From www.bloodjournal.org by guest on November 14, 2014. For personal use only. 1993 82: 877-883 Expression of the c-mpl proto-oncogene in human hematologic malignancies I Vigon, F Dreyfus, J Melle, F Viguie, V Ribrag, L Cocault, M Souyri and S Gisselbrecht Updated information and services can be found at: http://www.bloodjournal.org/content/82/3/877.full.html Articles on similar topics can be found in the following Blood collections Information about reproducing this article in parts or in its entirety may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#repub_requests Information about ordering reprints may be found online at: http://www.bloodjournal.org/site/misc/rights.xhtml#reprints Information about subscriptions and ASH membership may be found online at: http://www.bloodjournal.org/site/subscriptions/index.xhtml Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly by the American Society of Hematology, 2021 L St, NW, Suite 900, Washington DC 20036. Copyright 2011 by The American Society of Hematology; all rights reserved. From www.bloodjournal.org by guest on November 14, 2014. For personal use only. Expression of the c-mpl Proto-oncogene in Human Hematologic Malignancies By 1. Vigon, F. Dreyfus, J. Melle, F. ViguiB, V. Ribrag, L. Cocault, M. Souyri, and Similar to two other hematopoietic growth factor receptors, the c-fms (macrophage colony-stimulatingfactor receptor) and the c-kit genes, c-mpl has been discovered through the study of oncogenic retroviruses. Unlike c-fms and c-kif, which both belong to a subgroup of tyrosine kinase receptors, the c-mpl proto-oncogeneencodes a new member of the cytokine receptor superfamily. W e have studied the expression of c-mpl in a series of 105 patients with hematologic malignancies using Northern blot analysis. The levels of c-mpl transcripts in lymphoid malignancies and in chronic myeloproliferative disorders were not significantly different from those found in normal bone marrow cells, in which c-mpl was barely detectable. In S.Gisselbrecht contrast, c-mpl expression was increased in 26 of 51 patients with acute myeloblastic leukemia (AML) and in 5 of 16 patients with myelodysplastic syndromes. Amplification of the c-mplgene was detected in genomic DNA of one M4 AML patient. There was no significant correlation between c-mpl expression and the French-American-British classificationof AML. Patients with high c-mpl expression appeared to belong to a subgroup of AML with a low rate of complete remission and a poor prognosis, including secondary leukemia and AML with unfavorable cytogenetic abnormalities. 0 1993 by The American Society of Hematology. T HE PROLIFERATION and maturation of hematopoitive or myelodysplastic syndromes and in AML patients etic cells are tightly regulated by factors that positively with poor prognosis because of chemotherapy-resistant disor negatively modulate pluripotential stem cell proliferation ease, including most AML patient’s with deletions of chroand multilineage differentiation. These effects are mediated mosomes 5 and/or 7. through the high-affinity binding of extracellular factors to Although c-mpl expression was detected only in maligspecific cell surface receptors. Human leukemic cells have nancies with immature blast cell proliferation, its expresbeen shown to express functional receptors for a variety of sion did not uniformly correlate with the presence of immahematopoietic growth factors including interleukin-3 ture blast cells. Our results suggest that c-mpl expression in (IL-3), granulocyte-macrophage colony-stimulating factor AML patients could be of prognostic significance in predict(GM-CSF) granulocyte-CSF (G-CSF), macrophage-CSF ing response to induction therapy. (M-CSF), and stem cell factor.’-7Several investigators have shown that deregulated expression of growth factors and/or MATERIALS AND METHODS growth factor receptors could be involved in the pathogenePatients and cell line. Peripheral blood (PB) or bone marrow sis of human leukemias and myeloproliferative syn(BM) samples were obtained from 105 patients (104 adults and 1 dromes.8-12We have recently cloned complete cDNAs of a child) who had been referred to the Hematology Department at the new member of the cytokine receptor superfamily designed Hapita1 Cochin between 1988 and 1992. All specimens were colas c-mpl.I3This family includes not only cytokine receptors lected before the initiation of therapy and were obtained from 5 