Leukemia & Lymphoma

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Megakaryocytic Leukemia and Platelet Factor 4 Ryukichi Ryo, Mutsumi Yasunaga, Katsuyasu Saigo & Nobuo Yamaguchi To cite this article: Ryukichi Ryo, Mutsumi Yasunaga, Katsuyasu Saigo & Nobuo Yamaguchi (1992) Megakaryocytic Leukemia and Platelet Factor 4, Leukemia & Lymphoma, 8:4-5, 327-336, DOI: 10.3109/10428199209051011 To link to this article: http://dx.doi.org/10.3109/10428199209051011

Published online: 01 Jul 2009.

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Leukemia and Lymphoma, Vol. 8, pp. 327-336 Reprints available directly from the publisher Photocopying permitted by license only

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Megakaryocytic Leukemia and Platelet Factor 4 RYUKICHI RYO*, MUTSUMI YASUNAGA?, KATSUYASU SAIGOt and NOBUO YAMAGUCHIt

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*Blood Transfusion Service, Kobe University Hospital, and tDepartment of Laboratory Medicine, Kobe University School of Medicine, Kobe, Japan (Received 26 April 1992)

The de novo megakaryocytic leukemia fullfilling the FAB criteria is still an uncommonly recognized variant of acute leukemia. Many studies have shown that the megakaryocytic leukemic events may occur at a pluripotent stem cell level and clinical observations reveal that the megakaryocytic leukemias are diverse entities. The immunophenotyping using monoclonal antibodies against platelet specific surface antigens and the ultrastructural detection of platelet peroxidase reaction d o not provide sufficiently useful information to determine whether a megakaryocytic leukemia is chronic, acute, therapy-responsive or therapy-unresponsive. More sophisticated techniques are required to further characterize megakaryocytic leukemic cells. In this review, we emphasize that megakaryocytic leukemic cells can be categorized into two groups; one with the PF4 mRNA, and the other without it, and that the expression of PF4 mRNA in the blasts could be a useful marker for the identification of mature megakaryoblasts. It seems that the patients with blasts expressing PF4 mRNA will have a longer survival and a better response to chemotherapy than those without PF4. We further discuss the fact that the detection of mRNAs of the IL-6 receptor, P D G F A- and B-chains, and TGFBl in megakaryocytic leukemic cells will be useful to clarify the mechanisms involved in the proliferation of megakaryocytic leukemic cells and fibroblasts in the bone marrow. Furthermore, we reviewed data showing that megakaryocytic erythroid, and mast cell lineages share the nuclear transcription factor known as GF-1 (NF-El or Erf-1). We suggest that characterization of megakaryocytic leukemia should be performed using monoclonal antibodies against erythroid, megakaryocytic and mast cell lineages. KEY WORDS:

Megakaryocytic leukemia interleukin-6 receptor platelet-derived growth factor tumor growth factor b1

MEGAKARYOCYTIC LEUKEMIA Neoplastic hematologic disorders involving the megakaryocytic lineage have been recognized for the past 60 years'. Immunophenotyping using monoclonal antibodies against platelet specific antigens such as glycoprotein (GP)IIb/IIIa complex (CD4la) and GPIb (CD42b), and the ultrastructural detection Address for correspondence: Ryukichi Ryo, MD, Blood Transfusion Service, Kobe University Hospital, Kusunoki, Chuo-ku, Kobe 650, Japan. 321

platelet factor 4

of platelet peroxidase (PPO) activity have allowed the precise identification of immature megakaryoblasts2. In this respect it has been pointed out that the frequency of megakaryocytic leukemia might be much higher than previously recognized. In fact, among all patients with acute leukemia who were examined at the Mayo Clinic during the 36-month interval from May 1980 through April 1983, acute megakaryocytic leukemia comprised 8% of all acute leukemia cases and 12% of the cases of acute non-lymphoblastic leukemia3. In addition, Ruiz-Arguelles et aL4 reported that acute megakaryocytic leukemia constituted 8.4%

