Myeloid leukemia differentiation by phorbol ester and retinoic acid: A practical approach

Journal of Clinical LaboratoryAnalysis 4:342-349 (1990)

Myeloid Leukemia Differentiation by Phorbol Ester and Retinoic Acid: A Practical Approach V.M. Marques-Silva,' M.H.O. De Souza,2 M.C.L. Tei~eira,~ R.A. Arcuri,' and V.M. Rumjanek' 'Basic Research Centre, 2Bone Marrow Transplantation Unit, and 3HematologyService, lnstituto Nacional de Cancer, Rio de Janeiro, Brazil The effects of TPA (12-0-tetradecanoylphorbol-13-acetate) and RA (retinoic acid) were investigatedon the cell lines HL60 (acute promyelocytic leukemia) and K562 (erythroleukemia) and on cells from patients with several kinds of leukemia. There were 14 cases of acute lymphocytic leukemia (ALL), 2 cases of chronic lymphocytic leukemia (CLL), 23 cases of acute myeloid leukemia (M1-M7), 5 cases of chronic myelocytic leukemia in blast crisis (CML-BC) and 2 mixed leukemias. In almost all of the cases examined, after TPA exposure cells from patients with proven myeloid leukemiabecame adherent to the substrate, while lymphoid leukemia cells remained in suspension, allowing the differentiationof lymphoid from myeloid blasts. The only exception was in one case of CLL, which had cells that became adherent with long filamental projections. In addition, increased phagocytosis following TPA Key words:

INTRODUCTION

exposure permitted characterization of M7 as this was the only myeloid leukemia negative for phagocytosis. Further discrimination between the subtypes of myeloid leukemia could be based on the increased lysozyme production seen after TPA in M4 and M5. Esterase positivity allowed the discrimination of M I cells, which were negative before and after TPA treatment. In agreement with the results of other authors, TPA and RA led to independent ways of differentiation, granulocytic-like lineage and monocytic-like cells being favored by RA and TPA, respectively. The capacity of the same cell to differentiate into more than one lineage, depending on whether RA or TPA was used, was only seen in the present study with M3 cells. Our results show the usefulness of using differentiation inducers and functionaltests together with morphology to elucidate the nature of leukemic cells.

TPA, retinoids, cellular differentiation, cell lines, rnyelogenous leukemias

uration (10,13). RA, on the other hand, seems to induce maturation of acute promyelocytic leukemia (3), while other types The development of a malignant phenotype may be viewed of nonlymphocytic leukemia do not respond (8,9). Promyas a defect in the normal process of differentiation, and nuelocytes show that not only the nature of the cell, but also merous efforts have been dedicated to the study of substances the nature of the inducing agent, can determine the outcome in that can induce cell maturation, including phorbol diester (TPA) terms of maturation, as they differentiate into the monocytic (l), retinoic acid (RA) (2), dimethylsulfoxide (DMSO) (3), lineage with TPA treatment and into the granulocytic lineage and 1-a-25-dihydroxyvitamin D3 (4). Chemically induced after treatment with RA or DMSO (23). This fact suggests that, differentiation has been studied primarily in cell lines (5,6), in situations where the cell type is unknown, testing with more though study has been extended to a number of fresh tumors than one inducer might provide important information. Further(7), including fresh leukemic cells (8-13). Exposure of leumore, concomitant exposure to several different compounds kemic cells in vitro to various inducers was used to help remight induce some resistant cells to differentiate (24). veal the process of differentiation(10,14,15>or to allow better Our study examined the possibility, for diagnostic purclassificationof the type of cell involved ( 11,13,16). Despite poses, of employing a combination of four simple functional the FAB classification system (17-20), there are still cases in tests, available in most laboratories and associated with two which the differential diagnosis is difficult, including mixed different inducers, TPA and RA, in situations where the oriand undifferentiated leukemias (21,22). Among the differentiating agents, phorbol diesters have Received February 8, 1990; accepted April 16, 1990. been extensively used, since they are capable of inducing Address reprint requests to V.M. Rumjanek, Ph.D., Basic Research Cenmacrophage-like differentiationof human acute myelogenous tre, Instituto Nacional de Cancer, Praga Cruz Vermelha, 23-6" Andar, Cep cells (1 1,16) and of leading lymphocyteleukemic cells to mat- 20.230, Rio de Janeiro, Brazil. @ 1990 Wiley-Liss, Inc.

