Genetic abnormalities identified in leukemia associated SD.
Constitutional trisomy 21 or Down syndrome (DS) is the most common human genetic aneuploidy caused by the presence of all or part of an extra 21 chromosome. The incidence of DS is estimated at 1 per 700 births (Malinge et al., 2009) and is the most common genetic factor predisposing to childhood leukemia. People with DS present several clinical phenotypes, including cognitive impairment, craniofacial dysmorphy, gastrointestinal tract abnormalities, congenital heart defects, endocrine abnormalities, neuropathology leading to dementia and immunological defects. Concerning the hematopoietic system, children with DS frequently show abnormalities in platelet counts, macrocytosis and an increased prevalence of leukemia (Lange, 2000; Roizen & Amarose, 1993).
2. Manifestations of leukemia in Down Syndrome
The high frequency of leukemia in children with DS suggests that trisomy 21 is involved directly and functionally to the malignant transformation of hematopoietic cells. However, DS is not a classic genomic instability syndrome, since the overall risk of developing cancer, in particular solid tumors, including neuroblastoma and Wilms tumor, is lower in these people (Hasle, 2001; Malinge et al., 2009).
Newborns with DS have a risk 10 to 20 times higher of developing acute leukemia (AL) when compared with the incidence rates of leukemia in the general child population (Hitzler et al., 2003). The AL in children with DS presents an intriguing relationship between the age at onset of disease and the subtype of leukemia cell. DS children older than 4 years have predominantly acute lymphoblastic leukemia (ALL), whose incidence is approximately 20 times higher than in the general population. However the DS patients aged under 3 years are more likely to develop acute megakaryoblastic leukemia (AMKL), with an incidence 500 times higher than in children without DS(Hitzler et al., 2003; Issacs, 2003; Lange, 2000; Malinge et al., 2009).
The condition of patients with DS awakens, therefore, a special interest in studies on leukemogenesis not only by the high prevalence of AMKL, usually rare in the general pediatric population, but also by another form of clonal proliferation called transient myeloproliferative disorder (TMD) which affects between 5 and 10% of newborns with DS. The TMD is a clonal disease characterized by accumulation of immature megakaryoblasts in fetal liver and peripheral blood, a picture indistinguishable from AL (Hitzler et al., 2003; Malinge et al., 2009; Pine et al., 2007; Rainis et al., 2003; Zipursky, 2003). It is unclear whether all AMKLs are preceded by TMD, since several TMD cases are underdiagnosed. One study suggests that the prognosis for AMKLs preceded by TMD is better than de novo AMKL (Klusmann et al., 2008).
In contrast to AMKL, TMD usually evolves to spontaneous remission within the first three months of life and therefore is considered a pre-leukemic syndrome. This spontaneous remission can vary from 59 to 64% (Kanezaki et al., 2010; Massey et al., 2006). However, approximately 20% of children diagnosed with TMD will develop AMKL after 2 to 3 years of TMD spontaneous remission, which does not regress without chemotherapy (Malinge et al., 2009).
The biological mechanism of TMD spontaneous remission is not clear. Holt et al. (2002) showed that telomerase activity was decreased at the beginning of congenital leukemia and suggested that this deficiency could explain the spontaneous regression. Furthermore, the factors underlying the transformation of the TMD "benign" status for "evil" in AMKL are unknown (Izraeli et al., 2007; Malkin et al., 2000; Rainis et al., 2003).
In rare cases, the TMD is fatal due to poor prognostic factors such as liver fibrosis or liver dysfunction, manifested by jaundice, bleeding diathesis, fetal hydrops, cardiopulmonary failure, high white blood cell (WBC) and failure of spontaneous remission within the first 3 months (Malinge et al., 2009; Massey et al., 2006; Pine et al., 2007; Shimizu et al., 2008). Most of these variants were found in all reports. However, the risk factors for the progression to AMKL remain unclear (Kanezaki et al., 2010). Three studies in the United States, Japan and Europe reported the natural course of TMD in 264 children with DS. These studies confirmed the transient course of this disease that usually resolved spontaneously within the first 3 months of life. However, these studies revealed that the disease is not benign, since early deaths have been reported in 15 to 20% of the cases (Klusmann et al., 2008; Massey et al., 2006; Muramatsu et al., 2008). Kanezaki et al. (2010) also reported early death in 24.2% of the DS patients with TMD.