1 such as the IL-2 receptor 0 and y chains and the receptors patients with AML, 11 with acute lymphoblastic leukemia (ALL), for IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, GM-CSF, G-CSF, 19 with myeloproliferative disorders (MPD) in chronic phase, 8 with non-Hodgkin’s lymphoma (NHL), and 16 with myelodysplaserythropoietin, LIF, and oncostatin M, but also the growth tic syndromes (MDS) (Table 1). The diagnosis of each AML was hormone and prolactin receptors.’416c-mpl is the cellular established on the basis of morphologic and cytochemical staining, homologue of the viral oncogene transduced in an acute and patients were classified according to the criteria of the Frenchdefective murine retrovirus, the myeloproliferative leukeAmerican-British (FAB) committee.*’Two patients were classified mia virus (MPLV).I7 Within a few weeks, mice infected asFAB MO, 2 as MI, 13 asM2,5 as M3,8 asM4, I6 as M5,5 as M6, with MPLV develop an hepatosplenomegaly and their he7 as L2, and 4 as L3. The diagnosis of MDS was made according to matopoietic progenitors yield terminally differentiating the FAB criteria, and 1 I patients were classified as refractory anecells in vitro in the absence of any added growth f a c t ~ r . ’ ~ * ’ ~ Infection of bone marrow cells in vitro with MPLV generates autonomous growth factor-independent hematopoietic From the U363 INSERM, ICGM, and the Hematology Departcell lines belonging to various lineages.” Previously, we ment, H6pital Cochin, Paris: and the Laboratoire de Cytogknktique, showed that expression of the c-mpl proto-oncogene was H6tel Dieu, Paris, France. restricted to nonlymphoid hematopoietic tissues.” ThereSubmitted November 30, 1992; accepted March 15. 1993. fore, it seemed to be of interest to study the expression of Supported by grants from the Institut National de la Santk et de la this gene in patients suffering from different hematopoietic Recherche Mkdicale and Comitk de Paris de la Ligue Nationale contre le Cancer. I. V. received afellowshipfrom the Ligue Nationale malignancies. Very low or undetectable levels of c-mpl trancontre le Cancer. scripts were found in cells from patients with lymphoid maAddress reprint requests to F. Dreyfus, MD, Hematology Departlignancies or with myeloproliferative and myelodysplastic ment, H6pital Cochin, 27 rue du Faubourg, Saint-Jacques, 75014, syndromes in chronic phase. However, c-mpl expression Paris, France. was markedly detectable in 26 of 5 1 cases of acute myeloThe publication costs of this article were defrayed in part by page blastic leukemia (AML). In one case of AML, high exprescharge payment. This article must therefore be hereby marked sion was associated with a genomic amplification of the “advertisement” in accordance with 18 U.S.C. section I734 solely to lp34 region encompassing the c-mpl locus.2oc-mpl expresindicate this fact. sion was frequently observed in patients with secondary 0 1993 by The American Society of Hematology. AML developed during the progression of myeloprolifera0006-4971/93/8203-001I $3.00/0 Blood, Vol82, No 3 (August 1). 1993: pp 877-883 877 From www.bloodjournal.org by guest on November 14, 2014. For personal use only. VIGON ET AL Table 1. c-mpl Expression in Human Hematologic Malignancies Diagnosis Increase Over Normal c-mpl Expression AML (51 patients) MO MI M2 012 012 9/13 M3 M4 M5 215 318 7/16 M6 MDS (16 patients) 515 RSA RAE6 MPD (19 patients) 015 511 1 CML PV 017 011 1 ET ALL (11 patients) Q/1 L2 L3 NHL (8 patients) 017 014 018 mia with excess of blasts (RAEB), 5 as sideroblasticanemia (RSA). In the group of patients with MPD, 7 had chronic myelogenous leukemia (CML), 1 1 had polycythemia vera (PV), and 1 had an essential thrombocytemia (ET). As a control, PB and normal BM from volunteerswere obtained after an informed consent. A human erythroleukemia cell line (HEL), provided by W. Vainchenker (U362-IGR Villejuif, Villejuif, France), was cultured in (Y medium containing 10% fetal calf serum. AML patient population and treatment. The clinical characteristics ofthe 5 1 patients with AML are shown in Table 2. Of 5 1 AML patients, 47 were treated with doxorubicin and cytosinearabinoside (35 mg/m2/d for 3 days and 100 mg/m2/dfor 10 days, respectively). The 4 untreated patients included 2 elderly patients (unique patient number [UPN] 42 and 50: Table 2) and 1 patient with apoor performance status (UPN 51). One patient (UPN 49) refused therapy. Complete remission (CR) after