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Enzymatic differences between chronic lymphocytic leukemia and prolymphocytic leukemia
Leukemia Research Vol. 9, No. 10, pp. 1271-1275, 1985. Printed in Great Britain.
0145-2126/8553.00 + 0.00 © 1985 Pergamon Press Ltd.
ENZYMATIC DIFFERENCES BETWEEN CHRONIC LYMPHOCYTIC LEUKEMIA AND PROLYMPHOCYTIC LEUKEMIA* HOWARD RATECH, WILLIAM Z. BORER,~" CARL D. WIN'BERGand HENRY RAPPAPORT James Irvine Center for the Study of Leukemia and Lymphoma, Divisions of Anatomic Pathology and i'Clinical Pathology, City of Hope National Medical Center, Duarte, Californfa 91010, U.S.A. (Received 31 October 1984. Accepted 31 January 1985) Abstract--Chronic lymphocytic leukemia and prolymphocytic leukemia of the B-cell immunophenotype are closely related disorders, but differ in their cytomorphologic and clinical features. In an attempt to differentiate further between these two forms of leukemia, we measured adenosine deaminase and purine nucleoside phosphorylase activities by using a linked-enzyme spectrophotometric assay on peripheral-blood leukemic cells from seven patients with chronic lymphocytic leukemia, three patients with prolyrnphocytic leukemia, and one patient with prolymphocytoid transformation of chronic lymphocytic leukemia. By using discriminant analysis, we were able to distinguish the two groups only on the basis of purine nucleoside phosphorylase activity (F, ,,; p<0.001). The purine nucleoside phosphorylase activity in leukemic cells with prolymphocytic cytomorphology was significantly elevated (mean = 58.6 nM/min/mg protein) compared to the activity in leukemic cells with lymphocytic cytomorphology (mean = 25.6 nM/min/ mg protein). There was only one patient with chronic lymphocytic leukemia who was assigned to the prolymphocytic leukemia group on the basis of her purine nucleoside phosphorylase activity. Our study suggests that purine nucleoside phosphorylase activity in leukemic cells may be useful in the distinction of prolymphocytic leukemia from chronic lymphocytic leukemia, and that it may be an enzymatic marker for the early detection of prolymphocytoid transformation of chronic lymphocytic leukemia. Key words: Adenosine deaminase, purine nucleoside phosphorylase, lymphocytic leukemia. INTRODUCTION CMRON1C lymphocytic leukemia (CLL) and prolymphocytic leukemia (PRL) of the B-cell immuno-
*Supported by Grants CA-26422 and CA-09308 awarded by the National Cancer Institute, DHHS, and by Hematopathology Tutorials, Inc. Drs Borer, Winberg and Rappaport are members of the City of Hope Cancer Research Center which is supported by Grant No. CA-16434 awarded by the National Cancer Institute. This work was also made possible in part by support from the James A. Harless Research Fund. Howard Ratech has been supported by an American Cancer Society Regular Clinical Fellowship, a Public Health Service Fellowship and a Biomedical Research Support Grant. Approval for the use of human materials and of hospital records in this study was granted by the City of Hope National Medical Center Institutional Review Board. Abbreviations: CLL, chronic lymphocytic leukemia; PRL, prolymphocytic leukemia; PTCLL, prolymphocytoid transformation of chronic lymphocytic leukemia; ADA, adenosine deaminase; PNP, purine nucleoside phosphorylase. Correspondence to: Howard Ratech, Division of Anatomic Pathology, City of Hope National Medical Center, Duarte, California 91010, U.S.A.
phenotype differ in their clinical behavior [2, 8], cytologic features [2, 81, cytogenetic abnormalities [6, 19] and density of surface immunogiobulins [13, 14]. However, it is well established that CLL may transform to PRL [7, 13], suggesting an intimate relationship between these types of leukemia. Recently, adenosine deaminase (ADA) and purine nuclenside phosphorylase (PNP) have been shown to be important enzymes in normal lymphoid development, and to be possible markers for some lymphoid neoplasias (reviewed in [9]). Therefore, we measured ADA and PNP activities in leukemic cells from patients with CLL and PRL in an attempt to determine whether the activities of these enzymes may be of use in indicating a difference between these two closely related disorders. MATERIALS AND METHODS Preparation of patient leukemic cells Heparinized peripheral blood was obtained by venipuncture from seven patients with CLL, three patients with PRL, and one patient with prolymphocytoid transformation of chronic lymphocytic leukemia (PTCLL). Peripheral blood mononuclear cells were separated on Ficoll-Hypaque gradients [4], washed, and frozen in cell pellets (I0' cells), and stored at
1271
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HOWARD RATECH et aL
-70°C until enzymatic analysis was performed. Thawed cell pellets were suspended in 0.5 ml of cold 0.0l M sodium phosphate, pH 7.0, sonicated on ice for three 5s bursts with a Branson sonicator, and centrifuged at 7000 g for 5 rain in a Beckman microcentrifuge. The clear supernatant was then analyzed for enzymatic activity.