3. Mutations in
GATA1 gene and leukemogenesis in Down Syndrome
The analysis of megakaryocyte-specific knockdown of
Studies have shown that
Somatic mutations in the N-terminus activation domain of
The expression levels of GATA-1 isoforms are crucial for the proper development of erythroid and megakaryocytic cells and compromised GATA-1 expression is a causal factor in leukemia (Shimizu et al., 2008). These findings strongly suggest that the qualitative deficit of GATA-1 contributes to the genesis of TMD and AMKL (Kanezaki et al., 2010). The selection of mutations that retain GATA-1s may result in disruption of normal balance between GATA-1 and GATA-1s, which probably would be involved in regulating normal development of megakaryocytes (Izraeli et al., 2007), but pass to act as an oncogene directly in the presence of trisomy 21. Alternatively, GATA-1s may be required for survival of leukemic blasts and the oncogenic effect may be purchased by the loss of the heavy chain of GATA-1. Another possibility is that this type of mutation may reflect specific mechanisms of selection or generation of this mutation in the presence of trisomy 21 (Rainis et al., 2003).
According some evidences the arising of AL is due to the cooperation between one class of mutations which interferes with differentiation (class II mutations) and another class which confers a proliferative advantage to cells (class I mutations) (Deguchi & Gilliland, 2002). It has been shown that high level expression of exogenous GATA-1 lacking the N-terminus induced differentiation rather than decreased the aberrant growth of GATA1-null megakaryocytes (Kuhl et al., 2005; Muntean & Crispino, 2005). This observation suggested that abundant GATA-1s functions like a class I mutation in TMD blasts. In contrast, reducing GATA-1 expression leads to differentiation arrest and aberrant growth of megakaryocytic cells (Vyas et al., 1999). The present data suggest that GATA-1s is expressed at very low levels in TMD blasts with GATA-1s low mutations. These levels may not be sufficient to provoke normal maturation. Together, these findings suggest that the low expression of GATA-1s might function like class II mutations in TMD blasts. Additional class I mutations or epigenetic alterations might be more effective in the development of leukemia in blast cells expressing GATA-1s at low levels (Kanezaki et al., 2010).
Rainis et al. (2003) reported two patients with identical
Wechsler et al. (2002) analyzed the X chromosome inactivation in cell lysates from BM of women carrier from AMKL. Since the female leukemic cells showed the X chromosome inactivation due to monoclonality, and the mutant allele was detected only in leukemic cells, they predicted that the wild-type allele should be on the inactive X chromosome. As expected, only the truncated protein GATA-1s was observed. On the other hand Rainis et al. (2003) proposed that if there was no process of X chromosome inactivation,
Ahmed et al. (2004) described for the first time multiple independent
GATA-1s is no different from wild type in their ability to bind to DNA and interact with its co-factor friend of GATA-1(FOG-1), but shows a reduction in their ability to transcriptional activation since it was truncated to its activation domain N-terminal (Rainis et al., 2003; Wechsler et al., 2002).
FOG-1 binds specifically to the NF zinc finger motif of GATA-1, and is expressed abundantly in erythroid and megakaryocytic cells (Crispino et al., 1999). FOG-1 is encoded by the gene
A missense mutation in the
4. Other mutations associated with DS leukemia
The occurrence of mutations in exon 2 of
Based on numerous studies with mutations in
The identification of activating mutations in tyrosine kinase genes in TMD and AMKL specimens has provided new insights into the evolution of AMKL.
|Types of leukemia||Mutated gene||Localization||Frequencies recorded|
5. Trisomy 21 influence on hematopoiesis
The functional contribution of the trisomy 21 in hematologic malignancies is supported by several observations such as the high incidence of leukemia in DS patients, the fact that TMD and AMKL blasts present trisomy 21 (even in children without DS), and that acquired trisomy or tetrasomy of chromosome 21 is frequently observed in blasts of different types of leukemia, including hyperdiploid ALL and de novo AML (Vyas & Crispino, 2007).