induction chemotherapy was defined by a normocellular marrow with less than 5% blasts. We considered induction failure patients as those with resistant disease and those who died of infection or bleeding during induction therapy. Patients with resistant AML and with a good performance status received a second course of chemotherapywith mitoxantrone at 12 mg/m2/d for 3 days, etoposide at 200 mg/mz/d on days 8 through 10, and cytosine arabinoside at 500 mg/m2/d on days 1 through 3 and 8 through 10." mRNA preparation and Northern blot analysis. PB or BM cells were isolated after Ficoll-Hypaque density gradient centrifugation and washed three times in Hanks' balanced salt solution. RNA was extracted by the guanidium thiocyanate procedure.23After precipitation in ethanol at -2O"C, RNA pellets were resuspended in water. RNA concentrations were determined by optical density, and samples were frozen at -80°C until use. RNA samples ( 10 pg of total RNA) were denatured in glyoxal, dimethyl sulfoxide, and phosphate buffer at 65°C for 10 minutes, size-fractionated by electrophoresis through 1% agarose gel, and transferred to nylon membrane. Membranes were hybridized with random '2P-labeled probes and washed under stringent conditions as previously described." The c-mpl probe used in this study was a 280-bp-long fragment located in the human c-mpl extracellular domain. RNA loading was verified by hybridization of each filter with a murine 0 actin probe. The level of c-mpl expression was compared with that observed in HEL cells and in normal BM cells. The presence of megakaryocytes or platelets in BM and PB samples was detected with a glycoprotein IIb (GPIIb) cDNA given by Dr G. Uzan (U 2 I7-Grenoble, Grenoble, France). DNA preparation and Southern blot analysis. High molecular weight DNA was extracted by conventional methods. DNAs (10 pg) were digested with restriction enzymes under reaction conditions recommended by the manufacturers. Agarose gel electrophoresis, Southern blot transfer, and hybridization were performed as previously described.17Probes used as markers of the 1p32-34 chromosomal region were tal- 1, L-myc, c-jun (provided by Dr D. MathieuMahul, U 301, Pans, France), lck (provided by Dr R. Benarous, U 332, Paris, France), Rhesus (from Dr J.P. Cartron, CNTS, Pans, France), and a G-CSF receptor probe (given by Dr S. Gillis, Immunex, Seattle, WA). Cytogenetic analyses. The cytogenetic study was performed on 37 patients with AML at diagnosis on BM samples. Cells were cultured for 24 or 48 hours before chromosome preparation. When possible, 20 metaphases were photographed and analyzed after reverse heat G (RHG) banding of the chromosomes?5 Karyotypes were described according to the International System for Human Cytogenetic Nomenclature.26 Statistical analysis. The x2 test was used to determine significance of qualitative variable differences.Probabilities of c.05were considered statistically significant. The log-rank test was used to compare the groups of patients with respect to achieving CR and disease-free survival.27 RESULTS c-mpl is expressed in patients with AML or RAEB. RNAs isolated from PB or BM from 105 patients with hematologic malignancies described in Materials and Methods and Table 1 were studied for c-mpl expression by Northern blot analysis. We previously reported that c-mpl was detected as a major 3.7-kb and a minor 2.7-kb mRNA species in the human HEL cell line. Both transcripts have been cloned, and they potentially code for polypeptides that share common extracellular and transmembrane domains but differ in their cytoplasmic regions. These two mRNA probably derive from a unique c-mpl gene by alternative ~plicing.'~ To study c-mpl expression, we used as a probe a fragment located in the mpl extracellular domain that detects the two c-mpl transcripts in HEL cells (Fig I , lane a). As shown in Fig 1, no signal was detected in mRNA isolated from normal PB cells (Fig 1, lane b), and c-mpl was barely detectable as a faint 3.7-kb transcript in BM samples from healthy individuals (Fig 1, lane c). Very low c-mpl expression was detected in the 19 patients with lymphoproliferative disorders and in the 19 patients with MPD in chronic phase. In contrast, c-mpl mRNA levels appeared to be elevated in 26 of 5 1 ( 5 1%) AML patients and in 5 of 16 (31%) MDS patients. Typical results are shown in Fig 1. Variability in intensity was seen among patients despite equal loading (as