Leukemia Rearch28(2004)
315–319
Ca report
Pancytopenia prenting with monosomy7which disappeared
after immunosuppressive therapy
Masayuki Nagasawa∗,Daisuke Tomizawa,Youichirou Tsuji,Michiko Kajiwara, Tomohiro Morio,Shigeaki Nonoyama,Minoru Asada,Shuki Mizutani
Department of Pediatrics and Developmental Biology,Graduate School,Tokyo Medical and Dental University,5-45,
Yushima1-chome,Bunkyo-ku,Tokyo113-8519,Japan
Received4March2003;accepted25June2003
Abstract
Monosomy7syndrome in infant is considered as pre-leukemic condition of poor prognosis.However,it
ems controversial recently, becau some cas of monosomy7syndrome showed spontaneous remission.We report2-year-old girl with vere pancytopenia,who prented with monosomy7.Morphologically,there was little dysplasia in the trilineage hematopoiesis.Monosomy7clone of CD34positive cells,bone marrow mononuclear cells(BMMNC),and peripheral nuclear cells was4.0,40,and3.8%,respectively.Immunosuppressive therapy was effective along with the disappearance of monosomy7clone.WT1mRNA expression was not incread in monosomy7 clone.Pathogenesis of monosomy7and its relation to aplastic anemia is discusd.
©2003Elvier Ltd.All rights rerved.
Keywords:Monosomy7;Aplastic anemia;WT1;Cyclosporine A
1.Introduction
Loss of chromosome7(monosomy7)or deletions of its long arm(del(7q))is well-known chromosomal abnormal-ity found in hematologic disorders among early childhood. Usually this abnormality in children shows distinguished clinical features such as hepatosplenomegaly,leukocytosis, a high risk of transformation to myeloid leukemia,and poor prognosis[1–4].Monosomy7/del(7q)is also associate
感想英文d with treatment-related myelodysplastic syndromes both in chil-dren and adults[4,5].Monosomy7/del(7q)is identified in about30–40%of adults with condary AML or MDS,5% with de novo AML,and10–15%with de novo MDS.There is also a report of monosomy7in patient with vere aplas-tic anemia after G-CSF treatment[6].Becau abnormal clone is transformed to leukemia frequently during the clini-cal cour,this abnormality is considered to be pre-leukemic condition.When malignantly transformed,this dia is chemotherapy-resistant and therefore early stem cell trans-plantation is recommended[7–9].
广州新东方学校
Abbreviations:CsA,cyclosporine A;ATG,anti-thymocyte globulin; WT1,Wilm’s tumor gene1;JMML,juvenile myelomonocytic leukemia; MDS,myelodysplastic syndrome;G-CSF,granulocyte colony stimulating factor;PA-IgG,platelet-associated immunoglobulin G
∗Corresponding author.Tel.:+81-3-5803-5250;fax:+81-3-5803-5247. E-mail address:mnagasawa.ped@tmd.ac.jp(M.Nagasawa).
However,biological significance of monosomy7/del(7q) is somewhat controversial recently.It is reported that mono-somy7clone appeared after G-CSF treatment in aplastic anemia patients and disappeared after immunosuppressive treatment[10].Also,it is reported that there were mono-somy7
patients without characteristic features such as hep-atospenomegaly and leukocytosis,and prognosis of the patients was much better than that previously considered [11–13].
We describe a2-year-old girl with monosomy7who prented with pancytopenia.Immunosuppressive therapy improved pancytopenia and induced disappearance of mono-somy7clone.Sequential analysis of monosomy7clone, bone marrow colony assay,and WT1mRNA expression provide us with important information about pathogenesis and significance of monosomy7in hematological disorders.
2.Ca report
Two-year-old girl was referred to our hospital becau of petechiae,which had lasted for2weeks after common cold. Blood examination revealed that white blood cell count of 4500mm−3,hemoglobin of9.3g/dl,reticulocyte of0.72% and platelet count of0.5×104mm−3.Hemoglobin F was slightly incread to8.1%.Hepatosplenomegaly was not
0145-2126/$–e front matter©2003Elvier Ltd.All rights rerved. doi:10.1016/S0145-2126(03)00263-7
316M.Nagasawa et al./Leukemia Rearch 28(2004)
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Fig.1.Monosomy 7clone in the bone marrow FISH and chromosome analysis of the patient’s bone marrow.Arrow indicates chromosome 7.
