American Journal of Hematology 38:332-334 (1991)
Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia by Flow Cytometry With Monoclonal Antibodies Nobutaka lmamura and Atsushi Kuramoto Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima, Japan
To detect more precisely the minimal residual disease in acute lymphoblastic leukemia (ALL), two-color flow cytometric analysis for the detection of cell-surface antigen (CD10; CALLA) and nuclear terminal deoxynucleotidyl transferase (TdT) was performed in the six patients with CALLA-positive ALL coexpressing TdT. In all patients, the leukemic blasts coexpressed la (HLA-DR), CD9, CD19, CD20, CD24, and CDlO. Five of six patients achieved complete remission, but one has so far relapsed. No leukemic blasts (CDlO+, TdT') were detected at the time of complete remission. During maintenance chemotherapy, leukemic blasts coexpressed C10 and TdT were found 2.32% in the patient's peripheral blood by two-color analysis, whereas no obvious leukemic cells were recognized morphologically. The patient relapsed leukemia with the same phenotype 4 weeks after the examination. On the basis of our findings, we suggest that two-color flow cytometric analysis with the use of these antibodies is quite valuable to detect the minimal residual leukemic cells in a patient with ALL. The reduction of leukemic cells below the threshold of detection of methods currently available appears to be necessary to achieve a cure in ALL. Hence accurate diagnosis of ALLs with monoclonal antibodies (MAbs) should contribute substantially to the development of an effective form of therapy for their cure. Key words: minimal residual ALL, CALLA (CDlO), TdT
INTRODUCTION
Lymphoid progenitor cells express the nuclear enzyme terminal deoxynucleotidyl transferase (TdT), which catalyzes the random addition of free deoxynucleotides onto the 3'-hydroxyl terminus of a nucleotide without the presence of a primer [ 11. Since TdT is found in the nuclei of lymphoblasts of more than 90% of patients with acute lymphoblastic leukemia (ALL), the utility of measuring TdT to diagnose ALL has been well established [ 1-31. ALL cells closely resemble normal lymphocyte precursors in their expression of lineage-specific proteins and rearrangement of antigen receptor genes [2-4]. Indeed, no truly leukemia-specific proteins that could serve as markers for minimal residual disease have been identified. Recently, detection of low number of TdT-positive cells has gained importance in monitoring residual leukemia after intensive chemotherapy. However, detection of TdT alone without combination of other lineageassociated antigens was not sufficiently specific for the 0 1991 Wiley-Liss, Inc.
leukemic phenotypes to predict relapse in peripheral blood samples accurately [ 5 ] . Hence double marker studies are necessary to discriminate reliably between normal and residual leukemic cells [6]. Previously, we reported that common ALL antigen (CALLA)-positive ALL cells were also positive for TdT [ 3 ] . We describe here the surveillance of minimal residual ALL cells by means of two-color flow cytometric analysis. MATERIALS AND METHODS Diagnosis and Samples
The initial diagnosis of leukemias was established by means of examination of the peripheral blood and bone marrow smears prepared with May-Griinwald-Giemsa Received for publication June 10, 1991; accepted June 20, 1991. Address reprint requests to Nobutaka Imamura, MD, Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan.
Brief Report: Minimal Residual Leukemic Cells by Flow Cytometry
and myeloperoxidase (MPO) stains. The morphologic criteria for the ALL was based on the FAB classification. Sixty-six cases were diagnosed as ALL; the cells from all the cases were both MPO and Sudan black negative. Six patients whose blasts expressed CD 10 (CALLA) were further analyzed phenotypically by means of flow cytometry using a panel of monoclonal antibodies (MAbs). Cell Separation
333
with 50 p1 fetal calf serum (as a blocking serum) for 15 min, and then 10 pl of the rabbit anti-TdT-antiserum was added and the cells were incubated for 30 min at 4°C. After two wahses with PBS, FITC-goat antirabbit IgG (20 pl) was added for 30 min at 4"C, and two additional washes with PBS were performed prior to fluorescenceactivated cell sorter (FACS) analysis, as described previously [7-91. More than 200,000 cells were identified for the two-color analysis.