Leukemia Differentiation

gin of the leukemic cell is difficult to establish or the diagnosis is unclear.

MATERIALS AND METHODS Patients Forty-six leukemic patients from the Hematology Service at the National Cancer Institute in Rio de Janeiro were studied. Their diagnoses were based on clinical data and morphologic (FAB) and cytochemical criteria. In a few cases, classification by surface markers using monoclonal antibodies was also performed. There were 16 patients with lymphoid leukemia: 14 with acute lymphoblastic leukemia (ALL) and 2 with chronic lymphocytic leukemia (CLL). Twenty-eight patients were diagnosed as suffering from myeloid leukemia: four with myeloblastic leukemia without maturation (MI), three with hypergranular promyelocytic leukemia (M3), five with myelomonocytic leukemia (M4), nine with monocytic leukemia (M5), two with megakaryocytic leukemia (M7), five with chronic myelocytic leukemia in blast crisis (CML-BC), and two with mixed leukemias. Some of the lymphoid leukemias could not be diagnosed with certainty based solely on morphological grounds. Similarly, some myeloid leukemias, such as M1 and the megakaryocytic leukemia, could only be classified after they had been differentiated with TPA and RA.

Preparation of Leukemic Cells Heparinized peripheral blood was obtained from each patient, and the mononuclear cell fraction was isolated by the Ficoll-Hypaque gradient centrifugation technique. The final cell suspension was resuspended in RPMI medium (Sigma) with 5% fetal calf serum (FCS; Cultilab) at a concentration of 1 x lo6cells/ml. Adherent cells were removed by incubation in a plastic flask for 1 hr at 37°C in a humidified atmosphere of 5% COz.

Cell Lines The cell lines K562 (human erythroleukemia) and HL60 (human promyelocytic leukemia) were maintained in our laboratory in constant exponential growth in RPMI medium supplemented with antibiotics and 5% FCS. When used with the differentiating agents, the cell suspension was prepared at a concentration of I x lo6 cells/ml.

Treatment With Differentiating Agents The agents TPA ( 12-0-tetradecanoylphorbol13-acetate)and RA (retinoic acid) were both obtained from Sigma. TPA (10 mg) was dissolved in 40 ml of DMSO followed by 60 ml of RPMI to give a stock concentration of 100 pgiml. This was

343

aliquoted in small volumes, stored at - 70"C, and diluted with culture medium immediately before use to a final concentration of 10 ng/ml. The final concentration of DMSO showed no effect on cells tested. RA was dissolved in ethanol (500 pg/ml), aliquoted, stored in the dark at - 20"C, and diluted with culture medium immediately before use to a final concentration of 1 pM. The agents were added to 1 ml of the cell suspension distributed in (4-cm-diameter) plastic Petri dishes and incubated for 4 hr and 72 hr at 37°C in a humidified atmosphere of 5% C02. After the incubation period, morphological and functional changes were assessed.

Morphological Assessment After treatment with or without the differentiating agents, cells were assessed morphologically by phase microscopy or by staining. The nonadherent cells were assessed in a cytospin preparation and the adherent cells in the Petri dish. Staining was done with May-Griinwald-Giemsa (MGG) and nonspecific esterase using a-naphthyl acetate as the substrate, using the modified method of Rozenszajn et al. (25).

Functional Tests The following tests were performed after 4 hr or 72 hr of incubation: increased cell adherence to the substrate, phagocytic activity, and lysozyme production. Cell adherence was scored by counting 100 cells and calculating the percentage of cells adhering to the culture dish. Phagocytic activity was tested using Saccharomyces cerevisiae pretreated with AB fresh human serum. A suspension with a 10:1 ratio of yeast to leukemia cells was incubated at 37°C for 1 hr, and the percentage of cells completely ingesting one or more yeasts was determined microscopically, using the lysosomotropic substance acridine orange (26). The amount of lysozyme released into the medium was determined by the amount of decrease in turbidity of a cell suspension of the bacteria Micrococcus lysodeikticus measured at a transmittance of 540 nm at 25°C (27).