verified by 6-actin hybridization), but cmpl expression in patient samples was usually lower than in HEL cells, except for 1 patient (UPN 23 in Table 2) who presented with a diagnosis of AML, subtype M4 (Fig 1, lane g). Although the 3.7-kb c-mpl transcript was consistently expressed more than the 2.7-kb c-mpl mRNA species, as From www.bloodjournal.org by guest on November 14, 2014. For personal use only. c-mpl EXPRESSION IN LEUKEMIAS 879 Table 2. AML Patient Characteristics Remission Outcome (mo) Cell Blasts UPN Sex/Age FAB Sources (%) Karyotype Status 1 2 3 4 5 6 7' 8' 9 10 11 12' 13 14' 15' 16 17' 18 19 MO MO M1 M1 M2 M2 M2 M2 M2 M2 M2 M2 M2 M2 M2 M2 M2 M3 M3 M3 M3 M3 M4 M4 M4 M4 M4 M4 M4 M4 M5 M5 M5 M5 M5 M5 M5 M5 M5 M5 M5 M5 M5 M5 M5 M5 M6 M6 PB BM 100 93 85 93 65 67 90 95 45 84 93 80 61 64 43 83 38 82 80 80 90 65 87 72 90 93 92 60 65 75 90 61 94 92 43 66 100 81 62 87 48 58 90 80 80 91 60 34 CR CR CR F F CR F F F CR F F CR F CR CR F CR CR CR F CR F CR F CR F CR CR CR CR F F CR F F F F CR CR CR NT F F CR CR F F 49' 50' 51' MI49 F/81 F/56 M6 M6 M6 BM 46XX 46XY ? 50XX,+4,+6,+19,+21 ? 46XX,t(8;2 l)(q22;q22) ? 46XY.-5.-8,1 Zp+,+derl3,-17,-20,+marl ,+mar2,+mar3 43XY,de15(q31),- 11 ,-13,+14,-18,-22 46XX 46XX 44XY,-2,-3,-5,t(5;?)(qll -ql2,?),-7,-9,t(9;19)(~22.?),16,-18 46XX 46XX 46XX ? 48XY,1 p-,3q+,+8,t(9;22)(q34;ql l),lnvl7q,+l9 46XX.t(15;17)(q22;qll) 46XY,t(l5;17)(q22;qll) 46XY,t(l5;17)(q22;qll) 46XY,t(l5;17)(q22;qll) 46XY,t(l5;17)(q22;qll) ? ? 46XY.dell p.dell(q1 1),-5,de16,11 p+,t( 1 1,2 l)(q21,q?)- 17,-21 46XY ? 46XY ? ? 46XX.de17.lnvl6 49XX,-1 ,t(1 ,?)(p36?),de15(q13,q33),-8,+de18(q42),-21, 46XX,lnv3(q22;q26) ? ? ? 47XX,-7,+i(7p),+l4 46XY,t( 1;3)(p36;q21) 46XY 46XX 46XY ? ? 46XY 46XY 46XX.5q45XY.-7 42XY ,del 1(q1 1),-5,6p+,7p+,der(9),+ 1O,+der( 1 1)- 15,- 17,- 18, -19,-20 44XY.-3.de1(5)(ql2.q32).-7,de17,+i( 16p),-21,-22,+marl 45X.-X,del3(q22),-5,deI(5),del(6)ql4,- 18,-2 1 ,+marl ,+mar2 46XX,t(l0:21)(pl3,qll) A 24 A9 R 12 D D A9 D D D R9 D D A6 20 21 22 23 24' 25' 26 27 28' 29 30' 31 32' 33 34 35" 36 37 38 39 40 41 42 43 44 45 46 47 48 F/40 MI60 F/40 F/20 MI46 F/62 MI57 MI62 MI58 F/32 F/54 MI67 F/33 F/65 F/26 F/76 MI40 F/41 MI34 F/74 MI58 MI76 F/68 MI11 MI73 MI54 MI49 MI61 F/37 F/67 F/29 F/63 F/48 MI44 MI34 F/87 F/71 MI57 MI40 F/44 MI23 F/90 MI49 MI63 MI25 MI42 MI32 MI53 NT NT NT D D D BM BM PB BM PB PB BM PB BM PB BM BM BM BM BM BM BM BM BM BM PB PB PB BM BM BM BM PB BM BM PB PB BM BM PB PB BM BM BM BM PB PB P8 BM BM BM BM BM 34 61 39 c-mpl Expression D A 54 A 36 D A 18 A 10 A 50 D A 27 D A9 D A 12 D A 11 A8 A 12 A 74 D D R 19 D D D D A 24 A 26 A 18 D D D R 9 A9 D D Abbreviations: FAB. French-American-British classification; BM, bone marrow; PB, peripheral blood; CR, complete remission; F, failure; NT, not treated; R, relapse; D, dead; A, alive. Secondary leukemia. observed in normal BM and in HEL cells (in BM, the level of mpl expression is not high enough to detect the 2.7-kb transcript), the 2.7-kb c-mpl transcript was found to be quite abundant in the two cases shown in Fig 1, lanes g and i. In AML patients, it appeared that blast cell populations were heterogeneous with respect to the expression of c-mpl. No significant correlation was found between c-mpl expression and the FAB classification because c-mpl transcripts From www.bloodjournal.org by guest on November 14, 2014. For personal use only. VIGON ET AL 880 a b c d e f g h i j k Fig 1. Expression of c-mpl mRNA in human hematologic malignancies. Total RNA (10 pg) was denatured by glyoxal, size-fractionated by electrophoresis, and analyzed by blot hybridization using the c-mplprobe (A) or the@actin probe (B). Lane a, HEL cell line; lanes band c, PB and BM from healthy donors, respectively; lane d, patient with MPD; lane e, patient with ALL; lane f, patient with NHL; lane g, UPN 23; lane h, patient with RAEB; lane i, UPN 25; lane j, UPN 34; lane k, UPN 50. A B were found in patients with M2, M3, M4, M5, and M6 AML (Table I). However, it is noticeable that, although the percentage of blast cells were lower in M6 AML samples than in other AML subtypes, all patients with M6 leukemias were c-mpl positive. Of the 16 MDS patients, none of the 5 patients with RSA had increased c-mplexpression, whereas 5 ofthe I I patients with a diagnosis of RAEB showed increased c-mpl transcripts (Table I). The c-mpl gene is amplified in one patient with an M4 AML. To determine whether genomic alterations might be responsible for high expression, genomic