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of all the acute leukemias among their patients in the Cooperative group "Grupo de Hematologos de Puebla" in Mexico between June 1983 and May 1984. Based on such immunological phenotyping and ultrastructural techniques, the FAB group5 proposed criteria for the diagnosis of acute leukemia of megakaryocytic lineage (M7) in 1985. Recent studies"' also suggested that leukemia with megakaryoblastic involvement should be diagnosed immunologically using monoclonal antibodies against GPIITa (CD61) and CD4la. In our experience, however, de novo M7 AML, fulfilling the FAB criteria, is still an uncommonly recognized variant of acute leukemia. On the other hand, the megakaryoblastic transformation occurring in patients with chronic myelogeneous leukemia (CML)8 or myelodysplastic syndrome (MDS)9 seems to occur more frequently than previously expected. Indeed Cuneo et a/." reported that morphologic evidence of associated myelodysplastic features was detected in all the 12 pqtients with de novo M7, and 26.7% of the 15 patients fulfilling the FAB criteria for M7 included megakaryoblastic transformation of CML, refractory anemia with excess of blasts, and polycythemia Vera. Furthermore, the cytogenetic studies in the 15 patients with megakaryocytic leukemia revealed - 7/7q - and 8, alone or in combination with additional abberations, in three cases each. Rearrangements involving bands 3q21 or 3q26 were seen in two patients and +21, as a n additional abberation, in one. Other structural rearrangements all observed in a single patient were (16)(p13q22), t(13;20) (q13or14; ql I), del(20) (91 l), and der(7)t(7;17)(p14;q22). Many chromosome abnormalities may be related to mechanisms conferring a proliferative advantage to undifferentiated stem cells. According to cytologic and cytogenetic studies, they concluded that the megakaryocytic leukemic events may occur in a pluripotent stem cell. More recent studies" from the central and western part of Spain also confirmed that 13 out of 28 leukemic patients with megakaryocytic involvement were acute megakaryocytic transformations of chronic myeloproliferative disorders or MDS, and that megakaryoblasts coexisted with other hematopoietic components in 26 out of 28 cases. These studies confirmed the stem cell origin of megakaryocytic leukemia. Direct analyses" of A- and B- types of G6PD in the skin and the cells derived from CFU-GM in one megakaryocytic leukemic patient also provided evidence that M7 involved a pluripotent stem cell capable of differentiating along both the granulocytic and megakaryocytic pathways. Accord-

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ing to these studies and our own experience, we assume that most cases of megakaryocytic leukemia are clonal disorders that originate from a pluripotent stem cell. Another characteristic found in the patients with megakaryocytic leukemia is a diversity of clinical features. In some of them, the clinical pattern resembles that of acute myelofibrosis, which includes increased bone marrow reticulin, pancytopenia, the absence of leukoerythroblastosis or tear-drop poikilocytes, and no enlargement of liver or ~ p l e e n ' ~ . In '~. contrast, some chronic variants of megakaryocytic leukemia associated with increased platelet counts are also found'. A common feature of patients with acute leukemia and megakaryocytic transformation of myeloproliferative disorders is the poor prognosis, regardless of the chemotherapeutic regimen used3.' On the other hand, low-dose cytosine arabinoside4 or 6-mercaptopurine' were reported to be effective in controlling the megakaryoblastic proliferations. Furthermore, a transient megakaryoblastic proliferation which mimics congenital leukemia occurs in some neonates in Down's syndrome, which is known as the transient myeloproliferative disorder (TMD)I6j1'. The blast cells found in the patients with T M D possess the typical characteristics of the megakaryocytic lineage. In these patients, however, this leukemic feature disappears spontaneously within 4-6 weeks. These clinical observations imply that the manifestations of megakaryocytic leukemia are those of diverse entities comprising acute, chronic, malignant or benign proliferative disorders with the involvement of megakaryocytic lineages. To determine whether a manifestation is chronic or acute, or to obtain information for proper therapeutic indications, more sophisticated methods are required. In this review, we summarize the data relating to the characteristics of the proliferative megakaryoblasts in various patients. Platelet @-granule proteins such as platelet factor 4 (PF4), von Willbrand factor (vWF), platelet-derived growth factor (PDGF) and tumor growth factor /?l (TGFPl) are expressed in some of the leukemic cells derived from megakaryocytic lineages. Recently we examined the expression of platelet a-granule proteins in megakaryocytic leukemic cells and proposed the hypothesis that PF4 mRNA expressions in megakaryocytic leukemic cells may be evidence for a chronic type of megakaryocytic leukemia. We also stressed that the detection of PF4 mRNA is a good marker for defining the different characteristics of the megakaryocytic leukemic cells.

