CD5 Positive and Negative B-CLL Evidence Supporting Phenotypic Heterogeneity in B-Chronic Lymphocytic Leukemia (B-CLL) G. B. FAGUET,",' G. E. MARTI,b J. F. AGEE," AND P. BERT IN^
" Cancer Immunology Laboratory Veterans Aflairs Medical Center and Departments of Medicine, and Biochemistry and Molecular Biology Medical College of Georgia Augusta, Georgia 30910 Cellular and Molecular Biology Laboratory Division of Biochemistry and Biophysics Center for Biologics Evaluation and Research Food and Drug Administration Bethesda, Maryland
INTRODUCTION CD5 expression by B-CLL cells is thought so compelling that CD5- lymphoid malignancies are said not to be CLL. We and others, however, have encountered a relatively large subset of patients with CD5- B-CLL clones. In order to characterize the lineage heterogeneity in B-CLL and assess its potential biological significance, we examined the clinical, hematologic, and immunophenotypic profiles of CD5 B-CLL (n = 14) compared to CD5+ B-CLL (n = 48) tested to date. Immunophenotype was ascertained by fluorescence microscopy and flow cytometry using antibodies against a panel of surface antigens, including CD5, CD19, CD20, the common CLL antigen (cCLLa),' and monoclonal surface immunoglobulins (MosIgs). Other than a smaller tumor burden, there were no clinical, hematological, or immunophenotypic features distinguishing CD5- from CD5+ B-CLL, including coexpression and density of CDl9, CD20, cCLLa, and/or MosIg. These data support greater phenotypic heterogeneity in B-CLL than generally recognized and suggest that the search for the true cell of origin in this disease must go on. ~
METHODS OF PROCEDURE Patients B-CLL patients were diagnosed according to the International CLL Workshop criteria and staged according to the Rai staging system (extended to include preclinical disease2).Patient evaluation included routine hematologic tests, serum immunoglobu-
'Send correspondence to Guy B. Faguet, M.D., Chief, Hematology/Oncology, VA Medical Center (1 1lN), Augusta, GA 30910. 470
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lins and &-microglobulin, bone marrow aspirate and biopsy, and immunophenotype. Healthy individuals (n = 40) served as controls. Zmmunophenotype Assays MosIgs were assessed by direct immunofluorescence assay using fluorescein isothiocyanate (F1TC)-conjugated antisera specific for each heavy and light chain. Other surface determinants were ascertained by indirect microscopic fluorescence (using FITC-conjugated goat anti-mouse immunoglobulins against monoclonal antibodies (mAb) of the OKT and LEU series; My4, B1, M02, and J5; and anti-cCLLa mAb CLL,, CLL, or CLL,,') or by flow cytometry using reagents directly conjugated with either phycoerythrin (PE) or FITC (except for CLL,,, which was assayed indirectly).
RESULTS Clinical and Hematologic Profiles Except for a smaller average tumor burden in CD5- patients, the clinical, hematologic, and laboratory profiles of the CD5+ and CD5- patient subsets were comparable (TABLE1). Patient Segregation according to CD5 Expression When segregated according to whether B-CLL cells express CD5 (i.e. total CD5+ minus CD3+ cells by fluorescence microscopy), two distinct patient subsets emerged (TABLE1): one (n = 48 or 78%) whose CD5+ cells (mean 90%) appeared to include both remnant T cells (15%) and B-CLL cells (defined as MosIg+, CD19+, or cCLLa+ cells, mean 73%); the other (n = 14 or 22%), whose percentages of CD5+ and CD3+ cells were equal (mean 37%). Within each subset each patient exhibited the phenotype distribution characteristic for that subset: that is, all B-CLL cells in a given patient were either CD5+ or CD5-. Assessment of Marker Coexpression The following observations have been made from 30 B-CLL patients evaluated thus far by two-color flow cytometry: (1) cCLLa expression was confirmed in all cases including patients whose B-CLL cells were CD5-, MosIg-, or CD19-; (2) density of CD5 and of other CLL markers ranged from absent, to low density, to high density (FIG.1).
DISCUSSION Our observations indicating a higher incidence of CD5+ B-CLL than reported in the literature could be due to (1) inclusion of lymphoproliferative diseases other than B-CLL; (2) uncharacteristic B-CLL patient population; (3) fortuitous prevalence of CD5- patients in our series; (4) insensitive phenotype assays; or ( 5 ) underestimation of the true incidence of CD5- B-CLL in previous reports. The latter view is supported
..
A. B.
c-I
fluorescence are shown on the x-axis and y-axis, respectively. Panel A shows an example of CD5 expression approaching the level of CD5 seen on normal T lymphocytes. Panel B shows an example where only a portion of the CD20+ cells express the CD5 antigen with a density intermediate between normal T lymphocytes and negative cells. Panel C exemplifies CD5- B-CLL.
