Congenital amegakaryocytic thrombocytopenia (CAMT, OMIM 604498) is an autosomal recessive disorder characterized by absent or reduced number of megakaryocytes in the bone marrow (BM) since birth, elevated serum levels of thrombopoietin (TPO), and very low platelet count. Prognosis of CAMT patient

Alessandra Di Cesare-Merlone

Barbara Pressato

Carlo Dufour

Carlo L. Balduini

Chiara Cugno

Claudio Panarello

Cristina Morerio

Elisa Tassano

Emanuela Maserati

F Patitucci

Francesco Lo Curto

Francesco Pasquali

Franco Locatelli

Roberto Valli

English

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Letters to the Editorhaematologica | 2008; 93(8) | 1271 |11:1311-5.3. Rajkumar SV, Dispenzieri A, Kyle RA. Monoclonal gam-mopathy of undetermined significance, Waldenströmmacroglobulinemia, AL amyloidosis, and related plasma celldisorders: diagnosis and treatment. Mayo Clin Proc 2006;81:693-703.4. Wilder RB, Ha CS, Cox JD, Weber D, Delasalle K, AlexanianR. Persistence of myeloma protein for more than one yearafter radiotherapy is an adverse prognostic factor in solitaryplasmacytoma of bone. Cancer 2002;94:1532-7.5. International Myeloma Working Group. Criteria for the clas-sification of monoclonal gammopathies, multiple myelomaand related disorders: a report of the International MyelomaWorking Group. Br J Haematol 2003;121:749-57.6. Dimopoulos MA, Moulopoulos LA, Maniatis A, AlexanianR. Solitary plasmacytoma of bone and asymptomatic multi-ple myeloma. Blood 2000;96:2037-44.7. Zamagni E, Nanni C, Patriarca F, Englaro E, Castellucci P,Geatti O, et al. A prospective comparison of 18F-fluo-rodeoxyglucose positron emission tomography-computedtomography, magnetic resonance imaging and whole-bodyplanar radiographs in the assessment of bone disease innewly diagnosed multiple myeloma. Haematologica 2007;92:50-5.8. Schirrmeister H, Bommer M, Buck AK, Muller S, Messer P,Bunjes D, et al. Initial results in the assessment of multiplemyeloma using FDG-PET. Eur J Haematol Med 2002;29:361-6.9. Durie GM, Waxman AD, D’Agnolo A, Williams CM. Wholebody18F-FDG-PET identifies high-risk myeloma. J Nucl Med2002;43:1457-63.10. Nanni C, Zamagni E, Farsad M, Castelluci P, Tosi P, CanginiD, et al. Role of F18-FDG PET/Ct in the assessment of boneinvolvement in newly diagnosed multiple myeloma : prelim-inary results. Eur J Nucl Med Mol Imaging 2006;33:525-31.Clonal chromosome anomalies and propensityto myeloid malignancies in congenitalamegakaryocytic thrombocytopenia (OMIM 604498)Congenital amegakaryocytic thrombocytopenia (CAMT,OMIM 604498) is an autosomal recessive disorder charac-terized by absent or reduced number of megakaryocytes inthe bone marrow (BM) since birth, elevated serum levels ofthrombopoietin (TPO), and very low platelet count.Prognosis of CAMT patients is poor, because all develop inchildhood a tri-linear marrow aplasia that is always fatalwhen untreated.1,2 Mutations of the MPL gene (OMIM159530), coding for the TPO receptor,3 are responsible forCAMT.4,5 We report the cytogenetic investigations and theresults of analysis by fluorescent in situ hybridization (FISH)on 5 unrelated Italian patients whose clinical characteristicsand MPL gene mutations have already been reported.5Three patients were females and two males, age at diagno-sis ranging between 16 and 49 months. All children devel-oped pancytopenia at an age comprised between 22 and 49months. Patients’ designation (CAMT1-CAMT5 in Table 1)is as in Savoia et al.5Chromosome analyses were repeatedly performed onBM and peripheral blood (PB) PHA-stimulated cultureswith routine methods. Skin fibroblasts (SF) were culturedwith routine methods in patient CAMT2. QFQ-bandingtechnique was used for all chromosome analyses. FISHanalyses on interphase nuclei were repeatedly performedon all patients’ BM, on PB