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megakaryocytic leukemic cells varies from case to case. In general, the expression of CD61 is observed on most megakaryocytic leukemia cells, while the Platelet peroxidase (PPO) activity expression of CD42b is occasionally absent on Immature human megakaryoblasts do not contain megakaryocytic leukemic cells. Studies of the seany specific organelles such as a-granules and quential maturation of megakaryocytes using the in demarcation membranes which are found in the more vitro colony assays have shown that CD61 is first mature megakaryocytes and platelets. Blast cells in synthesized in megakaryocytic progenitors, followed megakaryocytic leukemia are usually classified as by a later appearance of CD42bZ3.The heterogeneity undifferentiated cytochemically positive for cc-naphtyl of surface membrane GPs expression may be linked acetate esterase activity and negative for myeloperox- to the level of differentiation of leukemic megaidase or cc-naphtyl butylate esterase activity. Breton- karyoblasts. It seems likely that leukemic megaGorius’ first reported that platelet peroxidase is a karyoblasts exhibiting CD61 or CD4la represent cells specific marker for the identification of immature at the most primitive stage of megakaryocytic megakaryoblasts. These are found in the perinuclear evolution, while expression of CD61 and CD42b on envelopes and endoplasmic reticulum, but never in the megakaryocytic leukemia cells reveals the clonal Golgi apparatus or in the granules, and may be evolution of more mature megakaryoblasts. CD4la is a calcium-dependent heterodimer of implicated in prostaglandin synthesis. Since then, the CD61 and GPIIb (CD4lb) belonging to a member of detection of PPO activity has been employed in the the cytoadhesion family. CD61 and CD4lb correidentification of leukemic megakaryoblasts. Although spond to the p-subunit of the vitronectin receptor and most leukemic megakaryoblasts possess PPO activity, to the cc-subunit of the platelet Arg-Gly-Asp-sensitive the same activity similar in it’s localization to P P O is integrin, which are the products of separate genes. The demonstrated in activated monocytes, mast cell kinetic studies of the formation of CD4la in the precursors, fibroblasts, hairy leukemic cells” and megakaryocytes demonstrated that early formation of proerythroblasts”. Recent studies have indicated that the nuclear transcription factor GF-1 (also known as proCD4lb and CD61 is an obligatory step in the NF-El or Eryf-1)21*zz,which is important in the maturation for the heterodimer of CD61 and CD4lb and its expression on the cell surfacez4. Clinically, the regulation of the transcription of globin and other monoclonal antibody against CD4la is generally used erythroid specific protein genes, may mediate for the determination of the presence of megacommitment to megakaryocytic lineages and mast karyocyte specific surface antigens on blasts, however, cells. It should be noted that the blasts with erythroid antibodies specific for CD61 and CD4lb, not CD4la and mast cell lineage also exhibit PPO-like activity, are more suitable to characterize the earlier leukemic thus it seems possible that GF-1 may participate in megakaryoblast. In addition, the same experiment the transcription of the PPO gene in erythroblasts, showed that a large pool of free GPIIIa in the megakaryoblasts and mast cells. Therefore, the megakaryocytes was not expressed on the surfaces. expression of P P O activity in blast cells does not Therefore, it is possible that the immunological always imply the precise identification of blasts as analysis of the leukemic megakaryoblasts using the cells of the megakaryocytic lineage alone. Other monoclonal antibody against CD61 is less sensitive to markers are also required in order to establish the recognize the presence of GPIIIa in the blasts than exact lineage. genomic determination of mRNA encoding GPIIIa. Many other surface proteins such as CD4 (receptor for H1Vz5), CD36 (thrombospondin re~eptor)’~, CD927, glycophorin A, DR antigen, CD33 (myeloid Platelet surface proteins marker)’* and P62 (a putative collagen receptor)’’ Megakaryocytes and platelets possess cytoadhesive are all reported to be expressed on some megaGPs on their membranes, which function as the karyocytic leukemic cells. The implications of the presence of megakaryocytic receptor for platelet aggregation and adhesion. Analysis of platelet membrane GPs by immunofluor- leukemic cells with phenotypic features of more than escence has demonstrated that GPIb (CD42b), CD4la one megakaryocytic lineage are as yet not clear. The and GPIIIa (CD61) are expressed on leukemic diverse phenotypic profiles of megakaryocytic leukemegakaryoblasts. However, the phenotypic profile of mia may reflect the clonal evolution of a mega-