It is assumed that the cells of DS complete or partial trisomy of Hsa21, approximately 33.7 Mb, promote an overexpression of at least one of the 364 known genes, 31 antisense transcripts, and five different miRNAs (miR-99a, let-7c, miR-155, miR-125b-2, and miR-802), which could cooperate with the loss of GATA-1 in the pathogenesis of AMKL. Mutations in several genes on chromosome 21 have been identified in leukemia, and many of them recognized as encoding transcription factors acting at various stages of hematopoiesis. There should be contribution of genes present on chromosome 21 that cooperate with mutations of the
The identification of the Down Syndrome Critical Region (DSCR) on the 21q22 band based in the genotype-phenotype correlations of partial trisomy in children suspected of having DS disclosed a list of genes potentially implicated in the clinical phenotype. However no specific genes have been certainly linked to the increased incidence of leukemia in DS. Few strong candidates include
Since the TMD is originated in a fetal liver progenitor and is restricted to children with DS (or to rare cases of acquired trisomy 21), it is presumed that trisomy 21 directly affects the development of hematopoietic cells during gestation. It has been shown that
The functional perturbations induced by trisomy 21 probably induce a highly susceptible cellular environment to additional transformations such as
6. Specific chromosome 21 genes in DS-associated leukemia
Two microarray studies comparing AMKL versus non-DS AML have recently been reported (Bourquin et al., 2006; Ge et al., 2006) and 76 genes were described that discriminate between DS AMKL and non-DS. For example, genes encoding erythroid markers, glycophorin A and CD36, were found meaningly overexpressed in AMKL, as conﬁrmed by immunophenotypic analysis of blasts (Langebrake et al., 2005).
Analysis of the gene expression data also revealed that there is an overall increase in expression of chromosome 21 genes in AMKL, relative to non-DS AMKL. By gene set enrichment analysis, 47 Hsa21 genes, including
6.1. Candidate leukemia oncogenes encoded by chromosome 21
Of the genes on chromosome 21, several are compelling candidate leukemia oncogenes. Of these, four such candidates are
Inherited hypomorphic mutations in
In different types of cancer, it has been shown that the
The ETS family member
6.2miRNAs encoded by chromosome 21
Hsa21 encode five miRNAs and overexpression of some of these has been observed in brain and heart tissues of people with DS and has been implicated in normal and pathologic hematopoiesis (Kuhn et al., 2008). For example, miR-99a is up-regulated during megakaryocytic differentiation of CD34+ cells, whereas miR-155 and let-7c are down-regulated (Garzon et al., 2006). Notably, miR-155 has been linked to myeloproliferative and B-lymphoproliferative disorders (Garzon & Croce 2008; O’Connell et al., 2008). Studies have implicated miR-125b-2, which is overexpressed in TMD and AMKL samples compared with normal megakaryocytes, in the megakaryocytic leukemia of DS (Klusmann, 2007).