Spectrophotometric ADA and PNP assays A linked spectrophotometric assay was used for the measuremerit of ADA [11] and PNP activities in leukemic-cell sonicates. In brief, in our assay of ADA activity, we used 0.9 ml of 3.89 mM adenosine (Sigma Co., St. Louis, MO) as substrate in 0.1 M sodium phosphate, pH 7.0. After addition of 50 1~1 of the leukemic-cell sample and 50 )xl of PNP and xanthine oxidase (Calbiochem-Behring, Los Angeles, CA), the rate of increase in absorption at 293 nm and 37* was measured with a Gilford recording spectrophotometer. The assay for purine nucleoside phosphorylase was similar; we used 0.9 ml of 0.11 mM inosine (Sigma Co., St. Louis, MO) as substrate in 0.1 M sodium phosphate, pH 7.0. After addition of 50 )d of the leukemic-cell sample and 50 ~tl of xanthine oxidase, the rate of increase in absorption at 293 nm and 37°C was measured with a Gilford recording spectrophotometer. Protein was determined by the method of Lowry [16]. Analysis of cell surface immunophenotype Peripheral blood mononuclear cells were stained with FITCconjugated rabbit antisera reactive with human IgM, IgD, leG, and IgA immunoglobulin heavy chains and with kappa and lambda immunoglobulin light chains (Tago, Burlingame, CA). Cells were enumerated under a Leitz Ortholux microscope equipped with epifluorescence and with a K-cube for fluorescein excitation. The results were interpreted according to established criteria for monodonality [31. The fluorescence intensity was graded on a scale from + to + + + +, where + = very faint, + + = faint, + + + = bright, and + + + + = very b~ight. Criteria for differentiating cases of chronic lymphocytic leukemia and prolymphocytic leukemia Cytomorphology. Peripheral-blood smears obtained from leukemic patients were stained by the May-Gr(lnwald Giemsa technique, and 200 cells were differentially counted. Lymphocytes and prolymphocytes were distinguished according to previously reported criteria [2, 8]. Leukemic lymphocytes as seen in CLL were defined as small ceils with condensed nuclear chromatin, inconspicuous nucleoli, and a relatively narrow cytoplasmic rim. Leukemic prolymphocytes as seen in PRL were defined as large lymphoid cells with nuclear chromatin coarser than that of lymphoblasts, but finer than that of lymphocytes, and with a single, prominent nucleolus having condensed chromatin around the nucleolar margins. The cytoplasm was invariably more abundant than that of the lymphocytes of CLL. We interpreted an approximately equal mixture of lymphocytes and prolymphocytes as suggestive of prolymphocytoid transformation of chronic lymphocytic leukemia (PTCLL; [7, 13]). Cell surface immunoglobulin fluorescence intensity. The density of cell surface immunoglobulin in cases of B-cell CLL is known to be much less than in cases of B-cell PRL [13, 14]. We, therefore, used a + to + + + + grading system in recording surface immunoglobulin fluorescence intensity of leukemic cells stained with fluoresceinated anti-
immunoglobulin reagents as a measure of surface immunoglobulin density (see Analysis of Cell Surface lmmunophenotype). All cases with dim fluorescence ( + Or + + ) corresponded to CLL cytomorphologically and all cases with bright fluorescence (+ + +) corresponded to PRL cytomorphologically. We used the criteria for immunoglobulin fluorescence intensity described by Kjeldsberg and Marty [13] in order to distinguish prolymphocytoid transformation of chronic lymphocytic leukemia from CLL and PRL. In PTCLL they observed an increase in density of surface immunoglobulin of the prolymphocytes compared to that of small lymphocytes.
