Peripheral blood or bone marrow of 24 patients with chronic myeloid leukemia (CML) were characterized for their surface membrane marker profiles using flow ...
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NEOPLASMA,42, l, 1995
Chronic myeloid leukemia: Correlation between purine metabolism enzyme activities and membrane immunophenotype A. MESÁROŠOVÁ, A. HRIVŇÁKOVÁ, 1M. KLOBUŠICKÁ, O. BABUŠÍKOVÁ Cancer Research Institute, Slovak Academy of Sciences, 812 32 Bratislava, Slovakia; lNational Cancer Institute, Bratislava, Slovakia
Received
August 8, 1994
Peripheral blood or bone marrow of 24 patients with chronic myeloid leukemia (CML) were characterized for their surface membrane marker profiles using flow cytometry and fluorescence microscopy. Purine metabolism enzyme activities were compared with membrane immunophenotype and cytochemical stains. CML subtypes were correlated with the expression of surface membrane antigens detected by the monoclonal antibodies. On the basis of immunophenotyping we found the following characteristic marker profiles: In stable phase of CML (CML-SP) - CDI5, CDIlb, CDw65, CDI3, in accelerated phase ofCML (CML-AP) - CDI5, CDw65, CDIIb, CD 13 and CD33, in myeloid blastic phase of CML(CML-BP-M) - CD 13, CD33, HLA-DR, CDIl b, CDI5, CDw65, in myeloid and lymphoid (mixed) blastic phase ofCML (CML-BP-M+L) - CDI3, CD33, CD34, HLA-DR, CDllb, CDIO and in chronic myelomonocytic leukemia (CMML) - CDI4, CDw65, CDllb, CD33 and HLA-DR. Analysis of purine metaboIism enzyme activities showed that there was a correlation between the values of adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) and various types of CML. ADA levels in CML-SP, CML-AP and CMML were comparable with those in normal cells. In CML-BP-M, which represents proliferation ofless mature myeloid cells (similar to less mature AML subtypes), ADA activity increased and PNP activity decreased. ADA activity was significantly different between control group and CML-BP-M (p < 0.0l), between CML-SP and CML-BP-M (p < 0.05). The values ofPNP activity were the highest in stable phase ofCML (125 pkat.ltľ'" cells) and the lowest (23 pkat.IO-6cells) in CML-BP-M+L. PNP activity in the other groups corresponded to control values. High ADAIPNP ratio was found in CML-BP-M and CML-BP-M+L (0.7 and 2.0, respectively) in comparison to CML-SP (0.2). It follows from our results that ADAIPNP ratio enables to discriminate between stable and blast phases ofCML (p < 0.01). The level of the cytochemical enzymes (CHAE, MPO, SBB,ANAE and 5' NT) varied and ref1ectedthe degree of cell differentiationand maturation.CHAE and MPO were characteristic enzymes for CML, ANBE for CMML and 5' NT for CML-BP-Iymphoid. Key words: Chronic myeloid leukemia, immunophenotype, cytochemical stains.
Chronic myeloid leukemia (CML) is a myeloproliferative disorder with a biphasic evolutive course [13]. The disease is presented by a chronic phase and subsequently evolves into a terminal phase -the blast crisis ofCML [23]. Most patients undergo transformation to blast crisis, often heralded by an intermediate period - the accelerated phase [5, 12]. Median survival usually observed for stable phase ranges from 3 to 4 years, in accelerated phase from 6 to 24 months and in blast crisis from 2 to 4 months, but great variability is ob served from case to case [5]. The simple finding of abnormal blood counts on routine testing frequently leads to the diagnosis ofCML in chronic phase. During the accelerated phase CI in ical symptoms
adenosine deaminase. purine nuc/eoside phosphorylase,
become more expressive. Erratic fluctuation in white blood cell (WBC) and plate let counts, increasing basophiles, rising leukocyte alkaline phosphatase (LAP), greater marrow immaturity and cytogenetic abnormalities may also accompany acceleration of the disease [5]. Blast crisis represents the evolution to acute leukemia. Two forms of blast crisis are generally seen: Myeloid in two-thirds of cases and lymphoid in fewer than one-third. Rarely, acute erythrocytic or megakaryocytic leukemias occur. The prognosis ofblast crisis is very poor, worse than de novo forms of AML or ALL [5]. CMML appears to be an overlap of various disorders, as it shows features ofboth myeloproliferative and myelodysplastic syndromes [20, 22].