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karyocytic progenitor capable of development along myeloid or erythroid pathways, or aberrant gene expressions commonly recognized in this leukemia. As mentioned above, megakaryocytic, erythroid and mast cell lineages have been found to share the transcription factor known as GF-1 (NF-El, or Erf- 1). Romeo et a1." revealed that CD42b, the platelet specific surface antigen, contains binding sites for GF-1 and these data strongly suggest a close association between the erythroid and megakaryocytic lineages, as well as mast cells. In fact, the clinical observations of several leukemic cell lines which coexpress erythroid and megakaryocytic markers support the concept that the erythroid and megakaryocytic lineages are partly regulated by the same transcription factor. Therefore, immunophenotyping of megakaryocytic leukemia should be performed using the monoclonal antibodies against erythroid, megakaryocytic and mast cell lineages. However, it is common not to find globin or glycophorin A mRNA in megakaryocytic leukemia cells, and another transcription factor seems to be required for regulation of the expression of megakaryocyte specific genes. Recent studies revealed that IL-6 is an important stimulator of the terminal differentiation of megakaryocytes3' and more recently, Navarro et aL3' showed that normal human megakaryopoiesis might be regulated in part by an IL-6 autocrine loop. Furthermore, the same group3' found that phorbol

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ester enhances expression of IL-6 and IL-6 receptor in most cell lines with erythroid markers, erythroid/megakaryocytic markers or megakaryocytic markers, and they suggested that the proliferation of these cells may be mediated by an IL-6 autocrine loop. The ability of IL-6 to exert its biological effect is believed to be linked to the expression of the corresponding receptor on the cells. Thus the binding of a cytokine to target cells through specific receptors is assumed to be an initial and important event in the regulation of cellular responses33. In order to clarify the mechanisms involved in the proliferation of megakaryocytic leukemic cells, and to characterize these cells, the detection of IL-6 receptor mRNA seems to be the most useful. In our experience, the amount of IL-6 receptor mRNA in leukemic megakaryocytic cells is too low to be detected by Northern blot analysis and RT-PCR analysis has been used to detect IL-6 mRNA. The locations of the PCR oligonucleotide primer for the IL-6 receptor cDNAJ4 are illustrated in Figure 1. However, some cells exhibited the IL-6 receptor mRNA, while others did not. The representative amplification of IL-6 receptor cDNA in CMK35is showed in Figure 2. cc-granule proteins

In megakaryocytes, a-granule proteins such as PF437, p-thromboglobulin (p-TG), vWF, fibrinogen, Factor V, CD4la, CMP140, PDGF, TGFbl, fibronectin and

Primer1 (456 - 475)

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cDNA

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Primer2 ( 9 4 0 - 9 5 9 )

Primer3 (695% 1 s t PCR Product

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1 504bps

2nd PCR !-P o,druct 265bps

Primer 1 5'-GTTTCAGAACAGTCCGGCCG-3'

Primer 2 Primer 3

5 ' - CTCCTGGATTCTGTCCAAGG- 3 '

5 ' - GTCTTGCCAGGTGACACTGAGCCAGC- 3 '

Figure 1 The location of the PCR oligonucleotide primers for IL-6 receptor

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MEGAKARYOCYTIC LEUKEMIA A N D PLATELET FACTOR 4

Figure 2 RT-PCR amplification of IL-6 receptor cDNA in C M K cells with megakaryocytic lineages. RT-PCR was performed as described p r e v i ~ u s l y ~The ~ . PCR products were separated on a polyacrylamide gel and stained with silver stains. The primer 1 and 2, as shown in Figure 2, were used for the first-round PCR. One tenth of the first- round PCR products were used as template for a second round of PCR. Lane 1; the second-round PCR product with primer 1 and primer 3. Lane 2; the second-round PCR product with primer 1 and primer 2.

