British /ournu/ of Haematologyy. 1992, 81, 45-51

Immunophenotypic subclassification of chronic lymphocytic leukaemia (CLL) A . S. KUREC, G. A. THREATTE, A. J,

GOTTLIEB,* J. R. SMITH,* J. ANDERSON-/ AND F. R . DAVEY Departments of Pathology and *Medicine, SUNY Health Science Center, Syracuse, New York, and TDepartment of Preventive and Societal Medicine. University of Nebraska Medical Center, Omaha, Nebraska

Received 22 August 1991: accepted for publication 7 January 1992

Summary. To determine the significance of the immunophenotypic heterogeneity of B-cell chronic lymphocytic leukaemia (CLL). surface immunoglobulins (Slgs), mouse rosette assays (MR). and a panel of monoclonal antibodies for B cells, T cells and myeloid cells were performed on peripheral blood samples from 61 newly diagnosed cases. Four groups were observed: group I (SIg+. M R + . CD19/20+. CD5+, T antigen (A@-: 2 7 cases): group I1 (SIg+, M R + , CD19/ 2 0 + , CD5+. T Ag+: 1 7 cases): group 111 (SIg+. M R + CDl9/20 , CD5 - , T AG - : 12 cases): and group IV (SIg- , MR + , CI)19/20 + , Cd5 , T Ag - ; 5 cases). Groups were compared according to French-American-British Cooperative Group subtypes, clinical and laboratory features, Rai staging, and survival. Typical CLL morphology ( > 90% small

lymphocytes) was present in 20/20 (100%)of group I cases and 23/27 (85%) group 11. 111 and IV cases (P=0.09). Expression of a rnyeloid antigen was seen in 5/27 group I cases (18%) and 1/16 group I1 cases (6%). but was not predictive of survival (P=0.36). The CD5- group 111 had a lower haemoglobin level ( P < 0*0001),higher Rai stage (P 10% and 10%large lymphocytes, < 10%prolymphocytes). While reduced survival is clear in cases of PLL (Melo et al. 1987: Vallespi et al, 199 1). the prognostic significance of the CLLIPLL and CLLlmixed subgroups is not known with certainty. Some evidence, however, suggests that survival in the CLL/PLL subgroup may not be as long as in CLL (Melo et al, 1987). In addition to the cytology of the lymphocytes, survival has also been predicted from clinical stage, pattern of bone marrow involvement. lymphocyte doubling time, and surface markers (Melo et al, 1987; Rozman et al, 1984: Montserrat et al, 1986: Vinolas et al, 1987: Hamblin et al, 1987; Kimby et aJ. 1988). Immunophenotypic differences are also evident in cases of CLL (Dadman & Cawley. 1988: Hamblin et al, 1987; Kimby et al, 1988:Melo et al, 1987;Orfao et al. 1989: Newman et al. 1990). While 95% of cases of CLL represent proliferations of B cell lineage, 5% are of T cell origin (Bennett et al, 1989).In

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Correspondence: Dr F. R. Davey. Department of Pathology, 7 5 0 East Adams Street, SUNY Health Science Center, Syracuse, New York 13210. U.S.A.