RESULTS Effect of Differentiating Agents on Cell Lines The cell line K562 did not respond in any of the functional assays tested, even after exposure to differentiating agents; on the other hand, the cell line HL60 responded to both agents functionally and morphologically. In this cell line, TPA and RA led to independent differentiation pathways (monocyticlike and granylocytic-like cells, respectively), in agreement with the results of other investigators (9,12). The concomitant addition of RA and TPA to the cells produced very similar results to those obtained by TPA alone (Table 1).

=

HL60(n

7)

4)

0 0 1 (0-6) 40 (17-60) 0

0 0 0 90(40-95) 5 (0-16) 92 (43-100)

0

0 0 14 (10-18) 0 17 (12-25)

RA TPA-RA None TPA RA

TPA-RA

40 (20-50)

0

0

91 (60-96)

10 (0-25) 85 (50-95) 10 (0-23)

0

0

0

0

0 0

0 0

72 hr

None TPA

4hr

72 hr

Treatment

4hr

Percentage of phagocytosis

0 0

0 0 0.1 (0.1-0.1) 2.3 (0.5-3.4) 2.1 (0.9-4.2) 2.8 (0.9-4.4)

0 0 0.1 (0-0.1) 1.3 (0.3-2.2) 1.2(0.5-2.3) 1.0 (0.2-2.3)

72 hr

0 0

4hr

Lysozyme (pg/ml/1O6cells)

+I+/-

+/+/-

+ + ++ +++ + + ++ +++

+/+/-

+/+/-

Esterase 4hr 72hr

Blast cells Blast cells with membrane projections Vacuolated blast cells Blast cells Promyelocytic blast cells Monocytic-like cells Band cells and granulocytic-like cells Monocytic-like cells

Morphology 172hr)

*

Results are expressed as the medians of the experiments; ranges are shown in parentheses. TPA treatment was at 10 nglml for 72 hr. RA treatment was at 1 p,M for 72 hr, ,very weak diffuse staining with no typical pattern.

=

K562(n

Cell line

Percentage of adherence

TABLE 1. Effect of DifferentiationInducers on K562 and HL60 Cell Lines

W

Leukemia Differentiation

Effect of DifferentiatingAgents on Lymphoid Leukemia Cells The lymphocytic leukemia cells cultured with the differentiating agents, either in isolation or in combination, did not show morphological changes or functional differences in the tests used in the present study when observed up to 72 hr in culture. The only exception was a case of CLL cells that became adherent to the substrate and showed long filamental projections. No phagocytic activity was detected in this case.

Effect of DifferentiatingAgents on Myeloid Leukemia Preliminary studies used only TPA as the inducer agent and adherence to the substrate as a functional test. With this LEUKEMIA TREATMENT TYPE NONE

-

345

approach, all 10 myeloid leukemias studied (one M4, six M5, and three CML) had cells that became adherent to the substrate after 4-hr incubation with TPA; the effect was much more obvious at 72 hr. Cells from lymphoid leukemias did not adhere. These results were encouraging as they clearly differentiated myeloid from lymphoid leukemias. The next step involved combining tests and agents in to distinguish the subtypes of myeloid leukemias. Cells from different patients than in the preliminary experiments were utilized; no frozen cells were ever tested in this study. As it can be seen in Figure I and Table 2, the various subtypes responded differently, to the stimuli functionally and morphologically. MI cells, obtained from four patients, were difficult to classify. They were negative for esterase and lysozyme production, and they had a very small quantity of adherent and

% ADHERENCE

I

TP4

M 1 RA

TPA/RA

NONE T P4 M3 R4 TP4 /RA

NONE TP 4

M4 R4 TPA/RA

NONE

R

T P4

MS R4 TP4 /R4

NONE T PA

M7 R4 TPP/RA

fig. 1. The effects of differentiationinducers TF'A (10 ngml), RA (1 (LM), and TPA-RA on cells from myeloid leukemia patients. The first bar of each group of two (representing the same treatment) shows the results

obtained after 4 hr of incubation, whereas the second bar shows results obtained after 72 hr.

Marques-Silva et al.