Mononuclear cells (PBMC and BMMC) were isolated from heparinized peripheral blood and bone marrow by a RESULTS density-gradient technique (Ficoll-sodium metrizoate Morphologically, all six cases were diagnosed as ALL, d = 1.077 g/cm3). They then were washed twice with phosphate-buffered saline (PBS) medium and resus- L1 or L2, as reported previously [ 3 ] .Phenotypes of these pended in the same medium. Subsequently, the cells ALL cells were analyzed with FACS IV using a panel of were used for flow cytometric analysis as described MAbs. The leukemic cells of all the patients coexpressed Ia (HLA-DR), CD9, CD19, CD20, CD24, and CD10. below. CD38 or CD71 antigen expression was found in one or Monoclonal Antibodies three of six cases, respectively. Negative reaction with All MAbs were obtained either commercially (Ortho MAbs C D l , CD2, CD3, CD4, CD7, CD8, and Diagnostic Systems K.K., Tokyo, Japan; Japan Scien- Ti(WT3 l ) , TiyA, GTCS 1, anti-TCR-y/G, and surface tific Instrument Co., Tokyo, Japan; Fujisawa Pharma- immunoglobulins was observed. Neither myeloid-monoceutical Co., Osaka, Japan) or through the courtesy of cyte-erythroid nor megakqocyte-related cell surface Drs. T. Hercend (Institute Gustave-Roussy, Villejuif, antigens were detected in this group. Cytoplasmic CD3, TCR, and MPO were also negative France), J. Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands), and T. Uchiyama (Kyoto Uni- by means of indirect cytoplasmic immunofluorescence versity, Kyoto, Japan). Phycoerythrin (PE)-conjugated testing, whereas nuclear TdT was positive in all cases. MAbs were used for two-color analysis. TdT was de- Coexpression of CDlO and TdT was demonstrated in all tected with a rabbit anti-TdT antibody (Cosmo Bio, the cases by the mean of two-color fluorescence analysis Tokyo, Japan) and a fluorescein isothyocianate (FITC)- as shown in Figure 1A. Five cases were successfully conjugated goat antirabbit IgG (Cosmo Bio, Tokyo, brought to complete remission by intensive chemotherJapan) as second antibody. Samples with normal rabbit apy with supportive therapy. No leukemic blasts serum were included as controls. In the double-staining (CDlO+, TdT+) were detected at the time of complete experiments, no cross reactivity of the FITC-goat anti- remission (Fig. 1B). During maintenance chemotherapy, rabbit IgG with the PE-labeled mouse MAbs was ob- leukemic blasts coexpressing CDI 0 and TdT constituted served. 2.32% of the patient's peripheral blood mononuclear cells by the two-color analysis, whereas no obvious Indirect lmmunofluorescence Testing leukemic cells were recognized morphologically (Fig. Cells (2 X lo5) were incubated with 10 pl of MAbs for IC). The patient relapsed with leukemia of the same 45 min at 4"C, washed twice with PBS medium, and then phenotype 4 weeks after the examination. were incubated with a FITC-conjugated F(ab'), fragment of sheep antimouse immunoglobulin (Ig) reactive with DISCUSSION gamma or mu (Tago, Burlingame, CA) for 30 min at 4°C. Much of the recent information on the immunophenoAfter incubation, the cells were washed twice and type of hematological malignancies has been obtained analyzed by flow cytometry. from flow cytometric studies. Major advantages of this Staining for Surface Antigens and TdT technique include the simultaneous analysis of several At first, the surface antigen was detected with a parameters in individual cell and a precise quantification PE-conjugated MAb. The cells were incubated at 4°C for of antigen expression. In addition, small populations of 30 min and washed twice with PBS afterwards. Cell malignant cells that may be missed by routine morphofixation was carried out in 1 ml of a solution containing logic examination can be detected by flow cytometry 3% paraformaldehyde in PBS for 10 min at 4°C; after [lo]. In our experiment, CDlO+, TdTt cells were washing with PBS, 0.1% saponin was added and the cells detected at only 0.001% in normal persons without any were incubated for 5 min at 4°C. After two washes with underlying hematologic disorders. This evidence supPBS, TdT was detected using a two-step immunofluo- ports a recent report by Smith and Kitchens [6] and rescence method: The resuspended cells were incubated suggests that two-color flow cytometric analysis with
334
Brief Report: lmamura and Kuramoto
these antibodies is quite valuable in detecting the minimal residual leukemic cells in a patient with ALL. The reduction of leukemic cells below the threshold of detection of methods currently available appears to be necessary to achieve a cure of ALL. Hence accurate diagnosis of ALLs with MAbs should contribute substantially to the development of an effective form of therapy.
max.
