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Clinical and Immunological Findings in Large B-Cell Chronic Lymphocytic Leukemia

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Clinical and Immunological Findings in Large B-Cell Chronic Lymphocytic Leukemia CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 46, 177- 185 (1988) Clinical and Immunological Findings in Large B-Cell Chronic Lymphocytic Leukemia ALBERTO~RFAO,* MARCOSGONZALEZ,*JESUSFERNANDO SANMIGUEL,* MARIA CONSUELOCA~ZO,* PURIFICACIONGALINDo,t MARIADOLORESCABALLERO,SRAMIROJIMENEZ,§ AND ANTONIO LOPEZBORRASCA* *Servicio de Hematologia, Hospital Vniversitario, Salamanca; fVnidad docente de Bioestadistica, Facultad de Biologia, Vniversidad de Salamanca; SServicio de Hematologia, Hospital Virgen Blanca, Leon; $Servicio de Hematologia, Hospital Virgen de la Vega, Salamanca, Spain In order to define the characteristics of B-CLL cases in which the predominant cell population is composed of large lymphocytes, we studied 97 patients with B-CLL, com- paring the cell morphological features with the clinical and biological findings and the immunological phenotype of the proliferating cells. Multivariant analysis showed that there were three significantly different morphological groups: Typical CLL, large lym- phocyte CLL (LLL), and CLL with prolymphocytes (CLL/PL). The LLL group showed a greater incidence of lymphadenopathies (P < 0.05) and higher percentages of both p,+6+ cells (P < 0.01) and Fmc/7 + cells (P < 0.001) than in typical CLL. The main differences between LLL and CLL/PL were the peripheral blood lymphocyte count and the percentage of Fmc/7+ cells (P < 0.002)-both higher in the CLL/PL group-and the percentage of mouse rosette-forming cells (P < O.Ol)-lower in CLU PL. Further studies including functional assays and survival analyses could contribute to elucidating whether these groups are different entities or a single disease with marked heterogeneity. 0 1988 Academic Press. Inc. INTRODUCTION For many years the term chronic lymphocytic leukemia (CLL) has included several different lymphoproliferative disorders such as B-CLL, prolymphocytic- leukemia (PLL), and hairy cell leukemia (HCL) (1, 2). Currently, these diseases are well differentiated from B-CLL. However, within this latter group of patients there continues to be considerable clinical and biological heterogeneity (3, 4). Accordingly, although in most cases the morphological appearance of the cells is that of typical small lymphocytes with clumped chromatin and scanty cytoplasm, it is not uncommon to find cases in which the predominant cell population is represented by large lymphocytes with abundant cytoplasm and mature clumped chromatin without a nucleolus (5, 6). These findings pose the question of consid- ering whether one is dealing with a single disease with a certain heterogeneity or whether there might be different disorders. In this sense, recently Melo er al. (7) suggested the possibility that some cases of B-CLL in prolymphocytic transfor- mation might correspond to a clinical, morphological, and immunological entity different from both B-CLL and B-PLL, whereas other cases would correspond to the intermediate form described by Enno et a/. (8). Immunological markers consistitute an additional tool for elucidating these 177 0090-1229/88 $1.50 Copyright 0 1988 by Academic Press. Inc. All nghts of reproduction in any form reserved. 178 ORFAO ET AL. aspects since they permit the phenotypic distinction between cells according to their stage of antigenic maturation. Ln order to define the characteristics of B-CLL cases in which the predominant cell population is composed of large lymphocytes, we studied peripheral blood (PB) cells of 97 patients with B-CLL, comparing the morphological features with the clinical and biological findings and the immunological phenotype of the prolif- erating cells. Four patients with B-PLL were also included in the analysis. MATERIAL AND METHODS One hundred and one patients were studied between June 1984 and January 1986. Ninety-seven were B-CLL and four were B-PLL. Cases in which lymph node biopsy findings revealed non-Hodgkin’s lymphoma were excluded from the study. The diagnostic criteria for B-CLL were those proposed by RAI et al. (9). The B-CLL cases were classitied according to Rai’s staging and Binet’s staging (9. 