DNAs of leukemic cells from patients expressing the highest levels of c-mpl mRNA were subjected to Southern blot analysis. No gross alteration of the c-mplgene was detected in these DNA samples, except in DNA from UPN 23, in which a c-mpl gene amplification was found (Fig 2A, lanes a and d). Although cytogenetic analysis was not available for this patient, these EcoRl a A b c Sac1 d e f results could be explained by either an amplification encompassing the c-mpl locus or by chromosome duplication. The c-mpl gene was previously located on human chromosome 1 ~ 3 4 . ~Therefore, ' probes for 6 genes located in the small arm of chromosome I (tal-1, L-mvc, c-jim. Ick, Rhesus, and G-CSF receptor)28were hybridized to DNA from UPN 23. No amplification was shown with any other probe tested (see, for example, hybridization with a L-myc probe Fig 2B), suggesting that the c-mpl locus was most likely amplified in the leukemic cells from this patient. Dot blot analysis performed on DNA dilutions indicated that the c-mpl gene was eightfold more abundant in DNA from UPN23 than in control DNA (data not shown). Correlation between c-mpl expression andclinicaland bioIogicSeatirres in AML. The characteristics of the AML population we studied are listed in Table 2. We found no significant correlation between c-mpl expression and characteristics such as sex, age, white blood cells, and platelets counts or the origin of sampling (PB or BM) (TaLes 2 and 3). The HEL cell line which has megakaryocytic and erythroid characteristics is the only human hematopoietic cell line we tested in which we detected c-mpl transcripts by Northern blot analy~is.'~ Consequently, we investigated the possibility of a correlation between GPIIb, glycophorin, and c-mpl expression in AML. By immunophenotyping, only M6 AML expressed the early erythroid glycophorin marker. Northern blots from AML patients were rehybridized with a GPIIb probe. No correlation was found between GPIlb and c-mpl expression (data not shown). Of the 51 AML cases we studied, 16 were presumably secondary leukemias; 12 were blast crises arising during the progression of either chronic MDS (UPN 7, 14, 15, 30, 50, and 5 1) or MPD (UPN 8, 12, 17,24, 28, and 35); I patient (UPN 49) had been professionally exposed to irradiation; and 3 patients (UPN 3,25, and 32) had received chemotherapy after diagnosis of a solid tumor. Sixty-eight percent ( 1 1 of 16) of these 16 secondary leukemias were c-mpl positive versus 43% (15 of 35) of the 35 AML patients presenting with a diagnosis of de novo AML (Table 3). Prognostic value of e-mpl expression in AML. Thirty seven patients with AML could be classified into prognostic groups based on the results of cytogenetic studies according to the prognostic cytogenetic criteria of Keating et a129and em-- B Fig 2. Southern blot analysis of DNA from patients with high mpl expression. Ten micrograms of high molecular weight DNA from UPN 2 3 (lanes a and d) and UPN 2 5 (lanes band e) or control PB DNA (lanes c and f) were digested with the restriction endonuclease FcoRl or Sad, electrophoresed in 0.8%agarose gel, blotted, and hybridized to the mpl probe (A) or to the L-myc probe (B). From www.bloodjournal.org by guest on November 14, 2014. For personal use only. c-mpl EXPRESSION IN LEUKEMIAS 881 Table 3. Relationship of c-mpl Expression to AML Patient Characteristics DISCUSSION Increase Over Normal Expression (+I (-J Age (vr) Sex ratio (M/F) White blood cell count (X 1 0 7 ~ ) Platelet count (X 1 Os&) Median Range Median Range 50.9 1.07 20-90 51.7 1.18 11-87 37.7 95.8 1.5-200 42.3 16-344 91.2 3.5-180 15-600 De novo AML (35patients) Secondary AML (1 6 patients) 20 (57%) 5 (31%) 15 (43%) 1 1 (68%) Good prognostic karyotype (20patients) 14 (70%) 6 (30%) Poor prognostic karyotype (1 7 patients) 4 (23%) 13 (76%) Schouten et al3' [good prognostic: normal karyotype, t(8,2 1) (q22q22), t( 15,17)(q22ql I), or inv( 16)(q22);poor prognostic: all others]. Among the 18 c-mpl-negative AML, 14 had a good prognostic karyotype [ I 1 had a normal karyotype, 3 had a translocation t( 15,17)(q22ql l)], and 4 had a poor risk karyotype. In contrast, among the 19 c-mpl-positive AML, only 6 had good prognostic characteristics: 3 had no cytogenetic abnormalities, 2 had a translocation t( 15,17)(q22q1 l), and 1 had a t(8,2 l)(q22q22). However, 13 patients had chromosomal abnormalities affecting chromosome 5 and/or 7 or chromosome deletions or additions. As