of CAMT4, and on SF ofCAMT2 with centromere-specific probes for chromo-somes 7 (D7Z1), and 8 (D8Z2) (Cytocell Technologies,Cambridge, UK) either with single fluorochromes or indual color combination. Nuclei from healthy subjects wereused as control.All the results are detailed in Table 1. The karyotype wasconsistently normal in patients CAMT1, CAMT3, andCAMT5, and parallel FISH analysis on interphase nucleifrom BM confirmed the normal disomies 7 and 8. DEB testperformed on PB of CAMT1, CAMT2, and CAMT3excluded Fanconi anemia (FA).Patient CAMT2 progressed to pancytopenia, with nor-mal BM and PB karyotype, at the age of 30 months(January 2005), but in a subsequent analysis on BM, inMay 2006, trisomy 8 was found in 1 mitosis out of 24 and5 nuclei out of 616. Analysis on fibroblasts from a skinbiopsy excluded a constitutional trisomy 8 mosaicism(Table 1). In patient CAMT4, the karyotype was normal inBM cells in 2001, at the age of 12 months, and in 2004when progression to pancytopenia was observed, but inMay 2006 a BM clone with monosomy 7 was found whichpersisted in the following analyses (Table 1). A risk of evolution into myelodysplastic syndrome(MDS) and acute myeloid leukemia (AML) has often beenassumed for CAMT, but in a search for bona fide CAMTpatients who developed MDS/AML, we found only 3 suchcases in the literature, and none of these was proved tocarry mutations of the MPL gene: they are one refractoryanemia with excess of blasts (RAEB) reported by King et al.,2and 2 cases mentioned by Alter,6 a male with acutemyelomonocytic leukemia (AMML) developed after anaplastic anemia phase, and a female with a pre-leukemiccondition. These 2 latter cases were never reported in moredetail, and were studied some decades ago.6 In addition, areport is available of a CAMT patient with MPL mutationswho developed a pre-B acute lymphoblastic leukemia.7A review of CAMT cases with chromosome anomaliesis even more difficult to carry out since cytogenetic resultsin the literature are often incorrectly mentioned, incom-plete, and probably questionable. Among the patients withMDS/AML mentioned above, clonal chromosome changesin the BM were reported to be present in 2: the child withRAEB2 with trisomy 21 in 10-15% of the cells, and thepatient with AMML6 with different anomalies of chromo-some 19 (monosomy, trisomy, deletion) in 11 cells out of55. As to CAMT without MDS/AML, King et al.2 reported2 patients with possible chromosome anomalies in PB, butthe case identified as CAMT9 showed a translocation onlyin one cell out of 19, whereas CAMT13 showed a super-numerary marker (not better defined) in 94% of the cells.The only case from the literature with a reliable clonalanomaly in the BM was reported by Steele et al.,8 whofound monosomy 7 in 12 out of 20 mitoses and 91 out of200 interphase nuclei. Mutations of the MPL gene wereidentified in these 3 patients.4,8In our 5 patients, BM clonal anomalies were found in 2, inthe absence of MDS/AML: one case of monosomy 7 andone of trisomy 8; the latter was demonstrated not to be aconstitutional mosaicism, as is the case in 15-20% ofpatients with MDS/AML and trisomy 8.9 While no exhaus-tive cytogenetic study on CAMT is available, our smallgroup of patients was monitored over time for possible clon-al chromosome anomalies in BM. We suggest that clonalchromosome changes are frequently acquired in the BM ofCAMT patients: they often seem to be the most typical ofMDS, monosomy 7 and trisomy 8. Interestingly, in both ourpatients the abnormal clones were found when the diseasehad already progressed to pancytopenia (Table 1), and thepatient reported by Steele and co-workers showed mono-somy 7 when CAMT had evolved to BM aplasia.8In both our patients, the abnormal clone was detectednot at the first chromosome analysis but after and