45

46

A. S. Kurec et a1

addition, there are immunophenotypic subgroups of B cell CLL and there is some evidence that these differences may have prognostic significance (Hamblin et al, 1987: Melo et al. 1987: Newman et al, 1990; Morabito et al, 1987). Thus, the purpose of this report is to describe our experience in a study of the significance of immunophenotypic subgroups of B-cell CLI,. We employed a panel of leucocyte cell markers (B cell, T cell, myeloid cell) in the evaluation of 61 de novo cases of CLL. Four distinct immunophenotypic subgroups of B-cell CLL were identified. These subgroups were then evaluated and compared according to clinical features, laboratory characteristics, the morphology of lymphoid cells, and survival. METHODS AND MATERIALS Sixty-one patients with CLL were clinically evaluated and . staged according to Rai et al(1975) at the time of diagnosis. Routine haematologic and immunologic studies were performed, including Wright-Giemsa stained peripheral blood smears. Peripheral blood smears were classified according to FAB criteria (Bennett et al, 1989). Heparinized peripheral blood samples were obtained and separated by Ficoll centrifugation techniques. Mononuclear cell suspensions were incubated with a standard panel of monoclonal antibodies (CD1-5; CD7: CD8: CDlO; CD14: CD15: CD19; CD20: CD33: HLA-DR: IgM IgG: kappa: lambda). One million cells were incubated with the appropriate FITC-conjugated antibody for 45 min at 4OC. Suspensions were washed with cold phosphate buffered saline (PBS) three times, resuspended in a 1%paraformaldehyde solution, and analysed on a FacScan flow cytometer (Becton Dickinson. San Jose, Calif.) (Davey et al, 1987). Negative controls consisting of non-immune mouse isotypic sera were subtracted from each respective antibody determination. Those cases with greater than 20% reactive cells were considered to be positive for the respective antibody. The mouse rosette assay was performed on lo5 mononuclear cells, admixed with mouse erythrocytes and fetal calf serum, centrifuged for 5 min at 500 g , and incubated overnight at 4OC (McGraw et al. 1982). Using a trypan blue dye-exclusion technique, the resuspended cell mixture was examined for the presence of rosettes. Cytocentrifuge preparations of cell suspensions were Wright-Giemsa stained and examined to confirm the presence of representative leukaemic cells in the cell suspension, thus co-expression of unexpected antigens (myeloid or T-cell associated) were inferred when greater than 20% of these cells stained positive. Mononuclear cell volume measurements were performed on a Coulter H4 Channelyzer (Coulter Electronics, Hialeah, Fla.) (Chapman et al, 1981). The mean cell volume and standard deviations were determined by counting at least 3 5 000 cells per sample. Serum immunoglobulin levels for IgG. IgM and IgA were determined by nephelometry (TDX Abbott Diagnostics, Abbott Park, Ill.) (Davey et al, 1987). Statistics were performed on data using standard nonparametric evaluations. Fisher's exact test and Wilcoxon rank test were used to compare clinical data and laboratory results among study groups (Fleiss, 1981). Kaplan-Meier curves and log-rank tests were used to compare survival (Peto et al, 1977).

RESULTS Sixty-one patients (36 males and 2 5 females) between the ages of 39 and 88 years were diagnosed with B-cell CLL by the detection of the presence of a sustained elevation ( > 5 .O x 109/1)of a population of mature lymphocytes in the peripheral blood, which formed mouse rosettes, and were CD19/CD20 positive, and TdT negative. Within this cohort of B-cell CLL patients were four distinct immunophenotypic subgroups (Table I). Group I possessed a typical B-cell CLL immunophenotype. Group I1 exhibited at least one T cell antigen in addition to the typical B-cell CLL immunophenotype. Co-expression of T-cell antigens on leukaemic cells were demonstrated by the presence of 20% or greater positivity of one or more T-cell associated antigens (CD1. CD2, CD3, CD4, CD8) in diagnosed cases of B-cell CLL. Cases were positive for CD2 in 17/17 cases, CD3 in 9/17 cases, CD4 in 2/17 cases, CD8 in 1/17 cases, and C D l in 1/17 cases. Group 111 possessed the typical B-cell CLL immunophenotype with the exception that CD5 was expressed on less than 20% of peripheral blood lymphoid cells. In group IV, surface immunoglobulins were present on less than 20% of lymphoid cells or not detectable, while all other markers of the typical B-cell CLL immunophenotype were present. In addition, lymphocytes from 6/42 patients (14%)expressed at least one myeloid associated antigen (2 1-47% of cells positive). Five of the latter patients were in group I and one was in group 11. The clinical features from these CLL patients are summarized in Table I1 according to their immunophenotype. The median age of the patients was 66 years and 59% were males. Lymphadenopathy was present in 10/40 (25%) cases, splenomegaly in 7/38 (18%),and hepatomegaly in 0/27 cases. While haemolytic anaemia (HA) was seen in 3/41 (7%) of cases, idiopathic thrombocytopenia purpura (ITP) was not observed in any. The laboratory data from these patients are presented in Table 111. The mean white cell count was 48 x 109/1(range 11-511 x 109/1),haemoglobin level was 13.3f 1.6 g/dl, platelet count was 235 f 104 x 109/1,and mean lymphocyte count was 7 6 f 16% (peripheral blood). Of the 61 cases, 22/ 40 (55%) showed a decrease in at least one serum immunoglobulin level. When classified according to Rai staging, 36/ 59 (61%) were stage 0: 8/59 (14%) stage I; 9/59 (1 5%)stage 11; 5/59 (8%)stage In; and 3/59 (5%) stage IV (Table II). Morphologic evaluation of 4 7 cases utilizing FAB criteria revealed 43 (91%)cases of CLL subtype, one (2%) CLLIPLL Table I. Immunophenotypic subgroups of B-cell chronic lymphocytic leukaemia Group

n

%

Immunophenotype

I I11

27 17 12

IV

5

44 28 20 8

SIg+, CD5 MR+, CD19/20+, T a l l Ag SIg+ , CD5 MR+,CD19/20+, T Cell Ag SIg+ , CD5 -, MR+, CD19/20+, T Cell Ag SIB-, CD5 , MR CD19/20 ,T Cell Ag -

