Francesco Lo Curto

Trisomy 8 in myelodysplasia and acute leukemia is constitutional in 15-20% of cases

The trisomy 8 found in malignancies may derive from a constitutional trisomy 8 mosaicism (CT8M), and in these cases the trisomy itself may be regarded as the first mutation in a multistep carcinogenetic process. To assess the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8, an informative sample of 14 patients was collected. The data ascertained included chromosome analyses of fibroblast cultures and of PHA‐stimulated blood cultures in patients with normal blood differential count, as well as possible CT8M clinical signs. One patient showed trisomy 8 in all cell types analyzed and undoubtedly has a CT8M; a second patient consistently showed trisomy 8 in PHA‐stimulated blood cultures when no immature myeloid cells were present in blood and should be considered as having CT8M; a third patient, with Philadelphia‐positive chronic myelocytic leukemia, was more difficult to interpret, but the possibility that she had CT8M is likely. A few clinical signs of CT8M were also present in these three patients. Our data indicate that the frequency of CT8M in hematological dysplastic and neoplastic disorders with trisomy 8 is approximately 15–20%.

Microhomologies and interspersed repeat elements at genomic breakpoints in chronic myeloid leukemia.

Reciprocal translocation t(9;22) is central to the pathogenesis of chronic myeloid leukemia. Some authors have suggested that Alu repeats facilitate this process, but supporting analyses have been sparse and often anecdotal. The purpose of this study was to analyze the local structure of t(9;22) translocations and assess the relevance of interspersed repeat elements at breakpoints. Collected data have been further compared with the current models of DNA recombination, in particular the single‐strand annealing (SSA) and the nonhomologous end joining (NHEJ) processes. We developed a protocol for the rapid characterization of patient‐specific genomic junctions and analyzed 27 patients diagnosed with chronic myeloid leukemia. Sequence analysis revealed microhomologies at the junctions of 21 patients of 27, while interspersed repeats were of relevance (P < 0.05) in at least 16 patients. These findings are more frequent than expected and give an indication that the main mechanisms involved in the t(9;22) translocation are the SSA and NHEJ pathways, both playing a role. Furthermore, our report is consistent with microhomologies facilitating the joining of DNA ends in the translocation process, and with both Alu and a variety of other repeat sequences pairing nonhomologous chromosomes during the SSA pathway.

The route to development of myelodysplastic syndrome/acute myeloid leukaemia in Shwachman-Diamond syndrome: the role of ageing, karyotype instability, and acquired chromosome anomalies

An investigation of 22 new patients with Shwachman‐Diamond syndrome (SDS) and the follow‐up of 14 previously reported cases showed that (i) clonal chromosome changes of chromosomes 7 and 20 were present in the bone marrow (BM) of 16 out of 36 cases, but if non‐clonal changes were taken into account, the frequency of anomalies affecting these chromosomes was 20/36: a specific SDS karyotype instability was thus confirmed; (ii) the recurrent isochromosome i(7)(q10) did not include short arm material, whereas it retained two arrays of D7Z1 alphoid sequences; (iii) the deletion del(20)(q11) involved the minimal region of deletion typical of myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML); (iv) only one patient developed MDS, during the rapid expansion of a BM clone with a chromosome 7 carrying additional material on the short arms; (v) the acquisition of BM clonal chromosome anomalies was age‐related. We conclude that karyotype instability is part of the natural history of SDS through a specific mutator effect, linked to lacking SBDS protein, with consequent clonal anomalies of chromosomes 7 and 20 in BM, which may eventually promote MDS/AML with the patients’ ageing.

Familial partial monosomy 7 and myelodysplasia: different parental origin of the monosomy 7 suggests action of a mutator gene.

Two sisters are reported, both with a myelodysplastic syndrome (MDS) associated with partial monosomy 7. A trisomy 8 was also present in one of them, who later developed an acute myeloid leukemia (AML) of the M0 FAB-type and died, whereas the other died with no evolution into AML. Besides FISH studies, microsatellite analysis was performed on both sisters to gather information on the parental origin of the chromosome 7 involved in partial monosomy and of the extra chromosome 8. The chromosomes 7 involved were of different parental origin in the two sisters, thus confirming that familial monosomy 7 is not explained by a germ-line mutation of a putative tumor-suppressor gene. Similar results were obtained in two other families out of the 12 reported in the literature. Noteworthy is the association with a mendelian disease in 3 out of 12 monosomy 7 families, which suggest that a mutator gene, capable of inducing both karyotype instability and a mendelian disorder, might act to induce chromosome 7 anomalies in the marrow. We postulate that, in fact, an inherited mutation in any of a group of mutator genes causes familial monosomy 7 also in the absence of a recognized mendelian disease, and that marrow chromosome 7 anomalies, in turn, lead to MDS/AML.