346

TABLE 2. Phenotypic Characteristicsof Myeloid Leukemia Cells Following 'keatment With DifferentiatingAgents Leukemia type

Treatment

Morphology

M1 (n = 4)

None TPA RA TPA-RA None TPA RA TPA-RA None TPA RA TPA-RA None TPA RA TPA-RA None TPA RA TPA-RA

Blast cells Few monocytic-like cells and large clumps of blast cells Blast cells Few monocytic-like cells and large clumps of blast cells Promyelocytic and blast cells Monocytic-like cells Band cells and granulocytic cells Monocytic-like cells Blast cells Blast and monocytic-like cells Promonocytic and blast cells Blast and monocytic-like cells Blast cells Monocytic-like cells Promonocytic and blast cells Monocytic-like cells Blast cells with blebs Blast and monocytic-like cells with blebs Blast cells with blebs Blast and monocytic-like cells with blebs

M3 (n

=

3)

M4(n

=

4)

M5 (n = 3)

M7 (n = 2)

phagocytic cells. M1 cells remained negative for esterase and lysozyme, and, although an increase was observed in adherence and phagocytosis after TFA or TPA plus RA, never more than 15% of the cells responded. Cell clumps were seen, but they were much bigger than the ones observed in lymphoid leukemia. Despite the poor phagocytosis, it was observed that most cells presented fluorescent red dots inside the isolated or clumped cells. These dots were never seen in cells from lymphoid leukemia. M3 untreated cells, obtained from three patients, contained mainly promyelocytes characterized by large nuclei, extensive cytoplasmic azurophilic granulation, and several nucleoli. These cells were not phagocytic and produced very small amounts of lysozyme. After treatment with TPA, 50% of the cells, which normally grow in suspension, attached to the surface of the Petri culture dish and spread their cytoplasm in a wide variety of shapes. This spreading increased with time, and the cells resembled cells of monocyte-macrophage lineage in many aspects, including being positive for nonspecific esterase. Up to 30% of the cells of the whole population became phagocytic, whereas lysozyme production remained unchanged. Phagocytosis was not seen after RA, when abnormal morphological characteristics of the granulocytic lineage could be observed in these cells with banded or segmented atypical nuclei. Lysozyme production was also unaffected. The combination of the two agents had basically the same results as seen after TPA alone. M4 cells, obtained from four patients, showed typical monocytic-likemorphology with nonspecificesterase-positive cells. The most marked characteristic of these untreated cells was their phagocytic capacity, with around 59% of the cells taking up S . cerevisiae in the absence of inducing agents. A proportion of untreated cells were also adherent, and they

Esterase

-

+ ++ + ++ + ++ + +++ + ++++ + ++++ + ++ + ++

had marked production of lysozyme. After TPA, there was an increase in all the parameters studied, while no functional change was observed following RA treatment. Morphologically, however, treatment with TPA, RA, or a combination led to differentiation within the macrophage lineage. M5 cells obtained from three patients showed a predominance of myeloblast forms growing in suspension and characterized by positivity for the nonspecific esterase reaction and lysozyme production, with a small proportion of cells showing phagocytic capacity. These cells differentiated into nondividing macrophage-like cells when exposed to TPA alone or in combination with RA, and they became adherent, developing long pseudopodia. They also increased lysozyme production, nonspecific acid esterase, and phagocytic ability. RA alone did not induce any significant changes. Cells from two M7 leukemia patients seemed to be in different stages of maturation. Although all the cells had grown in suspension and were heterogeneous in size, only cells from one patient showed characteristic nonspecific esterase staining. Following TPA treatment, up to 50% of the cells became attached to the substrate, acquiring a monocytic-like morphology and showing an increase in cytoplasmic blebs and a localized (cap-like) positive reaction to nonspecific esterase. No phagocytic activity or lysozyme production was seen before or after treatment. RA alone had no effect in any of the systems studied. In the second patient, classification could only be given after the cells had differentiated.