MESÁROŠOVÁ, HRIVŇÁKOV Á, KLOBUŠICKÁ,BABUŠÍKOV Á
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Enzymes of purine degradation metabolism have been of interest in the management ofleukemic disease for many years. Some reports have described the relationship of ADA and PNP activities to the immunological subclasses of acute lymphoblastic leukemia [l, 16,21]. In our previous study we found in patients with acute myeloid leukemia that ADA activity decreased and PNP activity increased with maturation ofcells [15]. Low activities of ADA were found in leukemic cells of chronic lymphatic leukemia (CLL) and CML in stable phase of the disease [ll]. In the present study we have examined leukemic cells in CML patients for the presence of conventional immunological markers and these characteristic s were correlated with enzyme activities of ADA and PNP and with cytochemical enzyme stains.
Material
and methods
Patients. A total of 24 CML patients were tested. Their age ranged from 16 up to 85 years. There were 14 male and 10 female patients. The median age was 57 years. Samples. Both the bone marrow (20%) and the peripheral blood (80%) from 24 patients with newly diagnosed untreated CML were referred to immunophenotyping study. The diagnosis was based on morphological examination and on cytochemical stains. Leukemic ce lIs were separated on Verografm gradient and in some cases on gelatine. Blast cells after separation were washed, processed for immunophenotypic and cytochemical studies, or frozen in cell pellets and stored at -196°C until enzymatic analysis was performed. The peripheral blood lymphocytes from normal controls were simultaneously investigated for enzyme activities. Analysis of cel! surface phenotype. Cel! surface antigens were detected by a standard indirect immunofluorescence assay with monoclonal antibodies (MoAbs) representative of cluster differentiation (CD) (Table l). MoAbs in our laboratory for myeloid leukemia immunophenotyping included antibodies against mature and immature myeloid cells. MoAbs detecting CDlO, CD24, CD7, Ia/HLA-DR and MoAb VIPLI (megakaryocytes) were also used. Some MoAbs were kindly donated by prof. W. KNAPP, Immunologic Institute, Vienna, Austria, others were either prepared at our institute (Dr. B. CHORVÁTH,Dr. K. POLÁKOVÁ) or commercially available. Indirect immunofluorescen ce assay was performed on viable cel!s in microplates using swine anti-mouse IgG labelled with FITC as a second antibody. Analysis ofmyeloid ce lis was done using a FACStar flow cytometer (Becton-Dickinson) and an Orthoplan (Leitz) epiilluminated fluorescence microscope. Cases with more than 20% ofmononuclear cells reactive with the appropriate antibody were considered positive.
Tab let.
Monoclonal antibodies used for CML pbenotype analysis
Antibody
CD
Antigen expression in normal cells
VIMD5
CDI5
monocytes, granulocytes
VIM2
CDw65
monocytes, granulocytes
VIM12
CDllb
monocytes, granulocytes
VIMJ3
CDI4
monocytes
My7
CDJ3
monocytes and precursors, granulocytes and progenitors
My9
CD33
monocytes and precursors, myeloid progenitors
Myl0
CD34
bone marrow progenitor cells, TdT+cells
Bra 30 (HLA-DR)
B cells, monocytes, macrophages, activated T cells, some Iympoid and myeIoid cells
VILAI
CDlO
pre-B cells, granuIocytes
Leu9
CD7
T cells and
VIBC5
CD24
B cells, granulocytes
VIPLl
NC
megakaryobIasts
NK cells
Determination of purine enzyme activities. Detennination of adenosine deaminase (ADA, E.C. 3.5.4.4.) and purine nucleoside phosphorylase (PNP, E.C. 2.4.2.1.) was performed by paper radiochromatography with quantification ofreaction products by a liquid scintillation counter as described in detail in our previous report [1]. The results were expressed in pkat.lO-6 cells. Differences between group s were evaluated for significanee by Studenťs t-test. Cytochemistry. Cytochemical study was performed on cell smears of freshly isolated mononuclear cells, Slides were stained by standard cytochemical techniques including chloracetate esterase (CHAE), myeloperoxidase (MPO), Sudan Black B (SBB), alpha-naphthylbutyrate este rase (ANBE) and 5'nucleotidase (5'NT) [6, 9, 17]. CHAE is considered to be granulocyte specific, AN BE is a characteristic selective marker of monocytes, MPO and SBB are specific .cytoplasmic markers of myeloid cells [17]. The differential diagnostic value of 5'NT is appreciated in the distinction between the myeloid and lymphoid phenotype in blast crisis of chronic myeloid leukemia [9].