Statistical analysis For statistical evaulation of the data, we performed discriminant analysis [15] to analyze age, sex, WBC, differential cell count, immunoglobulin heavy and light chains, ADA and PNP activities.
RESULTS On the basis of cytomorphologic and immunologic criteria, seven patients were classified as C L L and three patients were classified as P R L . Patient 9 had a peripheral blood smear conta:;ning a mixed population o f lymphocytes and prolymphocytes in nearly equal proportions, and his disease was classified as prolymphocytoid transformation of chronic lymphocytic leukemia ( P T C L L ) [7, 13]. This interpretation was supported by immunofluorescence microscopy which revealed similar proportions of small lymphocytes with dim fluorescence and larger lymphoid cells with bright immunofluorescence when stained by fluoresceinated anti-immunoglobulin reagents. In addition, the clinical course o f patient 9 was characterized by the appearance of massive splenomegaly without lymphadenopathy after two years o f quiescence, as has been reported in cases o f P T C L L [7, 131. We reported the age, sex, total peripheral-blood white-cell count, differential count, cell surface imm u n o p h e n o t y p e and immunofluorescence intensity, and leukemic-cell A D A and P N P activities (Table 1A, B). The patient with P T C L L No. (9) was included in the P R L group for analysis. By discriminant analysis, we were able to differentiate between leukemic cells from patients with C L L and those from patients with P R L on the basis of P N P activity only (F,,9 = 12.1; p < 0 . 0 0 1 ) (Table IA, B) (mean P N P activities = 25.6 n M / m i n / m g protein and 58.6 n M / m i n / m g protein, respectively). Only one patient (No. 5) was assigned to the opposite group by the discriminant function. No other significant differences between C L L and P R L were found with respect to A D A activity, age, sex, or total peripheral-blood white-cell count (.0>0.05) except for the expected differences in differential peripheral blood cell counts (i.e. lymphocyte and prolymphocyte counts) and in the immunofluorescence intensity of surface immunoglobulin. Each patient included in this study had a monoctonal population o f B cells circulating in the peripheral blood. Two patients with P R L expressed lgG heavy chains on
I .
ADA AND
57 59 62 62 68 72 83
1 2 3 4 5 6 7
F M F M F M M
Sex
20.6 189.0 115.8 383.0 12.9 26.7 29.7
WBC x 103
64 65 66 67
8 91 10 !1
M M F M
Sex
101.0 77.1 8.9 3.1
4.5 45 14 14
WBC x 10 ~ L y m p h
0.5 0 0 ! 0 0 0.5
Band
LYMPHOCYTIC
22 6 0 4.5 17 4 I 1.5
0 0 1 2
5.5 7 28.5 22
0 1.5 2 2
0.5 0.5 4 0
Eos
I 0 0.5 0.5 I 0 0.5
Baso
0 0 0 0 0 0 0
Baso K L K K K K L
M G M,D G
K K K K
Phenotype-heavy & light chain
D M M,D M,D M,D M,D M
+ + +/+ + + + + Mean
+ + + +
+ +
Mean
25.6
41.6 60.5 69.1 63.1 58.6
9.5 5.6 21.1 11.4 11.9
Enzyme activity~/ PNP ADA
! ! .4
+
+ + +
22.8 20.0 24.1 28. I 59.5 10.7 13.8
Enzyme activity J/ ADA PNP
22.8 2.5 12.3 NT 15.7 3.6 NT
+ + + + + + +
SIFIt
SlFlt
LEUKEMIA
Phenotype-heavy & light chain
AND PROLYMPHOCYTIC
I 0 0 0.5 0 0 0
Eos
LEUKEMIA
cell counts (%)* Seg Mono
Differential
IES IN CHRONIC
Differential cell counts (%)* Band Seg Mono
5.5 3 0.5 8 I 2 4
Pro
ACTIVN
89.5 ~6 50.5 60
Pro
70 91 99 85.5 81 94 84.5
Lymph
PNP
*200 cells counted. tSIFI = Surface immunoglobulin fluorescence intensity + = very faint; + + = faint; + + + = bright, + + + + = very bright. ~ln n M / m i n / m g protein; NT = not tested. QProlymphocytoid transformation of chronic lymphocytic leukemia.
Age (yr)
Patient No.
TAmE lB. PROLYMPHOCYTICLEUKEMIA
Age (yr)
Patient No.