obrved.Bone marrow aspiration prented hypocellular marrow with nuclear cell count of 68,000mm −3,megakary-ocyte count of 0mm −3and myeloid/erythroid ratio of 0.22.Bone marrow smear was subjected to the three different hematologists who were central reviewers of Japane MDS rearch group.Morphologically,there were no dysplastic changes in myeloid,erythroid and me
gakaryocyte ries.Erythropoietin was incread to 665mIU/ml (normal value <40mIU/ml),but PA-IgG was in the normal range.Con-cerning the post-infection effects on the hematopoiesis,there were no apparent infectious episodes before the patient was referred to our hospital.Furthermore,parvovirus could not be detected in the patient by PCR method at the time of admission.Following the examination described earlier,she was diagnod as aplastic anemia.After 2weeks’obrva-tion with platelet transfusions,immunosuppressive therapy (ATG,CsA,G-CSF)was started according to the AA-99protocol [14],becau no spontaneous improvement was found.After bone marrow chromosomal analysis revealed the prence of monosomy 7clones (Fig.1),administration of G-CSF was stopped,becau it might accelerate malig-nant transformation of monosomy 7clone [15].Sequential analysis of the bone marrow and peripheral blood cells re-vealed disappearance of monosomy 7clones along with the improvement of hematopoiesis (Table 1).After 6-month of immunosuppressive therapy,CsA was tapered and discontin-ued.Her hemogram is white blood cell count of 4500mm −3,hemoglobin of 12.3g/dl,reticulocyte of 0.72%and platelet
Table 1
Changes of bone marrow cells parameters
19June
18July 29August 7November 7March NCC (×104l −1) 6.810.99.824.121.2MgK (l −1)0015.678109M/E ratioaccessories是什么意思
0.220.30.82 3.5 3.0Monosomy 7clone FISH (%)
n.d.400
0WT1mRNA (copies per g RNA)n.d.
210020000130007300Chromosome
45,XX,del(7q)46,XX n.d.46,XX 46,XX 46,XX CFU-GM (±S.D.)(26.1±3.5)a
5.0±1.0
n.d.
3.3±0.6
26.0±3.0petite
24.0±3.6
Nuclear cell count (NCC),megakaryocyte (MgK),myeloid/erythroid (M/E).
a This is the mean ±S .D .of the data from the three different healthy controls.Not done (n.d.).
count of 9.5×104mm −3and stable without medication for more than 1year.
3.Materials and methods
3.1.Purification of bone marrow CD34positive cells Bone marrow mononuclear cells (BMMNC)were ob-tained from the heparinized bone marrow cells by gradi-ent centrifugation paration method.CD34positive cells were purified by using the anti-CD34antibody coated im-munobeads according to the manufacturer’s protocol (Dynal AS,Oslo,Norway).3.2.Colony assay
Colony assay was performed by standard methylcellu-lo method.Briefly,105cells were cultured in the prence of GM-CSF (10ng/ml),Epo (2u/ml),IL-3(10ng/ml),SCF (100ng/ml),and IL-6(100ng/ml).After 2weeks,colonies were counted as colony-forming units.3.3.Real time PCR
WT1mRNA was measured by quantitative real time PCR analysis as described [16].Briefly,RNA was extracted from BMMNC,and cDNA was constructed in the prence of RT
M.Nagasawa et al./Leukemia Rearch 28(2004)315–319317
primer and standard RNA.PCR reaction was performed in the prence of forward and rever primer that spans exon 7and exon 10of WT1genes and Taqman probe by 50cycles.
4.Results
CD34positive cells from patient and healthy control were obtained and colony forming activity was measured.Puri-fied CD34positive cells were assd for the chromosome 7by FISH analysis.As shown in Fig.2,patient’s CD34positive cells had poor colony forming activity although only 4%of the CD34positive cells was monosomy 7.Bone marrow mononuclear cells in which 40%was monosomy 7also had poor colony forming activity.Addition of cy-closporine A in the culture did not improve the colony forming activity (Fig.2).
As summarized in Table 1,WT1mRNA expression of bone marrow mononuclear cells was low when the dia was active,while it once became incread and then
de-
Fig. 2.Colony forming activity of the patient’s CD34positive cells and mononuclear cells from the bone marrow (BMMNC).CFU-GM and BFU-E colony forming activities of the bone marrow cells from the patient and healthy control were investigated (upper bar graph).BMMNC (105cells)or CD34positive cells (105cells)were obtained and colony assay was performed as described in Section 3.Effect of cyclosporine A (500ng/ml)on the colony forming activities of CD34positive cells and BMMNC from the patient was investigated (lower bar graph).Mean value of three different colony assays is prented (mean ±S .D .).Monosomy 7clone detected by FISH analysis shared 40%of BMMNC and 5.2%of CD34positive bone marrow cells.
