cells or B1 B cells. 9-13 In humans and mice, CD5+ B cells are the major population of B cells in fetal life, and their percentage decreases with age. Indeed, most human IgM+ cord blood B cells express CD5; however, in adults they represent approximately 10% to 25% of B cells in blood and may expand in early lymphoid recovery after hematopoietic stem cell transplantation and autoimmune diseases (such as systemic lupus erythematosus). 11,14-16 These findings may suggest the role of CD5+ B cells in lymphopoiesis and pathogenesis in immune dysregulation. CD5 expression on bone marrow hematogones was described in 1 case report in which there was significant hematogone hyperplasia in a 7-year-old girl with pancytopenia due to Shwachman-Diamond syndrome, shown by 2-color flow cytometric study and morphologic examination. 17 Such CD5 expression likely represents CD5 expression on transitional B cells in the immunologic literature. 11 The pattern of CD5 expression during the different stages of B-cell maturation in bone marrow, however, has not been systematically evaluated. Our studies demonstrate that CD5 is expressed on normal subsets of B cells in a continuum, specifically on stage 3 hematogones and mature B cells. Furthermore, a small subset of normal B cells coexpressed CD5 and CD10 (stage 3 hematogones). These CD5+ B cells may represent a developmental intermediate for mature B-cell generation. Exploration of the mechanism of this highly regulated CD5 expression may shed light on the pathogenesis of CD5+ and dual CD5+/ CD10+ B-cell lymphomas. Furthermore, awareness of the normal pattern of CD5 expression on B cells has clinical implications in the analysis of residual disease in CD5+ B-cell lymphomas such as CLL and mantle cell lymphoma. Our study indicates that differential antigen expression can distinguish normal CD5+ B cells from neoplastic CD5+ B cells. Features associated with CLL include dim CD20 expression, light chain restriction, and lack of CD10. Typically, precursor B lymphoblastic leukemia would not cause a diagnostic challenge in regard to CD5 expression because the vast majority of cases of B lymphoblastic leukemia express CD34 and terminal deoxynucleotidyl transferase and lack CD5, 4,6 an immunophenotype that mimics the normal counterpart of stage 1 hematogones. We show that CD5 is expressed on normal, polytypic B cells, predominantly on stage 3 hematogones and mature B cells. Awareness of this normal expression pattern has clinical implications in the analysis of minimal residual diseases of CD5+ B-cell lymphomas. From the Department of Pathology, University of Texas Southwestern Medical Center, Dallas. Address reprint requests to Dr Chen: Dept of Pathology, UT Southwestern Medical Center at Dallas, Dallas, TX 75390-9072. References 1. Loken MR, Shah VO, Dattilio KL, et al. Flow cytometric analysis of human bone marrow, II: normal B lymphocyte development. Blood. 1987;70:1316-1324. 736 Am J Clin Pathol 2009;132:733-737 736 DOI: 10.1309/AJCPU5E3NXEKLFIY
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