Table IA. Chronic lymphocytic leukemia
TABI.E
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HOWARDRATECHel al.
their leukemic cells, in contrast to none with CLL (Table 1A, B). 2.
DISCUSSION
3.
We have measured ADA and PNP activities in seven patients with CLL and in four patients with PRL, including one with PTCLL. The patients in each group had circulating monoclonal B cells in their peripheral blood and exhibited the cytologic features and cellsurface immunoglobulin fluorescence intensities characteristic of that disease group. The activity of PNP, but not that of ADA, was found by discriminant analysis to be significantly different in these two disorders. The mean values for PNP activity in CLL and PRL were 25.6 and 58.6 n M / m i n / m g protein, respectively. Our values for ADA and PNP activities in CLL agree with those published by others and referenced in a recent review article [10]. Gan et al. have reported values for ADA and PNP in CLL and in PRL [9]. It is of interest that, similar to our own data, there was no difference in ADA activities between these two groups. However, these authors found a bimodal distribution of PNP activities in CLL. In their two cases of PRL, the PNP activity was in the upper half of that determined in CLL. Although ADA activity has been measured in complement receptor-positive and -negative populations of peripheral-blood mononuclear cells from patients with CLL [12], a correlation with the immunophenotype has not been reported in CLL. In the present study, we did not detect a clear relationship between ADA or PNP activity and heavy-chain class. Activities of PNP measured by others in patients with acute lymphoblastic leukemia (ALL) and in normal peripheral-blood mononuclear cells were found to be greater than those measured in patients with CLL [1, 14, 17, 18]. We have found in the present study that the PNP activity in PRL is also greater than that in CLL. The higher values of PNP in PRL than in CLL may be related to differences in cytomorphology or maturation, or to the greater frequency of the IgG immunophenotype in PRL [51, Patient 5 is the only one with CLL in our study whose leukemic-cell PNP value falls within the range of values seen in PRL. It will be of interest to follow the clinical course of this patient to see whether high PNP activity is useful as an early marker of prolymphocytoid transformation. This might be anticipated in view of the high PNP activity measured in the leukemic cells of the patient with prolymphocytoid transformation of CLL
(patient 9). Acknowledgements--The authors thank Robert Hill, Biostatistics Department, City of Hope National Medical Center, Duarte, California for statistical analyses and Jan Maurer and Pamela Edwards for expert secretarial assistance.
REFERENCES I. Ambrogi F., Grass B., Simonetta R.-T., & Ronca G. (1977) Blood lymphocytes in chronic lymphocytic
4.
5.
6.
7.
8.
9.
I0.
leukaemia and Hodgkin's disease. Clin. exp. Immun. 28, 80. Bearman R. M., Pangalis G. A. & Rappaport H. (1978) Prolymphocytic leukemia: clinical, histopathological and cytochemical observations. Cancer 42, 2360. Bloomfield C. D., Gajl-Peczalska K. J., Frizzera G.. Kersey J. H. & Goldman A. I. 0979) Clinical utility of lymphocyte surface markers combined with the LukesCollins histologic classification of adult lymphoma. New EngL J. Med. 301, 512. Boyum A. (1968) Separation of leukocytes from blood and bone marrow (with special reference to factors which influence and modify sedimentation properties of hematopoietic cells). Scand. J. clin. Lab. Invest. 21, 77. Catovsky D. (1982) Prolymphocytic and hairy cell leukemias. In Leukemia, 4th edition (Gunz F. W. & Henderson E. S., Eds.), p. 759. Grune & Stratton, New York. Diamond L. W., Bearman R. M., Berry P. K., Mills B. J., Nathwani B. N., Weisenburger D. D., Winberg C. D., Teplitz R. L. & Rappaport H. (1980) Prolymphocytic leukemia: flow microfluorometric, immunologic and cytogenetic observations. Am. J. Hemat. 9, 319. Enno A., Catovsky D., O'Brien M., Cherchi M., Kumaran T. O. & Galton D. A. G. (1979) 'Prolymphocytoid' transformation of chronic lymphocytic leukemia. Br. J. Haemat. 41, 9. Galton D. A. G., Goldman J. M., Whitshaw E., Catovsky D., Henry K, & Goldenberg G. J. (1974) Prolymphocytic leukemia. Br. J. Haemat. 27, 7. Gan T. E., Hallam L. & Van der Weyden M. B. (1982) Purine and pyrimidine activities in acute and chronic lymphocytic leukemia: relation to cellular proliferative status. Leukemia Res. 6, 839. Hirschhorn R. & Ratech H. (1983) Genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase and their implications for therapy of leukemias. Current Topics Hemat. 4, 2.