showeda trend to expansion (Table 1). In particular, if we take intoaccount evaluations by interphase FISH, in patientLetters to the EditorCAMT2 the difference between BM samples of 23.05.06and 06.07.06 only indicates this trend (0.02<p<0.05), whilein patient CAMT4 the difference between the BM samplesof 24.05.06 and 06.07.06 is significant (p<0.01).The expansion of a BM clone with a chromosomeanomaly typical of MDS/AML, should be considered withrespect to the risk of hematologic malignancies in CAMTpatients. A plausible hypothesis is that a risk of progress-ing into MDS/AML is really part of the CAMT phenotype,although the short life expectancy and the use ofhematopoietic stem cell transplantation make it difficult todemonstrate this evolution in most patients. If this is thecase, the patients with clonal anomalies might be thosewho will progress to MDS/AML. We postulate that MPLmutations might cause karyotype instability through amutator effect, with emergence of abnormal clones in theBM possibly characterized by chromosome changes typi-cal of MDS: this mechanism was suggested also for otherMendelian BM failure syndromes, such as Shwachmansyndrome (SS), familial platelet disorder with propensity toacute myeloid leukemia (FPD/AML, OMIM 601399), andFA.10,11 In CAMT, the BM abnormal clone might stay qui-escent even for long periods, as in SS, a full-blown MDSpicture being the consequence of its expansion. In conclu-sion, we suggest that an appropriate cytogenetic follow-upof BM should be part of the clinical management ofpatients with CAMT, because the early detection of clonalanomalies might be crucial for treatment choice.Emanuela Maserati,1 Claudio Panarello,2 Cristina Morerio,2 RobertoValli,1 Barbara Pressato,1 Francesco Patitucci,1 Elisa Tassano,2Alessandra Di Cesare-Merlone,3 Chiara Cugno,3 Carlo L. Balduini,4Francesco Lo Curto,1 Carlo Dufour,2 Franco Locatelli,3Francesco Pasquali11Biologia e Genetica, Dipartimento di Scienze BiomedicheSperimentali e Cliniche, Università dell'lnsubria, Varese;2Dipartimento di Ematologia ed Oncologia Pediatrica, IRCCS IstitutoG. Gaslini, Genova; 3Oncoematologia Pediatrica, Università diPavia, Fondazione IRCCS Policlinico S. Matteo, Pavia; 4ClinicaMedica III, Università di Pavia, Fondazione IRCCS Policlinico S.Matteo, Pavia, ItalyFunding: FL and FP were supported by grants from MIUR(Ministero dell’Università e della Ricerca), CP by grants fromFondazione Gerolamo Gaslini, and FL from AIRC (AssociazioneItaliana Ricerca sul Cancro), CNR (Consiglio Nazionale delleRicerche), Istituto Superiore di Sanità (National Program on StemCells), European Community, and Fondazione IRCCS (Istituto diRicovero e Cura a Carattere Scientifico) Policlinico San Matteo.Key words: congenital amegakaryocytic thrombocytopenia, MDS,AML, trisomy 8, monosomy 7, mutator effect.Correspondence: Emanuela Maserati, Dipartimento di ScienzeBiomediche Sperimentali e Cliniche, Università dell'lnsubria,via J.H. Dunant 5, 21100 Varese, Italy. Phone: international+39.0332.217181; Fax: international +39.0332.217119.E-mail: emanuela.maserati@uninsubria.it Citation: Maserati E, Panarello C, Morerio C, Valli R, Pressato B,Patitucci F, Tassano E, Di Cesare-Merlone A, Cugno C, Balduini CL,Lo Curto F, Dufour C, Locatelli F, Pasquali F. Clonal chromosomeanomalies and propensity to myeloid malignancies in congenita-lamegakaryocytic thrombocytopenia (OMIM 604498). Haematologica2008; 93:1271-1273.  doi: 10.3324/haematol.12748References1. Federman N, Sakamoto KM. The genetic basis of bone mar-row failure syndromes in children. Mol Genet Metab 2005;86:100-9.2. King S, Germeshausen M, Strauss G, Welte K, Ballmaier M.Congenital amegakaryocytic thrombocytopenia: a retrospec-tive clinical analysis of 20 patients. Br J Haematol 2005;131:636-44.