Effect of Differentiating Agents on CML-BC Cells from CML were obtained from two patients. In one case, 50% of the cells were promyelocyte or myelocytes; after TPA, RA, or TPA-RA exposure, they behaved similarly

Leukemia Differentiation

to cells from a M4/M5 acute myeloid leukemia. In the other case, formerly a chronic myeloid leukemia, the first blast crisis showed a predominance of cells expressing lymphoid immunophenotypic markers and nearly none with myeloid markers (data not shown). The myelocytic nature of the cells only became apparent after TPA treatment, when approximately 40% of the cells became adherent, esterase-positive, and phagocytic, as well as producing lysozyme. RA, on the other hand, produced no effect. The patient nevertheless received therapy for lymphocytic leukemia and during treatment suffered from another blast crisis. A blood sample was then taken and tested. After TPA exposure, 90%of the cells showed functional characteristics of myeloid leukemia, conf i n g the presence of a myelocytic population, as observed in the f i t sample.

Effect of DifferentiatingAgents on Mixed Leukemias Two patients with acute leukemia had blasts that reacted with monoclonal antibodies specific for lymphoid or myeloid markers on different cells (data not shown). After TPA treatment, both cases reacted in very similar ways. There was a threefold increase in adherent cells and esterase positivity, corresponding to approximately 40% of the cells in the dish. The majority of the cells in the suspension remained esterasenegative. Phagocytic activity was present in all the adherent cells and in very few suspended cells; all other cells in suspension did not have fluorescent red dot markers. Lysozyme production increased nearly five times. RA exposure produced no effect.

DISCUSSION Peripheral blood from patients with myeloid leukemia often contains cells in different stages of maturation. The proportion of mature cells can be increased following exposure of leukemia cells in culture to a variety of physiological and pharmacological agents (28). In the present work, a number of systems used by other authors (5,9,11,12) were explored and a combination of two agents and four tests (based on functional and morphological criteria) was developed to obtain a system that would help the classification of immature leukemic cells or cells without definite diagnoses. The tests were done sequentially, therefore eliminating certain possibilities along the way. The first step involved the separation of myeloid from lymphoid leukemias based on substrate adherence after TPA incubation. Adherent cells were classified as myeloid leukemias, with the exception of two mixed leukemias, in which the myeloid component did adhere, and a CLL that showed morphology resembling the long projections characteristic of hairy-cell leukemia; a TPA-induced CLL differentiation into hairy-cell leukemia has been described (29). Another case of adherence by lymphoid cells following TPA has been described, involving cells from a patient suffering from ALL (1 1); the morphological changes,

347

however, differed from the ones seen in myeloid or hairy-cell leukemia, as no projection formation could be observed. In addition, a few normal lymphoid cells were also capable of adhering after TPA treatment; this adherent population seemed to possess granules and natural killer activity (30,3 1). In one large granular lymphocyte leukemia studied in our laboratory (data not shown), 30% of the cells adhered after TPA treatment. Despite the exceptional cases, adherence seems to be a clear identification step between myeloid and lymphoid blasts, as found by other authors (1 1,13). In some cases, however, adherence was not such an obvious characteristic. For example, less than 1% of the AMLs belong to the subtype M1, being very rare and easily confused with L2 cells. TPA differentiation in M1 cells produced a poor adherence, but these leukemic cells formed large clumps in suspension, and fluorescent dots were seen in both clumped and adherent cells during the phagocytic assay, suggesting the importance of using more than one test for classification. Moreover, the distinction between subtypes of myeloid leukemia could be done using other functional tests after differentiation. For instance, the absence of phagocytosis seen in M7 cells allowed its separation from other subtypes, in agreement with the results of Roth et al. (32). In our work, two cases of M7 leukemia were studied, and one of them could only be diagnosed after differentiation with TPA. Lack of lysozyme production was a characteristic of M7 cells shared by M3 cells. M3 cells, however, showed phagocytic activity after TFA and possessed a unique characteristic: they developed a granulocytic form following RA treatment, as has been previously described for promyelocytic cells (8,9,33). This finding was confiied by us using patients’ blood and HL60, a promyelocytic cell line. Myeloid leukemia M4 could be discriminated from M5 by the increased lysozyme production seen after TPA treatment in the latter. The basal levels produced by M4 subtype, despite being quite high, never reached the levels seen in M5 cells after stimulation. M6 leukemia cells were not available, but in the cell line K562, an erythroleukemia, no changes in the parameters studied were observed after TPA or RA treatment. This result was also described by Yu et al. (34), who induced a monocyticlike differentiation of the TPA-treated K562 cells only when those cells were transfected with the human c-fes gene. Some acute leukemias may express markers of myeloid and lymphoid characteristics. When the markers are expressed in the same cell, the leukemias are call biphenotypic, but when the markers are expressed in different blasts, as two populations, the leukemias are mixed leukemias (35). In the present work, two patients previously diagnosed as suffering from mixed leukemia were studied, who only possessed few cells with myeloid or lymphoid markers. In both cases, after TPA differentiation, an increase in myeloid and lymphoid cells could be seen, indicating that nonmarked undifferentiated cells belonged to both populations.