400
ACKNOWLEDGMENTS
A
The authors thank Ms. H. Ota for technical assistance, Ms. H. Sumida for typing the manuscript, and Ortho Diagnostic Systems K.K. (Tokyo), Japan Scientific Instrument Co. (Tokyo), and Fujisawa Pharmaceutical Co. (Osaka) for providing monoclonal antibodies.
max
600
REFERENCES 1. Bollum FJ: Terminal deoxynucleotidyl transferase as hematopoietic
B max.
l0OC
C Fig. 1. Two-color flow cytometric analysis of peripheral blood mononuclearcells from a patient with ALL coexpressing CDlO and TdT (representativecase). A: Coexpressionof CDlO-PE and TdT-FITC is evident at the time of diagnosis. B: Only less than 0.01% of cells were double positive during complete remission status. C: Leukemic cells with the same phenotype (arrow) as observed in A were found during the maintenance therapy (2.32% among lymphoidlblast cell populationi2.11 x 1O5 cells, gated).
cell marker. Blood 54:1203, 1979. 2. Foon KA, Todd RF Ill: Immunologic classification of leukemia and lymphoma. Blood 68: 1, 1986. 3. Imamura N, Kuramoto A: Analysis of peroxidase negative acute leukemias by monoclonal antibodies. Ill. Acute lymphoblastic leukemia. J Clin Lab Anal 3:88, 1989. 4. Nadler LM, Korsmeyer SJ, Anderson KC, Boyd AW, Slaugherihoupt B, Park E, Jensen J, Coral F, Mayer RJ, Sallan SE, Ritz J, Schlossman SF: B cell origin of non-T cell acute lymphoblastic leukemia. A model for discrete stages of neoplastic and normal pre-B cell differentiation. J Clin Invest 74:332, 1984. 5. Hetherington ML, Huntsman PR, Smith RG, Buchanan GR: Terminal deoxynucleotidyl transferase (TdT)-containing peripheral blood mononuclear cell during remission of acute lymphoblastic leukemia: low sensitivity and specificity prevent accurate prediction of relapse. Leukemia Res 11:537, 1987. 6 . Smith RG, Kitchens RL: Phenotypic heterogeneity of TdTf cells in the blood and bone marrow: Implications for surveillance of residual leukemia. Blood 74:312, 1989. 7. Imamura N , Tanaka R, Kajihara H, Kuramoto A: Analycis of peroxidase negative acute unclassifiable leukemias by monoclonal antibodies. 1 . Acute myelogenous leukemia and acute myelomonocytic leukemia. Eur J Haematol 41:420, 1988. 8. Imamura N, Kajihara H, Kuramoto A: Flow cytometric analysis of peroxidase negative acute leukemias by monoclonal antibodies. 11. Acute megakaryoblastic and acute pro-megakaryocytic leukemia. Leukemia Res 12:279, 1988. 9. Imamura N, Kusunoki Y, Kawa-Ha K, Yumura K, Hara J, Oda K, Abe K, Dohy H, Inada T, Kajihara H, Kuramoto A: Aggressive natural killer cell leukaemidlymphoma; Report of four cases and review of the literature. Possible existence of a new clinical entity originating from the third lineage of lymphoid cells. Br J Haematol 75:49, 1990. 10. Barlogie B, Raber MN, Schumann J, Johnson TS, Drewinko B, Schwartzendruber DE, Gohde W, Andreeff M, Freireich EJ: Flow cytometry in clinical cancer research. Cancer Res 43:3982, 1983.