I1. Hirschhorn R., Roegner V., Jenkins T., Seaman C., Piomelli S. & Borkowsky W. (1979) Erythrocyte adenosine deaminase deficiency without immunodeficiency: evidence for an unstable mutant enzyme. J. clin Invest. 64, 1130. 12. Huang A. T., Logue G. L. & Engdbrecht H. L. (1976) Two biochemical markers in lymphocyte subpopulations. Br. J. Haemat. 34, 631. 13. Kjeldsberg C. R. & Marty J. (1981) Prolymphocytic transformation of chronic lymphocytic leukemia. Cancer 48, 2,*47. 14. Koziner B., Kempin S., Passe S., Gee T., Good R. A. & Clarkson B. D. (1980) Characterization of B-cell leukemias: a tentative immunomorphological scheme. Blood 56, 815. 15. Lachenbruch P. A. (1975) In Discriminant analysis. Hafner Press, New York. 16. Lowry O. H., Rosebrough N. J., Farr A. L. & Randal R. J. (1951) Protein measurement with the Folin-phenol reagent. J. Biochem. 193, 265. 17. Ludwig H., Kuzmits R., Pietschmann H. & Muller M. M. (1979) Enzymes of the purine interconversion system in chronic lymphatic leukemia: decreased purine nucleoside
Enzymes in chronic lymphocytic leukemia and prolymphocytic leukemia
phosphorylase and adenosine deaminase activity.BIut 39, 309. 18. Mejer J. & Nygaard P. 0979) Adenosine deaminase and purine nucleoside phosphorylase levels in acute myeloblastic leukemia cells. Relationship to diagnosis and
1275
clinical course. Leukemia Rea. 3, 211. 19. Sadamori N., Han T., Minowada J., Bloom M. L., Henderson E. S. & Sandberg A. A. 0983) Possible specific chromosome change in prolymphocytic leukemia. Blood 62, 729.
0145-2126/8553.00 + 0.00 © 1985 Pergamon Press Ltd.
ENZYMATIC DIFFERENCES BETWEEN CHRONIC LYMPHOCYTIC LEUKEMIA AND PROLYMPHOCYTIC LEUKEMIA* HOWARD RATECH, WILLIAM Z. BORER,~" CARL D. WIN'BERGand HENRY RAPPAPORT James Irvine Center for the Study of Leukemia and Lymphoma, Divisions of Anatomic Pathology and i'Clinical Pathology, City of Hope National Medical Center, Duarte, Californfa 91010, U.S.A. (Received 31 October 1984. Accepted 31 January 1985) Abstract--Chronic lymphocytic leukemia and prolymphocytic leukemia of the B-cell immunophenotype are closely related disorders, but differ in their cytomorphologic and clinical features. In an attempt to differentiate further between these two forms of leukemia, we measured adenosine deaminase and purine nucleoside phosphorylase activities by using a linked-enzyme spectrophotometric assay on peripheral-blood leukemic cells from seven patients with chronic lymphocytic leukemia, three patients with prolyrnphocytic leukemia, and one patient with prolymphocytoid transformation of chronic lymphocytic leukemia. By using discriminant analysis, we were able to distinguish the two groups only on the basis of purine nucleoside phosphorylase activity (F, ,,; p<0.001). The purine nucleoside phosphorylase activity in leukemic cells with prolymphocytic cytomorphology was significantly elevated (mean = 58.6 nM/min/mg protein) compared to the activity in leukemic cells with lymphocytic cytomorphology (mean = 25.6 nM/min/ mg protein). There was only one patient with chronic lymphocytic leukemia who was assigned to the prolymphocytic leukemia group on the basis of her purine nucleoside phosphorylase activity. Our study suggests that purine nucleoside phosphorylase activity in leukemic cells may be useful in the distinction of prolymphocytic leukemia from chronic lymphocytic leukemia, and that it may be an enzymatic marker for the early detection of prolymphocytoid transformation of chronic lymphocytic leukemia. Key words: Adenosine deaminase, purine nucleoside phosphorylase, lymphocytic leukemia. INTRODUCTION CMRON1C lymphocytic leukemia (CLL) and prolymphocytic leukemia (PRL) of the B-cell immuno-
*Supported by Grants CA-26422 and CA-09308 awarded by the National Cancer Institute, DHHS, and by Hematopathology Tutorials, Inc. Drs Borer, Winberg and Rappaport are members of the City of Hope Cancer Research Center which is supported by Grant No. CA-16434 awarded by the National Cancer Institute. This work was also made possible in part by support from the James A. Harless Research Fund. Howard Ratech has been supported by an American Cancer Society Regular Clinical Fellowship, a Public Health Service Fellowship and a Biomedical Research Support Grant. Approval for the use of human materials and of hospital records in this study was granted by the City of Hope National Medical Center Institutional Review Board. Abbreviations: CLL, chronic lymphocytic leukemia; PRL, prolymphocytic leukemia; PTCLL, prolymphocytoid transformation of chronic lymphocytic leukemia; ADA, adenosine deaminase; PNP, purine nucleoside phosphorylase. Correspondence to: Howard Ratech, Division of Anatomic Pathology, City of Hope National Medical Center, Duarte, California 91010, U.S.A.