Shwachman syndrome as mutator phenotype responsible for myeloid dysplasia/neoplasia through karyotype instability and chromosomes 7 and 20 anomalies

An investigation of 14 patients with Shwachman syndrome (SS), using standard and molecular cytogenetic methods and molecular genetic techniques, showed that (1) the i(7)(q10) is not, or not always, an isochromosome but may arise from a more complex mechanism, retaining part of the short arm; (2) the i(7)(q10) has no preferential parental origin; (3) clonal chromosome changes, such as chromosome 7 anomalies and del(20)(q11), may be present in the bone marrow (BM) for a long time without progressing to myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML); (4) the del(20)(q11) involves the minimal region of deletion typical of MDS/AML; (5) the rate of chromosome breaks is not significantly higher than in controls, from which it is concluded that SS should not be considered a breakage syndrome; (6) a specific kind of karyotype instability is present in SS, with chromosome changes possibly found in single cells or small clones, often affecting chromosomes 7 and 20, in the BM. Hence, we have confirmed our previous hypothesis that the SS mutation itself implies a mutator effect that is responsible for MDS/AML through these specific chromosome anomalies. This conclusion supports the practice of including cytogenetic monitoring in the follow‐up of SS patients.

Familial platelet disorder with propensity to acute myelogenous leukemia: Genetic heterogeneity and progression to leukemia via acquisition of clonal chromosome anomalies

Familial platelet disorder with propensity to acute myelogenous leukemia, or FPD/AML (OMIM #601399), is a rare autosomal dominant condition, with only 12 families reported. It is characterized by qualitative and quantitative platelet defects and predisposition to the development of myeloid malignancies. Causal mutations have been identified in the RUNX1 gene (also known as AML1, CBFA2) in the 11 families so far analyzed. RUNX1 is a gene frequently involved in the pathogenesis of sporadic leukemia and myelodysplastic syndromes, through acquired chromosome rearrangements and point mutations. We report an Italian family with three members affected with FPD/AML, two sibs and their father, who developed myelodysplastic syndromes (which in one subsequently evolved into AML). Direct sequencing and polymorphisms haplotype analysis of the region of chromosome 21 where RUNX1 is mapped demonstrated that FPD/AML in this family was not caused by any mutation of the RUNX1 gene, thus providing evidence for the genetic heterogeneity of this disorder. Cytogenetic studies showed monosomy 7 in the marrow of all the three affected subjects, as well as an independent clone with trisomy 8 in the father. The importance of mutator effects in the pathogenesis of familial myeloid malignancies characterized by relevant chromosome changes, in the presence or absence of an underlying Mendelian disorder, has already been suggested. Our results and a review of the cytogenetic literature led us to postulate that mutations also causing FPD/AML may have a mutator effect that could give origin to myelodysplastic syndromes and acute myeloid leukemias through acquired chromosome changes.

Familial myelodysplastic syndromes, monosomy 7/trisomy 8, and mutator effects

A family is reported, in which two sisters presented with myelodysplastic syndrome (MDS), namely refractory anemia with excess of blasts in transformation (RAEB-t), and refractory anemia (RA). Bone marrow chromosome changes were present in both: trisomy and tetrasomy 8 (with a pericentric inversion of one chromosome 8) in the older sister, and monosomy 7 (with clones with additional trisomies 19 and 21) in the younger one. Molecular data were obtained on the parental chromosome involved in these numerical anomalies, which proved to be of paternal origin in these cases. The observations of this family, and a review of familial cases of MDS/acute myeloid leukemia (AML), led us to consider that they may be divided into two groups: those which arise on the basis of a Mendelian predisposing disorder exerting a mutator effect, often with the acquisition of monosomy 7, and those in which no specific Mendelian predisposing disease is recognized, as the familial monosomy 7 cases and the one reported here. We postulate that in these families an inherited mutator effect is present and that it causes a karyotype instability, which leads to MDS/AML, often through the acquisition of monosomy 7 and trisomy 8.