Detection of Minimal Residual Disease in Acute Lymphoblastic Leukemia by Flow Cytometry With Monoclonal Antibodies Nobutaka lmamura and Atsushi Kuramoto Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Hiroshima, Japan
To detect more precisely the minimal residual disease in acute lymphoblastic leukemia (ALL), two-color flow cytometric analysis for the detection of cell-surface antigen (CD10; CALLA) and nuclear terminal deoxynucleotidyl transferase (TdT) was performed in the six patients with CALLA-positive ALL coexpressing TdT. In all patients, the leukemic blasts coexpressed la (HLA-DR), CD9, CD19, CD20, CD24, and CDlO. Five of six patients achieved complete remission, but one has so far relapsed. No leukemic blasts (CDlO+, TdT') were detected at the time of complete remission. During maintenance chemotherapy, leukemic blasts coexpressed C10 and TdT were found 2.32% in the patient's peripheral blood by two-color analysis, whereas no obvious leukemic cells were recognized morphologically. The patient relapsed leukemia with the same phenotype 4 weeks after the examination. On the basis of our findings, we suggest that two-color flow cytometric analysis with the use of these antibodies is quite valuable to detect the minimal residual leukemic cells in a patient with ALL. The reduction of leukemic cells below the threshold of detection of methods currently available appears to be necessary to achieve a cure in ALL. Hence accurate diagnosis of ALLs with monoclonal antibodies (MAbs) should contribute substantially to the development of an effective form of therapy for their cure. Key words: minimal residual ALL, CALLA (CDlO), TdT
INTRODUCTION
Lymphoid progenitor cells express the nuclear enzyme terminal deoxynucleotidyl transferase (TdT), which catalyzes the random addition of free deoxynucleotides onto the 3'-hydroxyl terminus of a nucleotide without the presence of a primer [ 11. Since TdT is found in the nuclei of lymphoblasts of more than 90% of patients with acute lymphoblastic leukemia (ALL), the utility of measuring TdT to diagnose ALL has been well established [ 1-31. ALL cells closely resemble normal lymphocyte precursors in their expression of lineage-specific proteins and rearrangement of antigen receptor genes [2-4]. Indeed, no truly leukemia-specific proteins that could serve as markers for minimal residual disease have been identified. Recently, detection of low number of TdT-positive cells has gained importance in monitoring residual leukemia after intensive chemotherapy. However, detection of TdT alone without combination of other lineageassociated antigens was not sufficiently specific for the 0 1991 Wiley-Liss, Inc.
leukemic phenotypes to predict relapse in peripheral blood samples accurately [ 5 ] . Hence double marker studies are necessary to discriminate reliably between normal and residual leukemic cells [6]. Previously, we reported that common ALL antigen (CALLA)-positive ALL cells were also positive for TdT [ 3 ] . We describe here the surveillance of minimal residual ALL cells by means of two-color flow cytometric analysis. MATERIALS AND METHODS Diagnosis and Samples
The initial diagnosis of leukemias was established by means of examination of the peripheral blood and bone marrow smears prepared with May-Griinwald-Giemsa Received for publication June 10, 1991; accepted June 20, 1991. Address reprint requests to Nobutaka Imamura, MD, Department of Internal Medicine, Research Institute for Nuclear Medicine and Biology, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734, Japan.
Brief Report: Minimal Residual Leukemic Cells by Flow Cytometry
and myeloperoxidase (MPO) stains. The morphologic criteria for the ALL was based on the FAB classification. Sixty-six cases were diagnosed as ALL; the cells from all the cases were both MPO and Sudan black negative. Six patients whose blasts expressed CD 10 (CALLA) were further analyzed phenotypically by means of flow cytometry using a panel of monoclonal antibodies (MAbs). Cell Separation
333
with 50 p1 fetal calf serum (as a blocking serum) for 15 min, and then 10 pl of the rabbit anti-TdT-antiserum was added and the cells were incubated for 30 min at 4°C. After two wahses with PBS, FITC-goat antirabbit IgG (20 pl) was added for 30 min at 4"C, and two additional washes with PBS were performed prior to fluorescenceactivated cell sorter (FACS) analysis, as described previously [7-91. More than 200,000 cells were identified for the two-color analysis.