phenotype differ in their clinical behavior [2, 8], cytologic features [2, 81, cytogenetic abnormalities [6, 19] and density of surface immunogiobulins [13, 14]. However, it is well established that CLL may transform to PRL [7, 13], suggesting an intimate relationship between these types of leukemia. Recently, adenosine deaminase (ADA) and purine nuclenside phosphorylase (PNP) have been shown to be important enzymes in normal lymphoid development, and to be possible markers for some lymphoid neoplasias (reviewed in [9]). Therefore, we measured ADA and PNP activities in leukemic cells from patients with CLL and PRL in an attempt to determine whether the activities of these enzymes may be of use in indicating a difference between these two closely related disorders. MATERIALS AND METHODS Preparation of patient leukemic cells Heparinized peripheral blood was obtained by venipuncture from seven patients with CLL, three patients with PRL, and one patient with prolymphocytoid transformation of chronic lymphocytic leukemia (PTCLL). Peripheral blood mononuclear cells were separated on Ficoll-Hypaque gradients [4], washed, and frozen in cell pellets (I0' cells), and stored at
1271
1272
HOWARD RATECH et aL
-70°C until enzymatic analysis was performed. Thawed cell pellets were suspended in 0.5 ml of cold 0.0l M sodium phosphate, pH 7.0, sonicated on ice for three 5s bursts with a Branson sonicator, and centrifuged at 7000 g for 5 rain in a Beckman microcentrifuge. The clear supernatant was then analyzed for enzymatic activity.
Spectrophotometric ADA and PNP assays A linked spectrophotometric assay was used for the measuremerit of ADA [11] and PNP activities in leukemic-cell sonicates. In brief, in our assay of ADA activity, we used 0.9 ml of 3.89 mM adenosine (Sigma Co., St. Louis, MO) as substrate in 0.1 M sodium phosphate, pH 7.0. After addition of 50 1~1 of the leukemic-cell sample and 50 )xl of PNP and xanthine oxidase (Calbiochem-Behring, Los Angeles, CA), the rate of increase in absorption at 293 nm and 37* was measured with a Gilford recording spectrophotometer. The assay for purine nucleoside phosphorylase was similar; we used 0.9 ml of 0.11 mM inosine (Sigma Co., St. Louis, MO) as substrate in 0.1 M sodium phosphate, pH 7.0. After addition of 50 )d of the leukemic-cell sample and 50 ~tl of xanthine oxidase, the rate of increase in absorption at 293 nm and 37°C was measured with a Gilford recording spectrophotometer. Protein was determined by the method of Lowry [16]. Analysis of cell surface immunophenotype Peripheral blood mononuclear cells were stained with FITCconjugated rabbit antisera reactive with human IgM, IgD, leG, and IgA immunoglobulin heavy chains and with kappa and lambda immunoglobulin light chains (Tago, Burlingame, CA). Cells were enumerated under a Leitz Ortholux microscope equipped with epifluorescence and with a K-cube for fluorescein excitation. The results were interpreted according to established criteria for monodonality [31. The fluorescence intensity was graded on a scale from + to + + + +, where + = very faint, + + = faint, + + + = bright, and + + + + = very b~ight. Criteria for differentiating cases of chronic lymphocytic leukemia and prolymphocytic leukemia Cytomorphology. Peripheral-blood smears obtained from leukemic patients were stained by the May-Gr(lnwald Giemsa technique, and 200 cells were differentially counted. Lymphocytes and prolymphocytes were distinguished according to previously reported criteria [2, 8]. Leukemic lymphocytes as seen in CLL were defined as small ceils with condensed nuclear chromatin, inconspicuous nucleoli, and a relatively narrow cytoplasmic rim. Leukemic prolymphocytes as seen in PRL were defined as large lymphoid cells with nuclear