summarized in Table 3, 70% (14 of 20) of AML with a good prognostic karyotype were c-mpl negative, whereas 76% (1 3 of 17) of AML with a high-risk karyotype were classified as c-mpl positive. The correlation between high c-mpl expression and the presence of a poor prognosis karyotype was highly significant (P< .001). Of 5 1 AML patients, 47 were treated with doxorubicin and cytosine arabinoside as described in Materials and Methods. Of the 4 untreated AML patients, 1 was c-mpl negative (UPN 42) and 3 were c-mpl positive (UPN 49, 50, and 51), presenting with a diagnosis of M6 leukemia that evolved from a myelodysplastic disorder (UPN 49 and 50) or secondary exposure to irradiation (UPN 51). CR was obtained in 24 of 47 patients treated (51%). Eight of 23 c-mpl-positive AML (35%) achieved CR, whereas 16 of 24 c-mpl-negative AML (67%)reached CR (P= .O 1). Resistant disease was the main cause of induction failure in c-mplpositive AML (1 3 of 15) and was also observed in 3 of the 8 c-mpl-negative AML who did not achieve CR. Of the 13 c-mpl-positive patients who failed to enter CR, 5 patients with a good performance status were treated with a second course of chemotherapy, as indicated in Materials and Methods. None of these 5 patients reached CR. However, in our experience, this intensive therapy leads to CR in 45% of refractory AML, suggesting a primary resistance in c-mplpositive AML.22Because too few patients in the c-mpl-positive group achieved CR, a meaningful comparison of the duration of remission could not be performed. In this report, the expression of the c-mpl gene that encodes a new member of the cytokine receptor superfamily was examined by Northern blot analysis in cells from patients with various hematologic neoplasms. RNA blot analysis show that c-mpl transcripts were not detectable in normal PB and were expressed at very low levels in normal BM cells. We found c-mpl mRNA to be expressed above normal BM cell levels in 5 1% of AML and in 5 of 1 1 RAEB samples. No increase in expression was observed in acute leukemias of lymphoid origin, in lymphoma cells, or in samples of myelodysplastic or myeloproliferative disorders in chronic phase. This result contrasts with the biologic properties of MPLV, a murine retrovirus that has transduced a constitutively activated form of this receptor chain and induces the overproliferation of differentiating hematopoietic progenitors. Therefore, it seems very unlikely that c-mpl expression in AML is responsible for the impaired differentiation of AML blasts; but, because c-mpl is a component of a growth factor signal transduction pathway, expression of the c-mpl product could play a role in the proliferation of these cells. c-mpl expression in AML may be caused by an alteration of the c-mpl gene or may reflect the expansion of a cell population that normally expresses the c-mpl gene and represents only a small proportion of normal BM cells. Amplification of the c-mpl gene evidenced in AML DNA was probably responsible for high c-mpl expression in 1 M4 AML (UPN 23). Two AML had translocations involving the lp36 region, but 1 (UPN 38) did not express c-mpl. However, UPN 32 expressed a normal-sized c-mp13.7-kb transcript at relatively low levels (not shown). No gross alteration or amplification of the c-mpl gene was detected in the DNA of other c-mpl-positive AML. c-mpl expression in AML was associated with poor prognosis because, compared with c-mpl-negative AML, the cmpl-positive AML had a significantly lower rate of CR in response to chemotherapy. Chemotherapy resistance disease was the main cause of induction failure in c-mpl-positive AML. Therefore, we wondered whether c-mpl expression would correlate with other factors known to be of prognostic significance in AML. c-mpl expression did not significantly correlate with age, leucocyte count, or platelet count. Characteristics of the 26 patients with c-mpl-positive AML and of the 25 patients with c-mpl-negative AML were compared and showed that c-mpl was more frequently expressed in secondary than in de novo AML. Moreover, our results showed that c-mpl expression correlated with the presence of cytogenetic abnormalities known to be of major prognostic value in the outcome of AML in terms of both CR rate and long-term disease-free s u r ~ i v a l . ~We ~ ~noted ~" that 1 1 of the 12 AML patients with abnormalities leading to loss of the whole chromosome or part of chromosome 5 and/or chromosome 