| 1272 | haematologica | 2008; 93(8)Table 1. Results of chromosome analyses and FISH on interphase nuclei.Patient Date T/P1 Material2 Karyotype FISH on nuclei: D7Z1 probe, D8Z2 probe3CAMT1 03.11.04 T BM 46,XX[20] D7Z1: 450/467 normal4 (96.3%); D8Z2: 557/571 normal (97%)06.12.04 T PB-PHA 46,XX[100]20.01.05 T PB-DEB 46,XX[100]BM 46,XX[7] D7Z1: 450/458 normal (98.2%); D8Z2: 500/513 normal (97.4%)CAMT2 11.01.05 T BM 46,XX[8]PB-PHA 46,XX[100]PB-DEB 46,XX[100]18.11.05 T BM 46,XX[12]23.05.06 P BM 47,XX,+8[1]/46,XX[23] D8Z2: 5/616 with trisomy 8 (0.8%)06.07.06 P BM 47,XX,+8[5]/46,XX[22] D8Z2: 9/332 with trisomy 8 (2.7%)SF 46,XX[56] D8Z2: 1314/1342 normal (97.9%) CAMT3 12.01.06 T PB-PHA 46,XY[100]PB-DEB 46,XY[100]BM 46,XY[22] D7Z1: 450/461 normal (97.6%); D8Z2: 400/415 normal (96.4%)24.11.06 P BM 46,XY[35] D7Z1: 400/458 normal (98.2%); D8Z2: 500/515 normal (97%)CAMT4 17.05.01 T BM 46,XY[67] D7Z1 + D8Z25: 390/400 normal (97.5%)01.06.04 P BM 46,XY[26] D7Z1 + D8Z25: 11/400 with monosomy 7 (2.7%)24.05.06 P BM 45,XY,-7[22] D7Z1 + D8Z25: 121/400 with monosomy 7 (30.2%)30.08.06 P PB-PHA 46,XY[248] D7Z1 + D8Z25: 325/350 normal (92.9%)06.07.06 P BM 45,XY,-7[55]/46,XY[4] D7Z1 + D8Z25: 161/400 with monosomy 7 (40.2%)18.10.06 P BM 45,XY,-7[14]/46,XY[3]04.01.07 BM 46,XY[16]6CAMT5 14.03.02 T BM 46,XX[116] D7Z1 + D8Z25: 459/472 normal (97.2%)14.05.02 P BM 46,XX[48] D7Z1 + D8Z25: 397/400 normal (99.5%)02.10.03 P BM 46,XX[18]1T: thrombocytopenia; P: evolution into pancytopenia; 2BM: bone marrow direct preparations and 24-48h cultures; PB-PHA: peripheral blood PHA stimulated culture;PB-DEB: peripheral blood PHA stimulated cultures, with diepoxybutane (DEB); SF: skin fibroblast culture; 3FISH on normal controls showed an average of 97.2%nuclei with two signals for D7Z1, and of 97.4% for D8Z2; 4normal result means that no evidence of monosomy 7 and of trisomy 8 was reached in the few nuclei notshowing two signals, with data comparable to controls; 5dual color FISH with probes D7Z1 and D8Z2; 6after hematopoietic stem cell transplantation. Letters to the Editorhaematologica | 2008; 93(8) | 1273 |3. Ihara K, Ishii E, Eguchi M, Takada H, Suminoe A, Good RA,et al. Identification of mutations in the c-mpl gene in congen-ital amegakaryocytic thrombocytopenia. Proc Natl Acad SciUSA 1999;96:3132-6.4. Germeshausen M, Ballmaier M, Welte K. MPL mutations in23 patients suffering from congenital amegakaryocytic throm-bocytopenia: the type of mutation predicts the course of thedisease. Hum Mutat 2006;27:296-301.5. Savoia A, Dufour C, Locatelli F, Noris P, Ambaglio C, Rosti V,et al. Congenital amegakaryocytic thrombocytopenia: clinicaland biological consequences of five novel mutations.Haematologica 2007;92:1186-93.6. Alter B. Inherited bone marrow failure syndromes. In: NathanDG, Orkin SH, Look AT, Ginsburg D, editors. Nathan andOski’s hematology of infancy and childhood. W B Saunders:Philadelphia, PA, 2003, p. 314-5.7. Steinberg O, Gilad G, Dgany O, Krasnov T, Zoldan M, LoarR, et al. Congenital amegakaryocytic thrombocytopenia – 3novel c-MPL mutations and their phenotypic correlations. JPediatr Hematol Oncol 2007;29:822-5.8. Steele MacG, Hitzler J, Doyle JJ, Germeshausen M, FernandezCV, Yuille K, et al. Reduced intensity hematopoietic stem-celltransplantation across human leukocytes antigen barriers in apatient with congenital amegakaryocytic thrombocytopeniaand monosomy 7. Pediatr Blood Cancer 2005;45:212-6.9. Maserati E, Aprili F, Vinante F, Locatelli F, Amendola G,Zatterale A, et al. Trisomy 8 in myelodysplasia and acuteleukemia is constitutional in 15-20% of cases. GenesChromosom Cancer 2002;33:93-7.10. Minelli A, Maserati E, Rossi G, Bernardo ME, De Stefano P,Cecchini MP, et al. Familial platelet disorder with propensityto acute myelogenous leukemia: genetic heterogeneity andprogression to leukemia