I1

+,

+, +, +

+

+

+

+,

+,

+

T Cell Ag= CDl CD2 , CD3 , CD4 CD7+, and/or CD8 SIg =Surface immunoglobulins.MR =Mouse rosettes.

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Zmmunophenotypic Subclassification of CLL

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Table 11. Clinical features of B-cell chronic lymphocytic leukaemia

Group I (n=27)

Group I1 (n=17)

Group I11 (n=12)

Age (years) Median Range

67f12 32-88

66f12 52-87

66f10 50-82

Sex Male Female

17/27 (63). 10/27 (37)

8/17 (47) 9/17 (53)

8/12 (67) 4/12 (33)

3/5 (60) 2/5 (40)

4/17 (24) 2/17 (12) 0/17

2/12 (17) 2/12 (17) 2/13 (15)

3/8 (38) 3/7 (43) 1/8 (12)

1/3 (33) 0/2 0/3

3/12 (25)t 0/12 5/12 (42) 3/12 (25) 1/12 (8)

3/4 (75) 114 (25) 0/4 0/4 0/4

4/12 (33)$

0/5

Lymphadenopathy Splenomegaly Haemolytic anaemia Rai Stage 0 1 2 3 4

16/27 (59) 14/16 (88) 6/27 (22) 1/16 (6) 4/27 (15) 0/16 1/16 (6) 1/27 (4) 0/2 7 2/16 (12)

No. of deaths

2/27 (7)

Per cent survival probability at 5 years

1/17 ( 6 )

92

93

55*

Group IV (n=5)

64f10 50-76

100

*Figures in parentheses are percentages (%). t Pi0.002. * P < 0.02.

Table 111. Laboratory results from patients with B-cell chronic lymphocytic leukaemia ( m e a n f S D range) Group I WBC ( x 109/1) Mean Range Lymphocytes (%) MeanfSD

Group I1

Group I11

Group IV

22 11-43

88

15-51 1

11-395

23 19-30

80 f 20

74f 1 3

71f26

77f6

60

Mean size fSD 175f21 Observations 26 Haemoglobin (g/dl) Mean 13.7 f 1.4

172f20 17

172f37 11

171f17 4

1 3 . 9 f 1.6

1 1 . 2 f 2.1*

14.4f1.3

Platelets ( x 1OY/1) Mean

227f129

2 2 5 f 120

236f67

1 0 4 0 f 255 101 f 6 8 128f54

1215 f 4 9 7 103f85 253 f 207

1303 f567 83f58 177f8l

251 f l 0 l

Serum immunoglobulins (mg/dl) IgG mean 999f 340 IgM mean 8 O f 104 IgA mean 154f 1 14 ~

* P < 0~0001.

subtype, three (6%)CI,L/mixed subtype, and no cases of PLL. The mean cell volume of the peripheral blood lymphocytes from patients with CLI, was 172 fl with a range of 128-25 1 fl

(Table 111). There were no significant differences in FAB classification or mean cell volume of peripheral blood lymphocytes among the four immunophenotypic subgroups.

A. S. Kurec et al

48

-

1.o

DISCUSSION

"

0.9

0.7 O.%

0.3 0.2 0.1

I

I

I

I

I

I

0

1

2

3

4

5

Years Phenotype A Group I A Group11 0 GroupIII GroupN

Alive

Dead

Total

Median

25 16 8 5

2 1 4 0

27 17 12

UNDEF UNDEF UNDEF UNDEF

5

Fig 1 . Survival probability of patients with CLL according to immunophenotype. The median survival of each group is not yet defined. however, group 111 has a decreased survival rate at 5 years. (Group I =Classic CLL group I1 = T Ag B CLL: group I11 = CD5 - B CLL group IV = SMIg - B CLL).