10). The immunological markers and the morphological assessment were per- formed in all cases at the moment of diagnosis, prior to chemotherapy. The clinical and hematological data analyzed included age, sex, presence of lymphadenopathy in two or more anatomical regions, size of spleen, presence of anemia (hemoglobin < 10 g/dl) and/or thrombopenia (< 100 x IO9 platelets/liter), PB lymphocyte count, percentage of bone marrow (BM) lymphocyte infiltration, and the pattern of BM involvement (11). Morphology. Cell morphology was studied by light microscopy in PB films stained with May-Grunwald-Giemsa. According to the criteria of Melo et ctl. (7), with slight modifications, seven different types of lymphoid cells were estab- lished (Table 1). A total of 200 lymphoid cells were counted in each slide by one of us after a random sample of 40 cases had been independently assessed by two of the authors to test for the reproducibility of the criteria employed: simulta- neously, the diameter of the lymphocytes was measured with an occular microm- eter, counting 100 cells per patient. Immunological markers. Mononuclear cells from PB were obtained by Ficoll- Hypaque (Pharmacia Fine Chemicals) density gradient centrifugation and tested for (a) spontaneous rosette-forming cells with AET-treated sheep erythrocytes (SRFC) (12); (b) spontaneous rosette-forming cells with mouse erythrocytes (MRFC) (13); (c) surface immunoglobulins (sIg) were detected by direct immuno- fluorescence with rabbit F(ab’), antisera conjugated with fluorescein isothio- cyanate (FITC) polyvalent to human Igs and specific to human heavy and light chains (Behring and Nordic Lab); and (d) surface antigens assessed by indirect immunofluorescence with live monoclonal antibodies (McAb), GRBl (anti-class II MHC antigens) (14) Bl (anti-p35 antigen or CD20) (lS>, Fmc-7 (which recog- nizes a subpopulation of normal PB B lymphocytes and the majority of cells in B-PLL and HCL) (16), Fmc-8/Fmc-56 (anti-p24 antigen or CD9) (17), and Leu-1 (anti-p67 antigen or CD5) (18). According to the intensity, four degrees of fluores- cence were separated: undetectable, weak, moderate, and strong. The percentage of MRFC, sIg, Fmc-7, and Leul) positive cells was calculated from the total of B lymphocytes after removing the percentage of SRFC-T cells. Each case was con- TABLE 1 5: CRITERIA USEDFORTHEMORPHOLOGICALDEFINITIONOFLYMPHOID CELLS ii Cell Chromatin Amount of Other ? features Cell size pattern Nucleolus cytoplasm features 8 Small lymphocyte <2 erythrocytes Clumped Absent Scanty (high N/C) F: Large lymphocyte >2 erythrocytes Clumped Absent or very small Relatively abundant G and poorly defined (intermediate N/C) 5 Prolymphocyte Usually >2 Clumped Large, vesicular, Usually abundant erythrocytes prominent (intermediate N/C) ij Cleft lymphocyte >2 erythrocytes Clumped Absent Scanty (high N/C) Cleft nucleus 2 Lymphoplasmocyte >2 erythrocytes Clumped Absent or very small Abundant (low N/C) Slightly excentric $ nucleus 24 Basophilic 8 cytoplasm and halo perinuclear z3 Immunoblast >3 erythrocytes Finely dispersed Large, prominent, Very abundant (low Strongly basophilic usually >I N/C) cytoplasm L Granular >2 erythrocytes Clumped Absent Usually abundant Large azurophilic 2 lymphocyte (low N/C) cytoplasm granules z W 180 ORFAO ET AL. sidered positive for a marker when more than 10% of the cells displayed positivity for that marker. Statistical methods. To estimate the significance between means, the Mann- Whitney U or Student c test was used. A contrast for dichotomous variables was employed to estimate the significance between those parameters which were evaluated as presence/absence or positive/negative. To analyze whether the groups established were indeed different and, if so, which were the variables that most contribute to such a differentiation, the simultaneous HJ-Biplot technique (19, 20) was employed. The variables concerning clinical features were consid- ered as discontinuous variables, while the markers were considered both as con- tinuous and discontinuous, since they were introduced in two different analyses both as percentages of positive cells and as positive/negative cases. Low-range approximation was done according to the techniques proposed by Golub and Reinsch (21). RESULTS According to the morphology of lymphoid cells the 97 B-CLL patients were distributed as follows (Table 2): (a) typical CLL, 41 cases; (b) mixed CLL with small/large lymphocytes (CLL/LL), 28; (c) large lymphocyte CLL (LLL), 11; (d) CLL with prolymphocytes (CLL/PL), 7; and (e) CLL with cleft lymphocytes (Cleft LL), 10. No case of mixed large lymphocytes/prolymphocytes was de- tected. Mean lymphocytic diameters were significantly larger (P < 0.001) in the cases of LLL (11.3 -+ 0.8 km) than in those of typical CLL (7.7 -+ 1.2 pm). None of the cases had to be changed with respect to initial classification, in which erythrocyte size was employed as the comparative reference, after the measure- ment of lymphocytic diameter. The clinical and biological characteristics of the different groups are shown in Table 3. The LLL patients showed a significantly lower incidence of anemia and/ or thrombopenia (P < 0.01) with respect to the typical CLL. The finding of en- larged lymph nodes in two or more anatomical regions was more common in LLL (90%) than in the typical CLL cases (63%) (P < 0.05). All except one LLL patient were in stage I or II while 41% of typical CLL cases were in advanced stages, III/IV. No significant
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