via acquisition of clonal chromosomeanomalies. Genes Chromosom Cancer 2004;40:165-71.11. Dror Y. Shwachman-Diamond syndrome. Pediatr BloodCancer 2005;45:892-901.Variability of clinical manifestation of factor VII-deficiency in homozygous and heterozygoussubjects of the European F7 gene mutation A294VInherited factor VII deficiency (FVIID) is a rare autoso-mal bleeding disorder. Subjects with reduced FVII activityand identified F7 gene mutation are registered in theInternational Greifswald registry of FVIID.1 The mutationA294V is the most frequent among 717 FVIID patients inCentral and North-Eastern Europe. The high prevalenceof FVIID homozygous and heterozygous subjects, whoshare the same mutation (A294V), offers the unique pos-sibility to study the clinical variability in a relatively largenumber of individuals with the identical defect in the F7gene. In the reported study we have analyzed the clinicalvariability of 14 homozygous and 99 heterozygous sub-jects with F7 gene mutation A294V. Subjects with combi-nations with other bleeding disorders such as vonWillebrand Disease, deficiency of FIX, FII etc., wereexcluded.We collected data on factor VII clotting (FVII:C) activi-ty, FVII antigen (FVII:Ag), date of initial onset of bleedingand bleeding symptoms.1,2FVII:C was assayed locally with standard one-stagemethods using thromboplastin (mostly recombinant andhuman-derived, Thromborel S® Dade Behring). FVII:Agwas measured at some of the centers using an immunoen-zymatic method (Asserachrom, Diagnostica Stago,Asnieres, France). The following bleeding symptomswere evaluated according to published criteria:1-4 intracra-nial hemorrhage (ICH), gastrointestinal (GI) hemorrhage,hemarthrosis, subcutaneous hematoma as well as epis-taxis, oral bleeding, and menorrhagia in accordance withPeyvandi and Mannucci.5 Endogenous thrombin potential(ETP) was measured in platelet poor plasma from controlsand subjects homozygous for A294V in the presence ofactivated protein C.6 Citrated plasma samples from 8 con-trol subjects without FVLeiden or FVHR2 (factor V wild type;FVwt) and without A294V mutation were used as a refer-ence. We used the mutation H1299R as marker forFVHR2.7 The FVLeiden mutation was determined accordingto Bertina et al.8The 14 homozygous subjects for the F7 gene mutationA294V are characterized by mean levels of FVII:C of10±6% and of FVII:Ag of 52±11%. Nine of the homozy-gous patients (64%) had spontaneous bleeding symptoms(classified as symptomatic) and 5 (36%) did not (classifiedas asymptomatic). There was no significant difference inFVII level between symptomatic and asymptomatichomozygous A294V subjects.The spontaneous bleeding profile of the 9 symptomaticsubjects was characterized by GI (22%) bleeds,hemarthrosis (22%), epistaxis (33%), easy bruising (11%),gingival bleeding (22%), subcutaneous hematoma (11%),Table 1. Endogenous thrombin potential, bleeding symptoms and co-inheritance of FVHR2 in nine patients homozygous for the mutationA294V with available plasma samples. Endogenous thrombin potentialcompared to the referencesreference (100%)G-9125 6 8 hz asympt. 203%G-9297 18 25 ho asympt. 146%G-9130 15 4 wt mild 93%G-9623 32 8 hz mild 82%G-13255 10 7 wt mild 59%G-9773 59 6 wt moderate 56%G-13325 74 11 wt severe 65%G-12990 44 9 wt severe 57%G-10833 57 3 wt severe 47%PatientAgeFVII:C [%]FV HR2GIBleedHemarthrosisEasy BruisingEpistaxisGum BleedHematuriaMenorrhagiaHematomaSeverity*

Clonal chromosome anomalies and propensity to myeloid malignancies in congenital amegakaryocytic thrombocytopenia (OMIM 604498).

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Clonal chromosome anomalies and propensity to myeloid malignancies in congenital amegakaryocytic thrombocytopenia (OMIM 604498).

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