FIGURE 1. Summary of representative CD5 expression (CD5-PE displayed against CD20-FITC) on whole blood B-CLL lymphocytes. FITC and PE
I
I
5 N
473
FAGUET el al.: CDS HETEROGENEITY IN B-CLL TABLE 1. Clinical, Hematologic, and Laboratory Profiles in B-CLL
CD5 Positive
N Age Stage scores' B l d Counts: Hb (g/dL) WBC (X 1 0 3 4 ~ ) ALC~ (X 1 0 3 4 ~ ) PLTS~( x 1 0 3 4 ~ ) Immunophenotypeb CD3 (%) CD5 (%) CD19 (%) cCLLa (%) MosIg (%) CD5-CD3 (%)
CD5 Negative
Means
SEM
Means
SEM
48 63 1.41
NAf 8 0.2
64
14 0.6
NA 7 0.2
14.1 48.2 38.8 246
2.3 7.3 5.8 37
12.7 30.5 22.5 206
3.4 6.1 2.4 14
15 90 74 76 71d 75
2 14 11 11 11 11
37 37 51 51 50 0
100 98 100 94 22 76 62 36 29.5
NA NA NA NA NA NA NA NA 4.5
0 93 100 100 36
Clone Lineage' CD5 (%) CD19 (%) cCLLa (%) MosIg (%) IgG (%I fgM (%) K (%) A (9%) Clone size (X lo3/@,)'
NA NA NA NA NA NA NA NA 2.1
64 71 29 11.25
Average Rai stage numerals (preclinical CLL entered as - 1). Average percent of cells expressing the marker indicated. Percent of cases whose clones express the cell marker indicated. 77% (SEM 9) after excluding the 3 MosIg-negative patients. Calculated as ALC x %cCLLa+ cells. 'Not applicable. 8 Absolute lymphocyte count. Platelets.
by the fact that contrary to most other published reports, 2 90% of patients accrued to our study have been diagnosed, assayed, and followed long-term by the authors. This includes all CD5- patients. Whatever its real incidence, CDS- B-CLL appears clinically, hematologically, and phenotypically indistinguishable from CD5 B-CLL. Our data suggest that the CD5+ lymphocyte might not be the cell of origin of B-CLL and confirms that the cCLLa+ is the most reliable B-CLL marker. +
REFERENCES & G. B. FAGUET. 1986. Blood 68: 62. 1. AGEE,J. F., F. A. GARVER 2. FAGUET,G. B., G. E. MARTI& J. F. AGEE. 1992. Leukemia and Lymphoma. In press. 3. FAGUET, G. B. & J. F. AGEE. 1988. Blood 72: 679.
" Cancer Immunology Laboratory Veterans Aflairs Medical Center and Departments of Medicine, and Biochemistry and Molecular Biology Medical College of Georgia Augusta, Georgia 30910 Cellular and Molecular Biology Laboratory Division of Biochemistry and Biophysics Center for Biologics Evaluation and Research Food and Drug Administration Bethesda, Maryland
INTRODUCTION CD5 expression by B-CLL cells is thought so compelling that CD5- lymphoid malignancies are said not to be CLL. We and others, however, have encountered a relatively large subset of patients with CD5- B-CLL clones. In order to characterize the lineage heterogeneity in B-CLL and assess its potential biological significance, we examined the clinical, hematologic, and immunophenotypic profiles of CD5 B-CLL (n = 14) compared to CD5+ B-CLL (n = 48) tested to date. Immunophenotype was ascertained by fluorescence microscopy and flow cytometry using antibodies against a panel of surface antigens, including CD5, CD19, CD20, the common CLL antigen (cCLLa),' and monoclonal surface immunoglobulins (MosIgs). Other than a smaller tumor burden, there were no clinical, hematological, or immunophenotypic features distinguishing CD5- from CD5+ B-CLL, including coexpression and density of CDl9, CD20, cCLLa, and/or MosIg. These data support greater phenotypic heterogeneity in B-CLL than generally recognized and suggest that the search for the true cell of origin in this disease must go on. ~
METHODS OF PROCEDURE Patients B-CLL patients were diagnosed according to the International CLL Workshop criteria and staged according to the Rai staging system (extended to include preclinical disease2).Patient evaluation included routine hematologic tests, serum immunoglobu-
'Send correspondence to Guy B. Faguet, M.D., Chief, Hematology/Oncology, VA Medical Center (1 1lN), Augusta, GA 30910. 470
FAGUET el oL: CD5 HETEROGENEITY IN B-CLL
411
lins and &-microglobulin, bone marrow aspirate and biopsy, and immunophenotype. Healthy individuals (n = 40) served as controls. Zmmunophenotype Assays MosIgs were assessed by direct immunofluorescence assay using fluorescein isothiocyanate (F1TC)-conjugated antisera specific for each heavy and light chain. Other surface determinants were ascertained by indirect microscopic fluorescence (using FITC-conjugated goat anti-mouse immunoglobulins against monoclonal antibodies (mAb) of the OKT and LEU series; My4, B1, M02, and J5; and anti-cCLLa mAb CLL,, CLL, or CLL,,') or by flow cytometry using reagents directly conjugated with either phycoerythrin (PE) or FITC (except for CLL,,, which was assayed indirectly).