+

0.7

1

"i

3 g

0.4

a,

I-

O3

o.2 0.1

1

0

I

I

I

I

I

1

2

3

4

5

Years My+ A No A Yes

Alive

Dead

Total

Median

46 6

7 0

53 6

UNDEF UNDEF

Fig 2. Survival probability of patients with myeloid antigen positive (My ) CLL and myeloid antigen negative (My - ) CLL. The median survival of each group is not yet defined.

+

Immunophenotypic group 111 (CD5- ) had a significantly lower haemoglobin (P 10%of B cells displayed reactivity for that marker. However, in the current study a case was positive when 20% of the cells were reactive. Freedman et al(1989)have indicated that the expression of the CD5 antigen on B lymphocytes is a manifestation of transformation to an activated state. Following 12-O-tetradecanoylphorbal-1 3-acetate exposure, normal B lymphocytes are induced into a state in which CD5 is coexpressed along with B cell activation antigens B5, interleukin-2 receptor, and CD2 3. Several studies indicate that normal CD5 + and CD5 - lymphocytes (Zupo et a/, 1 9 9 1: Nawata rt al. 1990: Burastero rt a/, 1990) have differing functional characteristics such as immunoglobulin production and thymidine uptake as a response to activating agents. Our results may indicate that there is a clinically significant difference in aggressiveness between the neoplastic forms of these cell types. SIgs were detected on fewer than 20% of cells in 5/61 (8%) cases in this study. All other cell markers from these cases were typical of CLL. Although lymphocytes from cases of Bcell CLL characteristically exhibit weak expression of SIgs. previous studies have also noted in 10-20% cases, undetectable or minimal Slg expression (Tefferiet al. 1989: Freedman rt al. 1987). Tefferi et a1 (1989) indicated that patients with undetectable Slg have a very indolent clinical course and show little disease progression over a median 9-year followup period. These findings are similar to those in the present study where patients with undetectable SIB had early stage (0

49

and I) disease and no deaths occurred during the study period. Indolent forms of CLL have been recognized for many years (Han & Rai. 1990: Galton. 1966: French Cooperative Group on Chronic Lymphocytic Leukemia, 1990: Montserrat et al, 1988). It is possible that patients with an indolent form of CLL could be recognized by the identification of B-cells with undetectable SIg. However, to prove this possibility requires a longer study period, and a larger population of SIg negative patients. The finding of myeloid associated determinants on lymphocytes from patients with B-cell CLL has also been noted by previous investigators (Morabito et al, 1987: Wormsley et al, 1990: Newman et al, 1990). Morabito et al (1987) observed the presence of C D l l b (C3bi). CD13, CD14 and CD33 in a high percentage of cases with B-cell CLL. No correlation between the expression of these markers and clinical stage of disease or leucocyte count was apparent. Rather, a n association was noted between the presence of these antigens and the ability of these cells to produce IL- 1. Newman et al(1990) noted that CDI 1b was more often present in cases of CLL/ mixed and CLLIPLL than in the classic CLL subtype. Correlations between this subtype and its clinical features were not reported. Wormsley et al (1990) demonstrated that CDl l c was present on lymphocytes from 22% of cases of B-cell CLL. The clinical features of all cases were similar to cases of CLL or CLLIPLL. However, in the majority of cases (11/14), the peripheral blood lymphocytosis was composed of large lymphocytes ( > 14 pm in diameter) with clumped to moderately dispersed nuclear chromatin patterns without a prominent nucleolus. Hanson et a1 (1990) also noted the presence of C D l l c on a group. of patients who had a disease morphologically similar to CLL, but with cells of intermediate size and coarse, condensed chromatin. Since CD1 l c antigen is a member of the integrin family and is characteristically seen on hairy cell leukaemia (HCL), these authors suggested that this subgroup of CLL was developmentally between CLL and HCL. At the present time there is no convincing association between the presence of myeloid associated markers or cell-adhesion determinants and stage of disease or duration of survival in patients with CLL. This may be in contrast to cases of ALL where the presence of myeloid associated markers in some studies indicates a poor prognosis (Sobol et al. 1987: Wiersma et al, 1991). In summary, we have studied the immunophenotypic expression of 6 1 patients diagnosed with B-cell CLL. Four distinct immunophenotypic subgroups were identified. Patients with the typical B-cell CLL immunophenotype (SIg+. MR+, CD5+. CD19/20+), those with a t least one additional T-cell antigen or with myeloid antigen expression, and individuals with undetectable SIg had less advanced disease at presentation and longer survival at 5 years than those patients lacking CD5 antigens. These findings suggest that although no morphologic differences can be observed between CD5+ and CD5- CLL, there may be a biological difference resulting in a more aggressive disease in those patients which lack the CD5 antigen. A larger study with multivariate analysis is needed to demonstrate whether the absence of the CD5 antigen in CLL is an independent negative prognostic factor.