Mononuclear cells (PBMC and BMMC) were isolated from heparinized peripheral blood and bone marrow by a RESULTS density-gradient technique (Ficoll-sodium metrizoate Morphologically, all six cases were diagnosed as ALL, d = 1.077 g/cm3). They then were washed twice with phosphate-buffered saline (PBS) medium and resus- L1 or L2, as reported previously [ 3 ] .Phenotypes of these pended in the same medium. Subsequently, the cells ALL cells were analyzed with FACS IV using a panel of were used for flow cytometric analysis as described MAbs. The leukemic cells of all the patients coexpressed Ia (HLA-DR), CD9, CD19, CD20, CD24, and CD10. below. CD38 or CD71 antigen expression was found in one or Monoclonal Antibodies three of six cases, respectively. Negative reaction with All MAbs were obtained either commercially (Ortho MAbs C D l , CD2, CD3, CD4, CD7, CD8, and Diagnostic Systems K.K., Tokyo, Japan; Japan Scien- Ti(WT3 l ) , TiyA, GTCS 1, anti-TCR-y/G, and surface tific Instrument Co., Tokyo, Japan; Fujisawa Pharma- immunoglobulins was observed. Neither myeloid-monoceutical Co., Osaka, Japan) or through the courtesy of cyte-erythroid nor megakqocyte-related cell surface Drs. T. Hercend (Institute Gustave-Roussy, Villejuif, antigens were detected in this group. Cytoplasmic CD3, TCR, and MPO were also negative France), J. Borst (Netherlands Cancer Institute, Amsterdam, The Netherlands), and T. Uchiyama (Kyoto Uni- by means of indirect cytoplasmic immunofluorescence versity, Kyoto, Japan). Phycoerythrin (PE)-conjugated testing, whereas nuclear TdT was positive in all cases. MAbs were used for two-color analysis. TdT was de- Coexpression of CDlO and TdT was demonstrated in all tected with a rabbit anti-TdT antibody (Cosmo Bio, the cases by the mean of two-color fluorescence analysis Tokyo, Japan) and a fluorescein isothyocianate (FITC)- as shown in Figure 1A. Five cases were successfully conjugated goat antirabbit IgG (Cosmo Bio, Tokyo, brought to complete remission by intensive chemotherJapan) as second antibody. Samples with normal rabbit apy with supportive therapy. No leukemic blasts serum were included as controls. In the double-staining (CDlO+, TdT+) were detected at the time of complete experiments, no cross reactivity of the FITC-goat anti- remission (Fig. 1B). During maintenance chemotherapy, rabbit IgG with the PE-labeled mouse MAbs was ob- leukemic blasts coexpressing CDI 0 and TdT constituted served. 2.32% of the patient's peripheral blood mononuclear cells by the two-color analysis, whereas no obvious Indirect lmmunofluorescence Testing leukemic cells were recognized morphologically (Fig. Cells (2 X lo5) were incubated with 10 pl of MAbs for IC). The patient relapsed with leukemia of the same 45 min at 4"C, washed twice with PBS medium, and then phenotype 4 weeks after the examination. were incubated with a FITC-conjugated F(ab'), fragment of sheep antimouse immunoglobulin (Ig) reactive with DISCUSSION gamma or mu (Tago, Burlingame, CA) for 30 min at 4°C. Much of the recent information on the immunophenoAfter incubation, the cells were washed twice and type of hematological malignancies has been obtained analyzed by flow cytometry. from flow cytometric studies. Major advantages of this Staining for Surface Antigens and TdT technique include the simultaneous analysis of several At first, the surface antigen was detected with a parameters in individual cell and a precise quantification PE-conjugated MAb. The cells were incubated at 4°C for of antigen expression. In addition, small populations of 30 min and washed twice with PBS afterwards. Cell malignant cells that may be missed by routine morphofixation was carried out in 1 ml of a solution containing logic examination can be detected by flow cytometry 3% paraformaldehyde in PBS for 10 min at 4°C; after [lo]. In our experiment, CDlO+, TdTt cells were washing with PBS, 0.1% saponin was added and the cells detected at only 0.001% in normal persons without any were incubated for 5 min at 4°C. After two washes with underlying hematologic disorders. This evidence supPBS, TdT was detected using a two-step immunofluo- ports a recent report by Smith and Kitchens [6] and rescence method: The resuspended cells were incubated suggests that two-color flow cytometric analysis with
334
Brief Report: lmamura and Kuramoto
these antibodies is quite valuable in detecting the minimal residual leukemic cells in a patient with ALL. The reduction of leukemic cells below the threshold of detection of methods currently available appears to be necessary to achieve a cure of ALL. Hence accurate diagnosis of ALLs with MAbs should contribute substantially to the development of an effective form of therapy.