RESULTS Clinical and Hematologic Profiles Except for a smaller average tumor burden in CD5- patients, the clinical, hematologic, and laboratory profiles of the CD5+ and CD5- patient subsets were comparable (TABLE1). Patient Segregation according to CD5 Expression When segregated according to whether B-CLL cells express CD5 (i.e. total CD5+ minus CD3+ cells by fluorescence microscopy), two distinct patient subsets emerged (TABLE1): one (n = 48 or 78%) whose CD5+ cells (mean 90%) appeared to include both remnant T cells (15%) and B-CLL cells (defined as MosIg+, CD19+, or cCLLa+ cells, mean 73%); the other (n = 14 or 22%), whose percentages of CD5+ and CD3+ cells were equal (mean 37%). Within each subset each patient exhibited the phenotype distribution characteristic for that subset: that is, all B-CLL cells in a given patient were either CD5+ or CD5-. Assessment of Marker Coexpression The following observations have been made from 30 B-CLL patients evaluated thus far by two-color flow cytometry: (1) cCLLa expression was confirmed in all cases including patients whose B-CLL cells were CD5-, MosIg-, or CD19-; (2) density of CD5 and of other CLL markers ranged from absent, to low density, to high density (FIG.1).
DISCUSSION Our observations indicating a higher incidence of CD5+ B-CLL than reported in the literature could be due to (1) inclusion of lymphoproliferative diseases other than B-CLL; (2) uncharacteristic B-CLL patient population; (3) fortuitous prevalence of CD5- patients in our series; (4) insensitive phenotype assays; or ( 5 ) underestimation of the true incidence of CD5- B-CLL in previous reports. The latter view is supported
..
A. B.
c-I
fluorescence are shown on the x-axis and y-axis, respectively. Panel A shows an example of CD5 expression approaching the level of CD5 seen on normal T lymphocytes. Panel B shows an example where only a portion of the CD20+ cells express the CD5 antigen with a density intermediate between normal T lymphocytes and negative cells. Panel C exemplifies CD5- B-CLL.
FIGURE 1. Summary of representative CD5 expression (CD5-PE displayed against CD20-FITC) on whole blood B-CLL lymphocytes. FITC and PE
I
I
5 N
473
FAGUET el al.: CDS HETEROGENEITY IN B-CLL TABLE 1. Clinical, Hematologic, and Laboratory Profiles in B-CLL
CD5 Positive
N Age Stage scores' B l d Counts: Hb (g/dL) WBC (X 1 0 3 4 ~ ) ALC~ (X 1 0 3 4 ~ ) PLTS~( x 1 0 3 4 ~ ) Immunophenotypeb CD3 (%) CD5 (%) CD19 (%) cCLLa (%) MosIg (%) CD5-CD3 (%)
CD5 Negative
Means
SEM
Means
SEM
48 63 1.41
NAf 8 0.2
64
14 0.6
NA 7 0.2
14.1 48.2 38.8 246
2.3 7.3 5.8 37
12.7 30.5 22.5 206
3.4 6.1 2.4 14
15 90 74 76 71d 75
2 14 11 11 11 11
37 37 51 51 50 0
100 98 100 94 22 76 62 36 29.5
NA NA NA NA NA NA NA NA 4.5
0 93 100 100 36
Clone Lineage' CD5 (%) CD19 (%) cCLLa (%) MosIg (%) IgG (%I fgM (%) K (%) A (9%) Clone size (X lo3/@,)'
NA NA NA NA NA NA NA NA 2.1
64 71 29 11.25
Average Rai stage numerals (preclinical CLL entered as - 1). Average percent of cells expressing the marker indicated. Percent of cases whose clones express the cell marker indicated. 77% (SEM 9) after excluding the 3 MosIg-negative patients. Calculated as ALC x %cCLLa+ cells. 'Not applicable. 8 Absolute lymphocyte count. Platelets.
by the fact that contrary to most other published reports, 2 90% of patients accrued to our study have been diagnosed, assayed, and followed long-term by the authors. This includes all CD5- patients. Whatever its real incidence, CDS- B-CLL appears clinically, hematologically, and phenotypically indistinguishable from CD5 B-CLL. Our data suggest that the CD5+ lymphocyte might not be the cell of origin of B-CLL and confirms that the cCLLa+ is the most reliable B-CLL marker. +
REFERENCES & G. B. FAGUET. 1986. Blood 68: 62. 1. AGEE,J. F., F. A. GARVER 2. FAGUET,G. B., G. E. MARTI& J. F. AGEE. 1992. Leukemia and Lymphoma. In press. 3. FAGUET, G. B. & J. F. AGEE. 1988. Blood 72: 679.