50

A. S. Kurec et al.

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Zmmunophenotypic SubclassiJication of CLL Newman. R.. Davey. F.. Kornfeld. S.B.. Collins, H.. Brabyn. C., Bloomfield. C.D. & Royston. I. (1990) Correlation of immunophenotype and BCL-I gene rearrangement with morphology in B cell chronic lymphocytic leukemia. (Abstract). Blood. 76, Suppl. 1 , 304A. Orfao. A.. Conzaler. M.. San Miguel. J.F.. Canizo. M.C.. Calindo. P., Caballero. M.D., Jimenez, R. &Loper Borrasca. A.L. (1988) Clinical and immunological findings in large B-cell chronic lymphocytic leukemia. Clinical fmmuriologg and Immunopathology. 46, 177185. Orfao. A., Connalez, M.. San Miguel. J.F., Rios. A.. Canizo. M.C.. Hernandez. J., Maricato, M.1,. & Lopez Borrasca, A. ( 1 989) B-cell chronic lymphocytic leukaernia: Prognostic value of the immunophenotype and the clinico-hematological features. American lournal of Htmatology. 3 1, 26- 3 1 . Pdtrick.C.W..Libnoch. J.A.. Mi1son.T.. Kortright. K.H.&Keller. R.H. ( 1984) Common variant of B cell chronic lymphocytic leukemia is defined by the monoclonal antibody TQI. (Abstract). Blood. 64, Suppl. 1 , 195a. Peterson. L.C.. Bloomfield. C.D. & Brunning. R.D. ( 1980) Relationship of clinical staging and lymphocyte morphology to survival in chronic lymphocytic leukaemia. British journal of Haematology. 44, 563-567. Peto. R.. Pike, M.C.. Armitage. P.. Brewlow. N.E.. Cox. D.R.. Howard. S.V.. Mantel, N.. McPherson. K.. Peto. J. & Smith. P.G. (1977) Design and analysis of randomized clinical trials requiring prolonged observation of each patient. 11. Analysis and examples. British journal o$Caricrr. 3 5 , 1-39. Rai. K.K.. Sawitsky. A,. Cronkite. B.P.. Chanana. A.D., Levy. R.N. & Pasternack. B.S. ( 1 9 7 5 ) Clinical staging of chronic lymphocytic leukemia. Blood. 46. 219-2 34. Rozman. C.. Monsterrat. E.. Kodriquer-Fernander. J.M.. Ayats. R.. Vallespi. T.. Parody, R., Rios. A,. Prados. D..Morey. M.. Comis. F., Alcala. A,. Gutierrez. M.. Maldonado. 1.. Gonzalez. C.. Giralt. M.. Hernander-Nieto. I,.. Cabrera. A. & Fernandez-Ranada. 1.M. ( 1 984) Bone marrow histologic pattern-the best single

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Wiersma. S.R.. Ortega. 1.. Sobel. E. & Weinberg. K.I. (1991) Clinical importance of myeloid-antigen expression in acute lymphoblastic leukemia of childhood. New England]ournal of Medicine. 324,800808. Worman. C.P. & Cawley. J.C. (1982) B-CLL cells express true endogenous E receptor after culture with T-cells and mitogen. British journal of Haematology. 52, 205-2 10. Wormsley. S.B., Baird, S.M.. Gadol. N.. Rai. K.R.&Sobol. R.E. (1990) Characteristics of CDl I c + CD5 + chronic &cell leukemias and the identification of novel peripheral blood B-cell subsets with chronic lymphoid leukemia immunophenotypes. Blood. 76, 123130. Zupo. S.. Dono. W.. Azzoni. L.. Chiorazzi. N. & Ferrarini. M. (1991) Evidence for differential responsiveness of human CD5 + and CD5- B cell subsets to T cell-independent mitogens. European journal ofZmmuriology. 211. 351-359.