max.
400
ACKNOWLEDGMENTS
A
The authors thank Ms. H. Ota for technical assistance, Ms. H. Sumida for typing the manuscript, and Ortho Diagnostic Systems K.K. (Tokyo), Japan Scientific Instrument Co. (Tokyo), and Fujisawa Pharmaceutical Co. (Osaka) for providing monoclonal antibodies.
max
600
REFERENCES 1. Bollum FJ: Terminal deoxynucleotidyl transferase as hematopoietic
B max.
l0OC
C Fig. 1. Two-color flow cytometric analysis of peripheral blood mononuclearcells from a patient with ALL coexpressing CDlO and TdT (representativecase). A: Coexpressionof CDlO-PE and TdT-FITC is evident at the time of diagnosis. B: Only less than 0.01% of cells were double positive during complete remission status. C: Leukemic cells with the same phenotype (arrow) as observed in A were found during the maintenance therapy (2.32% among lymphoidlblast cell populationi2.11 x 1O5 cells, gated).
cell marker. Blood 54:1203, 1979. 2. Foon KA, Todd RF Ill: Immunologic classification of leukemia and lymphoma. Blood 68: 1, 1986. 3. Imamura N, Kuramoto A: Analysis of peroxidase negative acute leukemias by monoclonal antibodies. Ill. Acute lymphoblastic leukemia. J Clin Lab Anal 3:88, 1989. 4. Nadler LM, Korsmeyer SJ, Anderson KC, Boyd AW, Slaugherihoupt B, Park E, Jensen J, Coral F, Mayer RJ, Sallan SE, Ritz J, Schlossman SF: B cell origin of non-T cell acute lymphoblastic leukemia. A model for discrete stages of neoplastic and normal pre-B cell differentiation. J Clin Invest 74:332, 1984. 5. Hetherington ML, Huntsman PR, Smith RG, Buchanan GR: Terminal deoxynucleotidyl transferase (TdT)-containing peripheral blood mononuclear cell during remission of acute lymphoblastic leukemia: low sensitivity and specificity prevent accurate prediction of relapse. Leukemia Res 11:537, 1987. 6 . Smith RG, Kitchens RL: Phenotypic heterogeneity of TdTf cells in the blood and bone marrow: Implications for surveillance of residual leukemia. Blood 74:312, 1989. 7. Imamura N , Tanaka R, Kajihara H, Kuramoto A: Analycis of peroxidase negative acute unclassifiable leukemias by monoclonal antibodies. 1 . Acute myelogenous leukemia and acute myelomonocytic leukemia. Eur J Haematol 41:420, 1988. 8. Imamura N, Kajihara H, Kuramoto A: Flow cytometric analysis of peroxidase negative acute leukemias by monoclonal antibodies. 11. Acute megakaryoblastic and acute pro-megakaryocytic leukemia. Leukemia Res 12:279, 1988. 9. Imamura N, Kusunoki Y, Kawa-Ha K, Yumura K, Hara J, Oda K, Abe K, Dohy H, Inada T, Kajihara H, Kuramoto A: Aggressive natural killer cell leukaemidlymphoma; Report of four cases and review of the literature. Possible existence of a new clinical entity originating from the third lineage of lymphoid cells. Br J Haematol 75:49, 1990. 10. Barlogie B, Raber MN, Schumann J, Johnson TS, Drewinko B, Schwartzendruber DE, Gohde W, Andreeff M, Freireich EJ: Flow cytometry in clinical cancer research. Cancer Res 43:3982, 1983.