others3' are reported to be present. Many of a-granule proteins are found in megakaryocytes and platelets only, and not in other hematopoietic cells. The detection of a-granule proteins seems to be a good marker for recognizing the blasts as cells with megakaryocytic lineage. However, Rabellino et u I . showed that a small number of lymphoid-like cells, isolated from normal bone marrow, coexpressed GPs and a-granule proteins such as PF4, vWF and fibronectin. Immunofluorescence analysis of the cells sequentially obtained from the in uitro culture of CFU-Meg also demonstrated the simultaneous appearance of GPs and a-granule proteins such as vWF, PF4, fibrinogen and P D G F in the lymphoidlike mononuclear cells on day 5 of culture23. According to these studies, the detection of platelet GPs and a-granule proteins by immunofluorescence seems to be of the same degree of sensitivity as the definitions of the differentiating stages of leukemic megakaryoblasts and the identification of the cells with megakaryocytic characteristics. On the other hand, the immunophenotypic analysis of leukemic megakaryocytic cells does not support the idea that surface GPs and a-granule proteins are simultaneously expressed in megakaryoblasts. Koike4' and Erber et aL41 demonstrated that monoclonal antibody

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against vWF antigen did not stain any of megakaryocytic leukemic cells exhibiting CD4la and CD61 positivity, although CD4la and vWF antigens were simultaneously found in some megakaryocytic leukemic Furthermore, we encountered megakaryocytic leukemic patients having CD4la-positive cells with or without the expression of PF4 mRNA36. Thus it is likely that we can classify megakaryocytic cells into two groups, one exhibiting both surface antigens and a-granule proteins, and the other one exhibiting surface antigens only. We suppose that the detection of a-granule proteins will be helpful in further characterizing the nature of leukemic megakaryocytic cells. In many cases with megakaryocytic leukemia, marked myelofibrosis is observed and many investigators assume that the excretion of PDGF and TGFpl into the bone marrow may favour the proliferation of fibroblasts, resulting in myelofibrO S ~ Taking S ~ ~ . this into consideration, we reviewed the expression of PDGF and TGFpl in megakaryocytic leukemic cells. PDGF, one of the most potent physiological mitogens, is composed of two types of polypeptides, designated as the A- and B-chains of P D G F encoded by separate genes. PDGF stored in a-granule of platelet consists of AB heterodimers, though small amounts of BB homodimers are also present43. Therefore, leukemic megakaryocytic cells should express P D G F A- and P D G F B-chains (c-sis) reported that the c-sis mRNAs. In fact, Sunami et mRNA was detected in megakaryoblastic cells from a Down's syndrome patient with ~obtained ~ megakaryocytic leukemia. Since macrophages are also the source of PDGF, the expression of P D G F Aand P D G F B-chains (c-sis) mRNAs is not specific for the cells with megakaryocytic lineages. However, ~ ~ that leukePapayannopoulou et u I . demonstrated mia cell lines co-expressing erythroid and megakaryocytic markers secreted P D G F like substances. They also showed that phorbol ester increased P D G F A- and P D G F B-chains (c-sis) mRNAs in K562 cells in association with concomitant megakaryocytic ~ ~Colamonici et differentiation. Alitalo et u I . and also confirmed that megakaryocytic differentiation induced by phorbol ester was accompanied by the accumulation of P D G F A- and P D G F B-chains mRNAs in K562 cells. Furthermore, Alitalo et ~ 7 1 . ~ ~ have demonstrated that only the P D G F B-chain mRNA in K562 cells increased during erythroid differentiation induced by retinoic acid or sodium butyrate, whereas no accumulation of either the A- or B-chain mRNA was detected during erythroid

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differentiation induced by hemin. According to these data, it is probable that the simultaneous increase of PDGF A- and PDGF B-chains (c-sis) mRNAs is part of the megakaryocytic differentiation mechanism. On the other hand, phorbol esters appeared to induce the expression of PDGF B- chain mRNA, but not of the A-chain mRNA in HEL during megakaryocytic differentiation4'. Thus there seems to be a great variety in the expression of P D G F A- and PDGF B-chains (c-sis) mRNAs in megakaryocytic leukemic cells. To characterize the megakaryocytic cells, simultaneous analysis of PDGF A- and P D G F B-chains (c-sis) mRNAs may be required. TGFPl is a polypeptide growth modulator mostly stored in the a-granule of platelets. Recent study revealed that TGFPl induced connective tissue formation by fibroblasts and inhibited megakaryocytopoiesis as a negative feedback regulator of megakaryocyte proliferation. It is assumed that TGFPl may be implicated in myelofibrosis and abnormal megakaryopoiesis found in megakaryocytic leukemic patients. We have no reports on de novo megakaryocytic leukemia with TGFfll mRNA expression. However, Alitalo et d4'have shown that phorbol ester-induced megakaryoblastic differentiation was associated with the enhanced expression of T G F j l mRNA. This increase was slow and had a distinctly different kinetics from the accumulation of PDGF mRNAs. Thus the detection of TGFfll mRNA expression in human megakaryocytic cells will be helpful in understanding the diverse clinical features of the megakaryocyte leukemic patients, although the TGFBl mRNA is found to be expressed by tumors of meso-, endo-, and ectodermal origin as well as many other leukemic cell lines.