cread and stable along with the improvement of bone marrow hypoplasia.Colony forming activity of the bone marrow mononuclear cells became also improved after disappearance of monosomy 7clones (Table 1).5.Discussion
Monosomy 7/del(7q)is considered to be pre-leukemic condition.However,there is no direct evidence that links monosomy 7/del(7q)with malignant transformation so far.Most attracting linkage of monosomy 7/del(7q)with hema-tological malignancies is that of juvenile myelomonocytic leukemia (JMML)[17].According to the literature,more than 20%of JMML patients had monosomy 7/del(7q).Re-cently,pathogenesis of JMML was found to be hypernsi-tivity of GM-CSF signaling pa
thway,and the molecular basis of this phenomenon was determined to be dysregulation of ras-signaling pathway through GM-CSF stimulation [1,17].So far,mutations of ras or NF1gene that regulates GTPa were reported in JMML,but the genes were mapped to other than chromosome 7[18,19].Also there were no tumor suppressor genes mapped on the chromosome 7so far.Another noteworthy linkage was monosomy 7/del(7q)with chemotherapy-related myelodysplastic syndrome [4,5].However,there is no biological evidence that the monosomy 7/del(7q)induces the myelodysplastic syndrome.
According to the literature,there are eight cas of mono-somy 7/del(7q)reported,who showed spontaneous resolu-tion [10–13].Four cas were treatment-related MDS,and three of the others were RAEB.In one patient with t-MDS,CsA was effective clinically for improving the hematologic disorder including monosomy 7.However,preci investi-gations were not available in the cas and mechanism of spontaneous resolution is beyond speculation.
In our ca,monosomy 7clone of CD34positive cells,bone marrow mononuclear cells,and peripheral nuclear cells was 4.0,40,and 3.8%,respectively.The results indicate that monosomy 7clone proliferated predominantly in some stages of hematopoiesis,but could not differentiate into ma-ture cells.Possibility that monosomy 7clone was restricted to erythroid cells,which are denucleated after
maturation,was denied becau erythroid cell in the bone marrow was below 20%of whole BMMNC (data not shown).In the colony assay,purified CD34positive cells from the patient’BMMNC,most of which were not monosomy 7clones,had decread colony forming activities as well as whole BMMNC.Considering the results,both of monosomy 7and non-monosomy 7clones had decread colony forming activities.Furthermore,quential analysis of WT1mRNA expression in the BMMNC indicated that there was no pos-itive correlation between WT1mRNA expression and the number of monosomy 7clones.In the myelodysplastic syn-drome,WT1mRNA expression is reported to be correlated with the stages of malignant transformation [20,21].In this n,monosomy 7clone in this patient ems to have lower
318M.Nagasawa et al./Leukemia Rearch28(2004)315–319
potential for malignant transformation compared to tho of JMML and typical MDS.
Clinically,immunosuppressive therapy was effective in this patient.However,the mechanism how immunosuppres-sive therapy was effective in the eradication of monosomy7 clones is uncertain.In vitro colony assay,addition of CsA re-covered neither CFU-GM nor CFU-E.Also,analysis of pe-ripheral lymphocyte in the patient before treatment showed no increa of activated CD8T cells(data
not shown),which suppress hematopoiesis and induce aplastic anemia[22].In this n,it is not convincing that CsA worked as immuno-suppressant in the recovery of hematopoiesis.There is re-ported that aplastic anemia may be a clonal dia[23,24]. Concerning the karyotypic abnormalities,acquired aplastic anemia patients with trisomy of chromosome8,trisomy of chromosome6,or long arm deletion of chromosome13are reported,in which hematological disorders were clinically benign in nature and responsive to immunosuppressive therapy[24–27].Recently,it is reported that CsA modu-lates apoptosis by affecting the mitochondrial membrane depolarization or other mechanisms.Several reports sug-gest that cyclosporine A inhibits apoptosis by interfering with the mitochondrial membrane depolarization[28,29]. On the other hand,it is reported that CsA also works to induce apoptosis in some leukemic cells[30].It is possible that CsA affected on cell survival or death of the patient’s bone marrow.CsA might induce monosomy7clones to apoptosis and thereby normal hematopoiesis recovered in the abnce of interfering abnormal hematopoietic clones. Although limited,this ca is suggestive for the under-standing of the pathological significances of monosomy7in the childhood,and of the mechanisms of aplastic anemia. Acknowledgements
This work was supported in part by the Grant-in-Aid for Scientific Rearch from the Ministry of Education,Science and Culture,Japan(13670785to MN).
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