chromatin coarser than that of lymphoblasts, but finer than that of lymphocytes, and with a single, prominent nucleolus having condensed chromatin around the nucleolar margins. The cytoplasm was invariably more abundant than that of the lymphocytes of CLL. We interpreted an approximately equal mixture of lymphocytes and prolymphocytes as suggestive of prolymphocytoid transformation of chronic lymphocytic leukemia (PTCLL; [7, 13]). Cell surface immunoglobulin fluorescence intensity. The density of cell surface immunoglobulin in cases of B-cell CLL is known to be much less than in cases of B-cell PRL [13, 14]. We, therefore, used a + to + + + + grading system in recording surface immunoglobulin fluorescence intensity of leukemic cells stained with fluoresceinated anti-
immunoglobulin reagents as a measure of surface immunoglobulin density (see Analysis of Cell Surface lmmunophenotype). All cases with dim fluorescence ( + Or + + ) corresponded to CLL cytomorphologically and all cases with bright fluorescence (+ + +) corresponded to PRL cytomorphologically. We used the criteria for immunoglobulin fluorescence intensity described by Kjeldsberg and Marty [13] in order to distinguish prolymphocytoid transformation of chronic lymphocytic leukemia from CLL and PRL. In PTCLL they observed an increase in density of surface immunoglobulin of the prolymphocytes compared to that of small lymphocytes.
Statistical analysis For statistical evaulation of the data, we performed discriminant analysis [15] to analyze age, sex, WBC, differential cell count, immunoglobulin heavy and light chains, ADA and PNP activities.
RESULTS On the basis of cytomorphologic and immunologic criteria, seven patients were classified as C L L and three patients were classified as P R L . Patient 9 had a peripheral blood smear conta:;ning a mixed population o f lymphocytes and prolymphocytes in nearly equal proportions, and his disease was classified as prolymphocytoid transformation of chronic lymphocytic leukemia ( P T C L L ) [7, 13]. This interpretation was supported by immunofluorescence microscopy which revealed similar proportions of small lymphocytes with dim fluorescence and larger lymphoid cells with bright immunofluorescence when stained by fluoresceinated anti-immunoglobulin reagents. In addition, the clinical course o f patient 9 was characterized by the appearance of massive splenomegaly without lymphadenopathy after two years o f quiescence, as has been reported in cases o f P T C L L [7, 131. We reported the age, sex, total peripheral-blood white-cell count, differential count, cell surface imm u n o p h e n o t y p e and immunofluorescence intensity, and leukemic-cell A D A and P N P activities (Table 1A, B). The patient with P T C L L No. (9) was included in the P R L group for analysis. By discriminant analysis, we were able to differentiate between leukemic cells from patients with C L L and those from patients with P R L on the basis of P N P activity only (F,,9 = 12.1; p < 0 . 0 0 1 ) (Table IA, B) (mean P N P activities = 25.6 n M / m i n / m g protein and 58.6 n M / m i n / m g protein, respectively). Only one patient (No. 5) was assigned to the opposite group by the discriminant function. No other significant differences between C L L and P R L were found with respect to A D A activity, age, sex, or total peripheral-blood white-cell count (.0>0.05) except for the expected differences in differential peripheral blood cell counts (i.e. lymphocyte and prolymphocyte counts) and in the immunofluorescence intensity of surface immunoglobulin. Each patient included in this study had a monoctonal population o f B cells circulating in the peripheral blood. Two patients with P R L expressed lgG heavy chains on
I .