have suggested that PF4 inhibits megakaryocytopoiesis as a negative autocrine feedback. Therefore, the observation of the expression of the PF4 molecule in megakaryocytic leukemic cells may be helpful in understanding the clinical features in the patient with cloned the megakaryocytic leukemia. Poncz et d S 0 cDNA for PF4 from HEL cells with megakaryocytic specific antigens and subsequently localized the gene for PF4 to chromosome 4q12-q21. Using this cDNA as a probe, we have been characterizing human leukemic megakaryocytic cells. We summarize here our data concerning PF4 mRNA expression in megakaryocytic leukemic cells. Detection of PF4 mRNA in the leukemic cell lines with megakaryocytic markers In general, the expression of surface antigens are immunologically analyzed in order to characterize these megakaryocytic leukemic cells. However, immunophenotype of the leukemic megakaryoblasts does not always provide definitive information allowing for discrimination between chronic and acute megakaryocytic leukemia, or for distinguishing between therapy-responsive or -unresponsive megakaryocytic leukemias. More sensitive methods are obviously required. To determine the usefulness of the detection of PF4 mRNA in megakaryocytic leukemia cells, we studied its expression in three different kinds of cell lines with known megakaryocytic markers. As shown in Figure 3, no PF4 mRNA was found in the K562 and CMK cell lines, while only HEL cells

PF4 mRNA expression in megakaryocytic leukemia PF4, which is stored in the wgranules of platelets and m e g a k a r y o c y t e ~ is ~ ~ composed of 70 amino acid proteins with 4 cysteine residues. A repeating structure of two lysines followed by two hydrophobic amino acid residues is present near the C-terminus. PF4 is known to be a member of a multigene family, called the small inducible genes (SIG), that appears to be important in coagulation, inflammation, and cell growth. However, its exact biologic function has not yet been fully defined, although PF4 has been reported to be a potent heparin-neutralizing protein, a strong chemattractant for neutrophils, monocytes and fibroblasts, an immunoregulator, and an inhibitor of angiogenesis and c o l l a g e n a ~ e Several ~~. recent studies

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Figure 3 Detection of PF4 mRNA in K562, HEL and C M K cells using Northern blot analysis. The expression of PF4 mRNA was observed 0 and 48 hours after addition of 10 n M TPA. 1; HEL, 2; HEL treated with TPA, 3; C M K , 4; C M K treated with TPA, 5; K562, 6 ; K562 treated with TPA.

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expressed PF4 mRNA after the addition of TPA. These findings were confirmed using RT-PCR technique36. Thus the leukemic cells with megakaryocytic features could be categorized into two groups, one with the PF4 transcript and the other without it. Usefulness of the detection of PF4 mRNA for defining the developmental stages of megakaryoblasts * P F 4

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Even though megakaryocyte maturation is based on the expression of platelet-specific proteins or morphological features, the exact distinction between the different maturation stages of the immature leukemic megakaryocytes is difficult. To clarify the usefulness of the detection of PF4 mRNA for defining the different developmental stages of the blasts, we established two different types of megakaryocytic cell lines with immature and mature characteristics, as evaluated by morphological criteria and immunophen ~ t y p i n gThe ~ ~ .cell line with mature megakaryocytic characteristics, CMK 1 1-5, is morphologically and immunophenotypically different from the parent cell line which has immature features, designated CMK. The expression of PF4 and GPIIb mRNAs was analyzed during TPA-induced differentiation of CMK and CMK 11-5. Increase of GPIIb mRNA in both cell lines after TPA addition was observed, indicating that these cells underwent differentiation after TPA. In contrast, PF4 mRNA in CMK cells was not detected even after the addition of TPA, while TPA induced PF4 mRNA in CMK11-5 (Figure 4). The terminal maturation is believed to consist of cytoplasmic growth with the concomitant synthesis of platelet a-granule proteins. It is reasonable to assume that the synthesis of PF4 stored in a-granules occurs later than that of the membranous glycoproteins during megakaryocytic maturation. Thus enhanced PF4 mRNA expression may be a good marker for the identification of mature megakaryocytic leukemic cells. Detection of PF4 mRNA in megakaryoblasts from the megakaryocytic leukemic patients with an unusual clinical course and the megakaryocytic leukemic patient with a poor prognosis