Deletion of chromosome 20 in bone marrow of patients with Shwachman‐Diamond syndrome, loss of the EIF6 gene and benign prognosis

Bianchi, V., Robles, R., Alberio, L., Furlan, M. & Lammle, B. (2002) Von Willebrand factor-cleaving protease (ADAMTS13) in thrombocytopenic disorders: a severely deficient activity is specific for thrombotic thrombocytopenic purpura. Blood, 100, 710–713. Coppo, P., Bengoufa, D., Veyradier, A., Wolf, M., Bussel, A., Millot, G.A., Malot, S., Heshmati, F., Mira, J.P., Boulanger, E., Galicier, L., DureyDragon, M.A., Fremeaux-Bacchi, V., Ramakers, M., Pruna, A., Bordessoule, D., Gouilleux, V., Scrobohaci, M.L., Vernant, J.P., Moreau, D., Azoulay, E., Schlemmer, B., Guillevin, L. & Lassoued, K. (2004) Severe ADAMTS13 deficiency in adult idiopathic thrombotic microangiopathies defines a subset of patients characterized by various autoimmune manifestations, lower platelet count, and mild renal involvement. Medicine (Baltimore), 83, 233–244. Froehlich-Zahnd, R., George, J.N., Vesely, S.K., Terrell, D.R., Aboulfatova, K., Dong, J.F., Luken, B.M., Voorberg, J., Budde, U., Sulzer, I., Lammle, B. & Kremer Hovinga, J.A. (2011) Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura. Haematologica. Epub ahead of print. doi: 10.3324/haematol.2011.051433. Hovinga, J.A., Vesely, S.K., Terrell, D.R., Lammle, B. & George, J.N. (2010) Survival and relapse in patients with thrombotic thrombocytopenic purpura. Blood, 115, 1500–1511; quiz 1662. Legendre, C.M., Babu, S., Furman, R.R., Sheerin, N.S., Cohen, D.J., Gaber, O., Eitner, F., Delmas, Y., Loirat, C., Greenbaum, L.A. & Zimmerhackl, L.B. (2010) Safety & efficacy of eculizumab in aHUS patients resistant to plasma therapy: interim analysis from a phase II trial. Journal of the American Society of Nephrology, 21, 93A. Mache, C.J., Acham-Roschitz, B., Fremeaux-Bacchi, V., Kirschfink, M., Zipfel, P.F., Roedl, S., Vester, U. & Ring, E. (2009) Complement inhibitor eculizumab in atypical hemolytic uremic syndrome. Clin J Am Soc Nephrol, 4, 1312– 1316. Remuzzi, G. (2003) Is ADAMTS-13 deficiency specific for thrombotic thrombocytopenic purpura? No. J Thromb Haemost, 1, 632– 634. Remuzzi, G., Galbusera, M., Noris, M., Canciani, M.T., Daina, E., Bresin, E., Contaretti, S., Caprioli, J., Gamba, S., Ruggenenti, P., Perico, N. & Mannucci, P.M. (2002) von Willebrand factor cleaving protease (ADAMTS13) is deficient in recurrent and familial thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Blood, 100, 778–785. Salmon, J.E., Heuser, C., Triebwasser, M., Liszewski, M.K., Kavanagh, D., Roumenina, L., Branch, D.W., Goodship, T., Fremeaux-Bacchi, V. & Atkinson, J.P. (2011) Mutations in complement regulatory proteins predispose to preeclampsia: a genetic analysis of the PROMISSE cohort. PLoS Med, 8, e1001013. Tsai, H.M. (2003) Is severe deficiency of ADAMTS-13 specific for thrombotic thrombocytopenic purpura? Yes. Journal of Thrombosis and Haemostasis, 1, 625–631.

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

Evaluating chromosomal mosaicism by array comparative genomic hybridization in hematological malignancies: the proposal of a formula

Array-based comparative genomic hybridization (aCGH) has proven indispensable to the study of unbalanced constitutional and acquired chromosomal anomalies, but its sensitivity for detecting mosaicism is still not well established. On the basis of the ADM2 algorithm used for microarray image analysis with one of the most widely used oligomer-based aCGH platforms [the whole genome 244K system by Agilent Technologies (Santa Clara, CA)] we suggest a formula to infer the percentage of cells bearing a chromosome imbalance in cases with constitutional or acquired mosaicism. Three examples of acquired mosaicism in which this formula was applied are reported together with parallel fluorescence in situ hybridization (FISH) to interphase nuclei with informative probes. Although some approximation affects both the results inferred from aCGH and FISH data, the proposed formula was successful in the three patients studied.