348

Marques-Silva et al.

Chronic LymphocyticLeukemia, R Polliack, D Catovsky, eds. H a r d Academic Publishers, London, 1988, pp 325-352. 11. Ohta M, Takaku F, Miura Y, Kitahawa S-I, Saito M. Clinical application of phorbol diester-induced leukemic cell differentiation for the definite diagnosis of acute leukemias. Jpn J Cuncer Res 79:350-358, 1988. 12. Pegoraro L, Abrahm J, Cooper RA, et al: Differentiation of human leukemias in response to 12-0-tetradecanoilphorbol-13-acetatein virro. Blood 55:859-862,1980. 13. Polliack A, Leizerowitz R, Korkesh A, Gurfel D, Gamliel H, Galili U: Exposure to phorbol diester (TPA) in vitro as an aid in the classification of blast in human myelogenous and lymphoid leukemias. Am J Hemutol13:199-211, 1982. 14. Girard PR, Stevens VL, Blaskshear PJ ,et al: Immunocytochemicalevidence for phorbol ester-induced directional translocations of protein kinase C in HL60,K562, CHO and E75KS cells: Possible role in differentiation. Cancer Res 47:2892-2898, 1987. 15. Thompson BY, Sivam G, Britigan BE, Rosen GM, Cohen MS: Oxygen metabolism of the HL60 cell line: Comparison of the effects of monocytoid andneutrophilicdifferentiation. JLeubcyte Bio143:140-147,1988. 16. Fibach E, Rachmilewitz EA Tumour promoters induce macrophage differentiation in human myeloid cells from patients with acute and c h n i c myelogenous leukaemia. Br J Huemutol47:203-210, 1981. 17. Bennett JM, Catovsky D, Daniel MT, et al: Proposals for the classification of acute leukaemias.Br JHuematol33:451-458, 1976. 18. Bennett JM, Catovsky D, Daniel MT, et al: Proposals for the classificationofthemyelcdysplastic syndromes.BrJHuemtolS1:189-19!9,1985. 19. Bennett JM, Catovsky D, Daniel MT, et al: Criteria for the diagnosis of ACKNOWLEDGMENTS acute leukemia of megakaryocyte lineage (M7). Ann Intern Med 103: 1985. We thank Dr. L.C. 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The importance of early recognition of another population that could lead to possible relapse was underlined by a case of CML-BC in which the majority of blasts had lymphoid markers. However, an important component of myeloid cells could also be seen after incubation with TPA. After lymphoid therapy, this patient suffered from a new blast crisis, in which 100% of the blasts belonged to myeloid lineage confirming the finding observed after TPA treatment. It is important to better classify the cell type involved in leukemias so that more precise diagnosis and treatment may be offered. The FAB classification, based mainly on morphological criteria, attempted to classify cell types, but difficulties relating to the original criteria have been pointed out (36). Many of the classification problems could be overcome by the combined use of immunological and cytogenetic techniques; however, such techniques are impractical for many laboratories due to their complexity and high cost. The tests discussed in this article, combined with maturation inducers, may help to distinguish the cell types involved, are accessible to most workers, and permit evaluation of the potential for lineage deviation.

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the human c-fes gene acquire the ability to undergo myeloid differentiation. JBiol Chem 264:10276-10281, 1989. 35. Stass SA, Mirro J Jr: Lineage heterogeneity in acute leukaemia: Acute mixed-lineage leukaemia and lineage switch. Clin Haematol 15311827, 1986. 36. Bloomfield CD, Brunning RD: The revised French-American-British classification of acute myeloid leukemia: Is new better? Ann Intern Med 103:614-616, 1985.