Wes previously proposed the hypothesis that patients with megakaryocytic leukemia cell expressing PF4 mRNA will have a longer survival and a good response to chemotherapy. In order to confirm our hypothesis, we examined the expression of PF4

~~K

~

~

K

l

~

-

Figure 4 Northern blot analysis of PF4 and GPlIb mRNA expression during TPA-induced differentiation of CMK and CMKl1-5 cells (from Adachi et U L , ~ ' 1991).

mRNA in the megakaryoblasts from the megakaryocytic leukemic patients with a different clinical course using the RT-PCR method. A transient increase of megakaryoblasts in some neonates with Down's syndrome can spontaneously resolve without chemotherapy, thus indicating that these megakaryoblasts are not leukemic. In fact, ultrastructural observations5' have also demonstrated obvious differences between the megakaryoblasts in T M D and megakaryoblastic leukemia cells obtained from a de novo megakaryocytic leukemia in a patient with Down's syndrome. The latter have been reported to have a higher incidence of megakaryoblastic leukemia. Most of the blasts isolated from T M D possessed features of megakaryocytes, basophils, eosinopils and erythroblasts, while those isolated from the de novo megakaryoblastic leukemia had features of immature blasts without granules. Firstly, the expression of PF4 mRNA was analyzed and found to be present in the megakaryoblasts from the Down's syndrome patient with TMD. PF4 mRNA was also present in megakaryoblasts, which appeared in a patient with agnogenic myelofibrosis and myeloid metaplasia for at least 15 years. Her clinical course was unusual as reported p r e v i o u ~ l yThe ~ ~ . patient has remained reasonably well for at least 24 months after developing a megakayoblastic leukemic crisis. On the other hand, the cells from patient with megakaryoblastic leukemia transformed from refractory anemia lacked the expression of PF4 mRNA. His clinical course was described in a previous reportg

~

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and the patient died about 3 months after clonal evolution to megakaryoblastic leukemia. These studies provide further evidence for the fact that patients with the blasts expressing PF4 mRNA have a longer survival and a better response to chemotherapy. The nature of the differences between megakaryoblasts with PF4 mRNA and those which lack it remains to be clarified. However, the terminal maturation of megakaryocytes is believed to consist of cytoplasmic growth, with the concomitant synthesis of platelet @-granuleproteins, although some immunocytochemical studies do not support this con~ e p t ~Therefore, ~ , ~ ~ we . assume that the leukemic megakaryoblasts expressing PF4 mRNA represent a clonal evolution at a more differentiated stage resulting in better responsiveness to therapy and a longer survival. Recent showed that PF4 inhibits the proliferation of megakaryocyte progenitor cells and suggested a role as a negative autocrine regulator of human megakaryocytopoiesis. It is possible that the leukemic megakaryoblastic cells expressing PF4 mRNA may inhibit the proliferation of their own precursor megakaryoblastic cells by secreting PF4 into the bone marrow. Therefore, the mild proliferation of blast cells expressing PF4 mRNA may account for the longer survival of these leukemic patients. The numbers of cases, tested for the expression of PF4 mRNA, is too small to conclude whether the detection of PF4 mRNA in the leukemic megakaryoblasts is useful in practice and more studies are obviously required. Recent studies showed that like CD4lb, the PF4 promoter has a strong GF-1 (transcription factor)-binding site22. The authors speculated, that by analogy to the involvement of GF-1 in globin gene expression in erythroid cells, GF-1 may participate in the transcription of the PF4 gene in megakaryocytes, erythroid cells and mast cells. In fact, some cell lines with erythroid and megakaryocytic lineages are reported to express PF4 mRNA or PF4 antigens5’. The precise mechanism of expression and transcription pathways of PF4 mRNA should be investigated in the future in order to clarify the significance of the determination of expression of PF4 mRNA in the blasts.

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