Results Immunophenotyping. In this study we have examined leukemic cells in CML patients for the presence of conventional immunological markers and these characteristics were correlated with enzyme activities of ADA and PNP. Antigen expressions in CML types are sh own in Table 2. Patients in the stable phase of CML showed characteristic
CHRONIC MYELOID Tab I e monoclonal
II
LEUKEMIA
2. Reactivity antibodies
pattern of chronic
myeloid
leukemia
with
antigen expressions (more than 50% of cases positive) for CDI5, CDllb, CDw65, CDI3 and CD33. Two patients in FAB subtypes accelerated phase ofCML had a high express ion ofCD15, CDllb, CDw65, CD13 and CD33. The patients with CML CML-SP CMML CML-AP CML-BP-M CML-BP-M+L in myeloid blastic phase demonstrated antigen expression n 10 3 2 7 2 (more than 50%) of CDI3, CD33, HL A-DR, CDw65, (%) 42 13 8 29 8 CDll band CDl5. The blasts of the patients with CMML Marker had a higher expression (more than 60%) ofCD14, CDw65, 6/9' 0/2 2/7 CDl5 4/7 0/2 CDllb, CD33, HLA-DR and low value of CDI3 (33%). 66b 100 O O 57 Two patients demonstrated mixed myeloid (CD34, CD13, CDw65 2/3 6/10 2/2 5/7 0/2 CD33, CDllb) and lymphoid (CDlO) cell phenotypes (Ta60 66 100 71 O ble 2). CD34 expressivity was the highest in patients with CD II 6/10 3/3 2/2 6/7 1/2 CML-BP-M+L. HLA-DR was strongly expressed in 60 100 100 86 50 CMML, CML-BP-M and CML-BP-M+L patients, whereas CD14 1110 3/3 1/2 1/6 Oli the lowest value of this marker was detected in CML-SP 10 100 50 17 O patients. COl3 4/5 1/3 2/2 3/3 1/1 Purine metabolism enzyme activities. The further meth80 33 100 100 100 odo logical approach to CML examination was the determiCD33 3/5 2/2 2/2 ll! 3/3 nation ofpurine enzyme activities. ADA levels in CML-SP, 100 60 100 100 100 CML-AP and CMML were comparable to those in normal CD34 1/3 0/2 0/2 1/1 0/2 cells (up to 30 pkat. 10-6 ce lis), while ADA level in both 33 O O O 100 CML-BP subtypes (myeloid and mixed myeloid + lymHLA-DR 1/8 3/3 1/2 5/5 2/2 phoid) was higher (average 60 and 51 pkat.l 0-6 celIs, 13 100 50 100 100 respectively). ADA activity was significantly different beCD24 3/6 1/2 liľ 0/3 tween control group and CML-myeloid BP (p < 0.01), 50 50 100 O between CML-SP and CML-BP-M (p < 0.05). l/8 CDlO 0/2 O/l 2/2 0/6 PNP activity in CML-SP was the highest (average 125 13 O O O 100 pkat.IO-6 cells). The values of others besides CML-BP-M+L a - Number of positive/number of cases; b - percent of positive cases. group corresponded with control group. PNP activity of CML-BP-M+L subtype was the lowest (23 pkat.l0-6 cells). PNP activity was not significantly different between the groups. 160 ADA/PNP ratio is shown in Fig. 2. This ratio was not very different between CML-SP, CML-AP and CMML (0.2 and 0.3 and 0.5, respecvl 120 tively), which corresponded with the control äJ group. On the other hand ADA/PNP ratio inu
