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

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 patients is poor, because all develop in childhood a tri-linear marrow aplasia that is always fatal when untreated.1,2 Mutations of the MPL gene (OMIM 159530), coding for the TPO receptor,3 are responsible for CAMT.4,5 We report the cytogenetic investigations and the results of analysis by fluorescent in situ hybridization (FISH) on 5 unrelated Italian patients whose clinical characteristics and MPL gene mutations have already been reported.5 Three patients were females and two males, age at diagnosis ranging between 16 and 49 months. All children developed pancytopenia at an age comprised between 22 and 49 months. Patients’ designation (CAMT1-CAMT5 in Table 1) is as in Savoia et al.5

Chromosome analyses were repeatedly performed on BM and peripheral blood (PB) PHA-stimulated cultures with routine methods. Skin fibroblasts (SF) were cultured with routine methods in patient CAMT2. QFQ-banding technique was used for all chromosome analyses. FISH analyses on interphase nuclei were repeatedly performed on all patients’ BM, on PB of CAMT4, and on SF of CAMT2 with centromere-specific probes for chromosomes 7 (D7Z1), and 8 (D8Z2) (Cytocell Technologies, Cambridge, UK) either with single fluorochromes or in dual color combination. Nuclei from healthy subjects were used as control.

All the results are detailed in Table 1. The karyotype was consistently normal in patients CAMT1, CAMT3, and CAMT5, and parallel FISH analysis on interphase nuclei from BM confirmed the normal disomies 7 and 8. DEB test performed on PB of CAMT1, CAMT2, and CAMT3 excluded Fanconi anemia (FA).

Patient CAMT2 progressed to pancytopenia, with normal BM and PB karyotype, at the age of 30 months (January 2005), but in a subsequent analysis on BM, in May 2006, trisomy 8 was found in 1 mitosis out of 24 and 5 nuclei out of 616. Analysis on fibroblasts from a skin biopsy excluded a constitutional trisomy 8 mosaicism (Table 1). In patient CAMT4, the karyotype was normal in BM cells in 2001, at the age of 12 months, and in 2004 when progression to pancytopenia was observed, but in May 2006 a BM clone with monosomy 7 was found which persisted in the following analyses (Table 1).

A risk of evolution into myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) has often been assumed for CAMT, but in a search for bona fide CAMT patients who developed MDS/AML, we found only 3 such cases in the literature, and none of these was proved to carry mutations of the MPL gene: they are one refractory anemia with excess of blasts (RAEB) reported by King et al.,2 and 2 cases mentioned by Alter,6 a male with acute myelomonocytic leukemia (AMML) developed after an aplastic anemia phase, and a female with a pre-leukemic condition. These 2 latter cases were never reported in more detail, and were studied some decades ago.6 In addition, a report is available of a CAMT patient with MPL mutations who developed a pre-B acute lymphoblastic leukemia.7

A review of CAMT cases with chromosome anomalies is even more difficult to carry out since cytogenetic results in the literature are often incorrectly mentioned, incomplete, and probably questionable. Among the patients with MDS/AML mentioned above, clonal chromosome changes in the BM were reported to be present in 2: the child with RAEB2 with trisomy 21 in 10–15% of the cells, and the patient with AMML6 with different anomalies of chromosome 19 (monosomy, trisomy, deletion) in 11 cells out of 55. As to CAMT without MDS/AML, King et al.2 reported 2 patients with possible chromosome anomalies in PB, but the case identified as CAMT9 showed a translocation only in one cell out of 19, whereas CAMT13 showed a supernumerary marker (not better defined) in 94% of the cells. The only case from the literature with a reliable clonal anomaly in the BM was reported by Steele et al.,8 who found monosomy 7 in 12 out of 20 mitoses and 91 out of 200 interphase nuclei. Mutations of the MPL gene were identified in these 3 patients.4,8

In our 5 patients, BM clonal anomalies were found in 2, in the absence of MDS/AML: one case of monosomy 7 and one of trisomy 8; the latter was demonstrated not to be a constitutional mosaicism, as is the case in 15–20% of patients with MDS/AML and trisomy 8.9 While no exhaustive cytogenetic study on CAMT is available, our small group of patients was monitored over time for possible clonal chromosome anomalies in BM. We suggest that clonal chromosome changes are frequently acquired in the BM of CAMT patients: they often seem to be the most typical of MDS, monosomy 7 and trisomy 8. Interestingly, in both our patients the abnormal clones were found when the disease had already progressed to pancytopenia (Table 1), and the patient reported by Steele and co-workers showed monosomy 7 when CAMT had evolved to BM aplasia.8

In both our patients, the abnormal clone was detected not at the first chromosome analysis but after and showed a trend to expansion (Table 1). In particular, if we take into account evaluations by interphase FISH, in patient CAMT2 the difference between BM samples of 23.05.06 and 06.07.06 only indicates this trend (0.02<p<0.05), while in patient CAMT4 the difference between the BM samples of 24.05.06 and 06.07.06 is significant (p<0.01).

Table 1.Results of chromosome analyses and FISH on interphase nuclei.

The expansion of a BM clone with a chromosome anomaly typical of MDS/AML, should be considered with respect to the risk of hematologic malignancies in CAMT patients. A plausible hypothesis is that a risk of progressing into MDS/AML is really part of the CAMT phenotype, although the short life expectancy and the use of hematopoietic stem cell transplantation make it difficult to demonstrate this evolution in most patients. If this is the case, the patients with clonal anomalies might be those who will progress to MDS/AML. We postulate that MPL mutations might cause karyotype instability through a mutator effect, with emergence of abnormal clones in the BM possibly characterized by chromosome changes typical of MDS: this mechanism was suggested also for other Mendelian BM failure syndromes, such as Shwachman syndrome (SS), familial platelet disorder with propensity to acute myeloid leukemia (FPD/AML, OMIM 601399), and FA.10,11 In CAMT, the BM abnormal clone might stay quiescent even for long periods, as in SS, a full-blown MDS picture being the consequence of its expansion. In conclusion, we suggest that an appropriate cytogenetic follow-up of BM should be part of the clinical management of patients with CAMT, because the early detection of clonal anomalies might be crucial for treatment choice.

Footnotes

• Funding: FL and FP were supported by grants from MIUR (Ministero dell’Università e della Ricerca), CP by grants from Fondazione Gerolamo Gaslini, and FL from AIRC (Associazione Italiana Ricerca sul Cancro), CNR (Consiglio Nazionale delle Ricerche), Istituto Superiore di Sanità (National Program on Stem Cells), European Community, and Fondazione IRCCS (Istituto di Ricovero e Cura a Carattere Scientifico) Policlinico San Matteo.