ADA AND
57 59 62 62 68 72 83
1 2 3 4 5 6 7
F M F M F M M
Sex
20.6 189.0 115.8 383.0 12.9 26.7 29.7
WBC x 103
64 65 66 67
8 91 10 !1
M M F M
Sex
101.0 77.1 8.9 3.1
4.5 45 14 14
WBC x 10 ~ L y m p h
0.5 0 0 ! 0 0 0.5
Band
LYMPHOCYTIC
22 6 0 4.5 17 4 I 1.5
0 0 1 2
5.5 7 28.5 22
0 1.5 2 2
0.5 0.5 4 0
Eos
I 0 0.5 0.5 I 0 0.5
Baso
0 0 0 0 0 0 0
Baso K L K K K K L
M G M,D G
K K K K
Phenotype-heavy & light chain
D M M,D M,D M,D M,D M
+ + +/+ + + + + Mean
+ + + +
+ +
Mean
25.6
41.6 60.5 69.1 63.1 58.6
9.5 5.6 21.1 11.4 11.9
Enzyme activity~/ PNP ADA
! ! .4
+
+ + +
22.8 20.0 24.1 28. I 59.5 10.7 13.8
Enzyme activity J/ ADA PNP
22.8 2.5 12.3 NT 15.7 3.6 NT
+ + + + + + +
SIFIt
SlFlt
LEUKEMIA
Phenotype-heavy & light chain
AND PROLYMPHOCYTIC
I 0 0 0.5 0 0 0
Eos
LEUKEMIA
cell counts (%)* Seg Mono
Differential
IES IN CHRONIC
Differential cell counts (%)* Band Seg Mono
5.5 3 0.5 8 I 2 4
Pro
ACTIVN
89.5 ~6 50.5 60
Pro
70 91 99 85.5 81 94 84.5
Lymph
PNP
*200 cells counted. tSIFI = Surface immunoglobulin fluorescence intensity + = very faint; + + = faint; + + + = bright, + + + + = very bright. ~ln n M / m i n / m g protein; NT = not tested. QProlymphocytoid transformation of chronic lymphocytic leukemia.
Age (yr)
Patient No.
TAmE lB. PROLYMPHOCYTICLEUKEMIA
Age (yr)
Patient No.
Table IA. Chronic lymphocytic leukemia
TABI.E
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HOWARDRATECHel al.
their leukemic cells, in contrast to none with CLL (Table 1A, B). 2.
DISCUSSION
3.
We have measured ADA and PNP activities in seven patients with CLL and in four patients with PRL, including one with PTCLL. The patients in each group had circulating monoclonal B cells in their peripheral blood and exhibited the cytologic features and cellsurface immunoglobulin fluorescence intensities characteristic of that disease group. The activity of PNP, but not that of ADA, was found by discriminant analysis to be significantly different in these two disorders. The mean values for PNP activity in CLL and PRL were 25.6 and 58.6 n M / m i n / m g protein, respectively. Our values for ADA and PNP activities in CLL agree with those published by others and referenced in a recent review article [10]. Gan et al. have reported values for ADA and PNP in CLL and in PRL [9]. It is of interest that, similar to our own data, there was no difference in ADA activities between these two groups. However, these authors found a bimodal distribution of PNP activities in CLL. In their two cases of PRL, the PNP activity was in the upper half of that determined in CLL. Although ADA activity has been measured in complement receptor-positive and -negative populations of peripheral-blood mononuclear cells from patients with CLL [12], a correlation with the immunophenotype has not been reported in CLL. In the present study, we did not detect a clear relationship between ADA or PNP activity and heavy-chain class. Activities of PNP measured by others in patients with acute lymphoblastic leukemia (ALL) and in normal peripheral-blood mononuclear cells were found to be greater than those measured in patients with CLL [1, 14, 17, 18]. We have found in the present study that the PNP activity in PRL is also greater than that in CLL. The higher values of PNP in PRL than in CLL may be related to differences in cytomorphology or maturation, or to the greater frequency of the IgG immunophenotype in PRL [51, Patient 5 is the only one with CLL in our study whose leukemic-cell PNP value falls within the range of values seen in PRL. It will be of interest to follow the clinical course of this patient to see whether high PNP activity is useful as an early marker of prolymphocytoid transformation. This might be anticipated in view of the high PNP activity measured in the leukemic cells of the patient with prolymphocytoid transformation of CLL
(patient 9). Acknowledgements--The authors thank Robert Hill, Biostatistics Department, City of Hope National Medical Center, Duarte, California for statistical analyses and Jan Maurer and Pamela Edwards for expert secretarial assistance.
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