7 were c-mpl positive. AML with deletion of chromosomes 5 and/or 7 represent a subset of AML characterized by poor prognosis usually because of chemotherapy-resistant disease. Although c-mpl did not correlate with the FAB classification, the fact that 5 erythroleukemias (FAB M6) were all From www.bloodjournal.org by guest on November 14, 2014. For personal use only. VIGON ET AL 882 c-mplpositive raised questions concerning the possible influence of the phenotype of these leukemias o n c-mpl expression. Alternatively, because 3 M6 AML patients had secondary AML and 4 of 5 had abnormalities of chromosomes 5 and /or 7 frequently associated with c-mpl-positive AML, these M6 leukemias could represent a subset of AML in which c-mpl is frequently expressed. Another significant prognostic indicator of response to therapy is the expression of the glycoprotein CD34 antigen ( M Y ~ O ) . ~High ' - ~ ~incidence of secondary AML versus de novo AML and chromosomal abnormalities covariated ~' with CD34 expression in a large multivariate a n a l y ~ i s .Our preliminary results indicate that, although CD34 and c-mpl expression both correlate with the same poor prognostic factors, their expression does not completely overlap because some CD34+ AML did not overexpress c-mpl, and, conversely, some c-mpl-positive AML were CD34- (data not shown). Analysis of a larger series of patients would indicate to what extent these two markers covanate. Interestingly, our study o n MDS patients shows that, similar to AML, elevated c-mpl expression could also be associated with a poor prognosis. Indeed, 5 of 1 1 RAEB patients had increased c-mpl expression versus none of the 5 RSA patients. A survey of c-mpl expression in a larger panel of MDS patients is currently being performed in an attempt to define the precise relationship between c-mpl expression and the emergence of a blastic cell population. Further studies on the c-mpl protein will show whether c-mpl is a ligand binding chain or a part of a multimeric complex. Because c-mpl has the overall structure of cytokine receptor, one could suppose that expression of the cmpl gene product may promote the proliferation of myeloid blast cells in vivo. ACKNOWLEDGMENT We thank F. Ajchenbaum, F. Picard, M. Sitbon and A. Turhan for critical reading of the manuscript and V. Chauvin for expert secretarial support. REFERENCES 1. Budel LM, Touw IP, Delwel R, Clark S, Lowenberg B: IL-3 and GM-CSF receptors on human acute myelocytic leukemia cells and the relationship to the proliferative response. Blood 74:565, 1989 2. Park LS, Waldron PE, Friend D, Sassenfeld HM, Price V, Anderson D, Cosman D, Andrews RG, Bernstein ID, Urdal DL: Interleukin-3, GM-CSF, and G-CSF receptor expression on cell lines and primary leukemia cells: Receptor heterogeneity and relationship to growth factor responsiveness.Blood 74:56, 1989 3. Begley CG, Metcalf D, Nicola NA: Primary human myeloid leukemia cells: Comparativeresponsivenessto proliferativestimulation by GM-CSF or G-CSF and membrane expression of CSF receptors. Leukemia I:l,1987 4. Dubreuil P, Torres H, Courcoul MA, Birg F, Mannoni P: c-fms expression is a molecular marker of human acute myeloid leukemias. Blood 72:1081, 1988 5. Ikeda H, Kanakura Y, Tamaki T, Kuriu A, Kitayoma H, Ishikawa J, Kanayama Y, Yonezawa T, Tarui S, Griffin JD: Expression and functional role of the proto-oncogene c-kit in acute myeloblastic leukemia cells. Blood 78:2962, 1991 6. Lerner NB, Nocka KH, Cole SR, Qiu F, Strife A, Ashman LK, Besmer P Monoclonal antibody YB5.B8 identifiesthe human c-kit protein product. Blood 77:1876, 1991 7. Broudy VC, Smith FO, Lin N, Zsebo KM, Egrie J, Bemstein I D Blasts from patients with acute myelogenous leukemia express functional receptors for stem cell factor. Blood 80:60, 1992 8. Depper JM, Leonard WJ, Kranke M, Waldmann TA, Greene WC: Augmented T cell growth factor receptor expression in HTLV-1 infected human leukemic T cells. J Immunol 133:169I , 1984 9. Rambaldi A, Wakamiya N, Vellenga E, Horiguchi J, Warren MK, Kufe D, Griffin J D Expression of the macrophage colony stimulating factor and c-fms genes in human acute myeloblastic leukemic cells. J Clin Invest 81:103O, 1988 IO. Mitjavila MT, le Couedic JP, Casadevall N, Navarro S, VileVal JL, Dubart A, Vainchenker W Autocrine stimulation by erythropoietin and autonomous growth of human erythroid leukemic cells in vitro. J Clin Invest 88:789, 1991 1 I. Eaves AC, Eaves CJ: Erythropoiesis in culture. Clin Haemato1 13:371, 1984 12. Prchal JF, Axelrad AA: Bone marrow responsesin polycythemia vera. N Engl J Med 290: 1382, 1974 13. Vigon I, Mornon JP, Cocault L, Mitjavila MT, Tambourin P, Gisselbrecht S, Souyri M: Molecular cloning and characterization of h-mpl, the human homolog of the v-mpl oncogene: Identification of a new member of the hematopoietic growth factor receptor superfamily. Proc Natl Acad Sci USA 89:5640, 1992 14. Cosman D, Lyman SD, Idzerda RL, Beckmann MP, Park LS, Goodwin RG, March CJ: A new cytokinereceptor superfamily. Trends Biochem Sci 15:265, 1990 15. Miyajima A, Hara T, Kitamura T Common subunits of cytokine receptors and the functional redundancy of cytokines. Trends Biochem Sci 17:378, 1992 16. Renauld JC, Druez C, Kermouni A, Houssiau F, Uyttenhove C, Van Roost E, Van Snick J: Expression cloning of the murine and human interleukin 9 receptor cDNAs. Proc Natl Acad Sci USA 895690, 1992 17. Souyri M, Vigon I, Penciolelli JF, Heard JM, Tambourin P, Wendling F A putative truncated cytokine receptor gene transduced by the myeloproliferativeleukemia virus immortalizeshematopoietic progenitors. Cell 63: l 137, 1990 18. Wendling F, Vigon I. Souyri M, Tambourin P Myeloid progenitor cells transformed by the myeloproliferative leukemia virus proliferate and differentiate in vitro without the addition of growth factors. Leukemia 3:475, 1989 19. Wendling F, Charon M, Penciolelli JF, Tambourin P Factor-independent erythropoietic precursor cells in leukemia induced by the myeloproliferativeleukemia virus. Blood 73:1161, 1989 20. Le Conniat M, Souyri M, Vigon I, Wendling F, Tambourin P, Berger R The human homolog of the myeloproliferative virus maps to chromosome band lp34. Hum Genet 83:194, 1989 21. Bennet JM, Catowsky D, Daniel MT, Flandrin G, Galton DAG, Granlick H, Sultan C: Proposals for the classificationof the acute leukemia. Br J Haematol 33:451, 1976 22. Archimbaud E, Leblond V, Michallet M, Cordonnier C, Fenaux P, Travade P, Dreyfus F, Jaubert J, Devaux Y, Fiere D: Intensive sequentialchemotherapywith mitoxantrone and continuous infusion etoposide and cytarabine for previously treated acute myelogenous leukemia. Blood 77: 1894, 1991 23. Chirgwin JM, Przybyla AE, MacDonald RJ, Cowan NJ, Rutter WJ, Kirschner MW: Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294, 1979 24. Frachet P, Uzan G, Thevenon D, Denarier E, Prandini MH, From www.bloodjournal.org by guest on November 14, 2014. For personal use only. c-mpl EXPRESSION IN LEUKEMIAS Marguerie G: GpIIb and GpIIIa amino acid sequences deduced from human megakaryocytescDNAs. Mol Biol Res 14:27, 1990 25. Dutrillaux B, Lejeune J: Sur une nouvelle technique d'analyse du caryotype humain. C R Acad Sci 111 272:2638, 1971 26. ISCN: An international system for human cytogenetic nomenclature, in Harnden DG, Klinger HP (eds). Published in collaboration with Cytogenet Cell Genet, Basel, Switzerland, Karger, 1985 27. Kaplan EL, Meier P Nonparametric estimation for incomplete observation. J Am Stat Assoc 53:457, 1958 28. Collins A, Keats BJ, Dracopoli N, Shields DC,Morton NE: Integration of gene maps: Chromosome I . Proc Natl Acad Sci USA 89:4598, 1992 29. Keating MJ, Cork A, Broach Y , Smith T, Walters RS, McCredie KB, Trujillo J, Freireich EJ: Toward a clinically relevant cytogenetic classification of acute myelogenous leukemia. Leuk Res 11:119, 1987 30. Schouten HC, van Putten WW, Hagemeijer A, Blijham GH, 883 Sonnenveld P, Willemze R, Slater R, van de Lely J, Verdonck LF, Lowenberg B The prognostic significanceof chromosomal findings in patients with acute myeloid leukemia in a study comparing the efficacy of autologous and allogeneicbone marrow transplantation. Bone Marrow Transplant 8:377, 1991 31. Vaughan WP, Civin CI, Weisenburger DD, Karp JE, Graham ML, Sanger WG, Grierson HL, Joshi SS, Burke PJ: Acute leukemia expressing the normal human hematopoietic stem cell membrane glycoprotein CD34 (My 10). Leukemia 2:661, 1988 32. Borowitz MJ, Gockerman JP, Moore JO, Civin CI, Page SO, Robertson J, Bigner SH: Clinicopathologicand cytogenetic features of CD34 (My 10)-positiveacute nonlymphocytic leukemia. Am J Clin Path01 91:265, 1989 33. Geller RB, Zahurak M, Hunvitz CA, Burke PJ, Karp JE, Piantadosi S, Civin CI: Prognostic importance of immunophenotyping in adults with acute myelocytic leukaemia: The significance of the stem-cell glycoprotein CD34 (My 10). Br J Haematol76:340, I990
© Copyright 2024