Klusmann et al. (2010) showed that miR-125b-2 is an oncogene potentially involved in the pathogenesis of trisomy 21-associated leukemia. They demonstrated in mice and human that overexpression of miR-125b-2 led to specific hyperproliferation and enhanced self-renewal capacity of megakaryocytic progenitor (MPs) and megakaryocytic/erythroid progenitors (MEPs), without affecting their normal differentiation. The miR-125b was highly expressed in AMKL blasts, whereas the identified target genes of miR-125b were down-regulated. Thus, miR-125b-2 has a role in regulating megakaryopoiesis and in the pathogenesis of trisomy 21-associated TMD and AMKL, in cooperation with GATA1s. The miR-125b-2 exerts its oncogenic potential by at least two different mechanisms: blocking post-transcriptional miRNA processing through repression of
7. Methods of leukemia diagnosis in DS
The diagnosis of TMD usually occurs during the first weeks after birth and is observed as hydrops fetalis. The elevated blood count associated with hepatomegaly is the common symptom in an asymptomatic neonate. Infants with TMD can also display occasionally jaundice and bleeding diatheses, respiratory distress coupled with ascites, pleural effusion, signs of heart failure, and skin inﬁltrates. There is megakaryocytic inﬁltration and liver ﬁbrosis, likely caused by excess cytokines secreted from the megakaryoblasts. The full clinical TMD may develop only at the second or third week of life. Laboratory tests are signiﬁcant for either thrombocytosis or thrombocytopenia accompanied by elevated leukocytes with excess of blasts. The blood smear may show nucleated red cells, giant platelets and megakaryocytic fragments, and, most signiﬁcantly, typical deeply basophilic blasts with blebs characteristic to megakaryocytic blasts. The differential diagnosis includes leukoerythroblastic reaction associated with prematurity, sepsis, or asphyxia. However, the blasts of TMD usually persist for several weeks, and
AMKL is preceded in 20 to 60% of cases by an indolent prephase of myelodysplasia (MDS), characterized by thrombocytopenia and dysplastic changes, BM aspiration is often dry, and ﬁbrosis is detected in BM biopsy (Creutzig et al., 1996; Lange et al., 1998). This MDS can last several months or years before progressing to leukemia. In contrast to MDS in non-DS children, which requires stem-cell transplantation for cure, MDS in children with DS present a highly favorable response to chemotherapy alone (Lange et al., 1998). Therefore, Hasle et al. (2003) suggested that all cases of MDS and overt myeloid leukemia in DS, children should be classified as one disease entity, and referred to as ‘‘acute myeloid leukemia of Down syndrome’’ or ML DS. As this is a unique disease, it should be classified separately from other cases of AML in the WHO-classification.
Immunophenotyping characterizes the hematopoietic lineage involved and their degree of maturation by monoclonal antibodies labeled with fluorochromes. Flow cytometry reveals that blasts are positive for CD34, CD33, CD41, CD61, glycophorin A, and often CD7 and CD36 (Langebrake et al., 2005, Massey et al., 2006). Savasan & Ravindranath (2003) observed that blasts of DS children with AMKL express CD36, in contrast to the low or no expression of CD36 in AML without DS. If 25% of blast cells are not detected, the diagnosis of AMKL can be given by the megakaryocytic markers CD41, CD61 and CD42a. The immunophenotype of the blasts in AMKL is generally similar to TMD, except that the percentage of CD34 cells may be lower in AMKL (Langebrake, 2005; Malinge et al., 2009).
Pine et al. (2005) demonstrate the possibility of using specific
This approach serves as a valuable tool in monitoring the spontaneous remission of TMD and in assessing response to treatment of AMKL subcytologic level. In addition, the MRD based GATA-1s mutations has been much in demand as a prognostic parameter for newborns with TMD. One may speculate, for example, that every group of newborns showing apparent remission of TMD can be divided into two subgroups: one in which the size of the clone of blasts in TMD after morphological remission continues to decline to become undetectable versus a second group, in which a clone of blasts in the TMD remains detectable submicroscopic level. It is interesting to correlate these patterns of MRD kinetics in TMD with the probability of developing AMKL later (Hitzler & Zipursky, 2005).
Additional copies of chromosome 8 and 21 in addition to the constitutional trisomy 21 are the most frequent in AMKL, and are found in approximately 10 to 15% for each chromosome. Cytogenetic findings associated with a high rate of relapse in non-DS AML, such as monosomy 7 and deletion 5/5q- also occur in DS patients but do not seem to have a negative impact on prognosis in the rare cases (Gamis et al., 2003, 2005; Rainis et al., 2003).
The approach of molecular techniques including: PCR amplification of
Until recently, there were no reports on the expression levels of GATA-1s in TAM blasts, and the risk factors for the progression to AMKL. In 2010, Kanezaki et al. tested whether the spectrum of transcripts derived from the mutant
Nevertheless, neither mice nor humans with germline mutations expressing GATA-1s develop TMD or AMKL without trisomy 21 (Hollanda et al., 2006; Li et al., 2005). Therefore, the role of the trisomy 21 in the cellular transformation in AMKL seems to be fundamental (Klusmann et al., 2010).It remains unknown which factors on chromosome 21 cooperate with the oncogenic GATA-1s and which factors are involved in this transition from preleukemia to AMKL in only a part of these children (Kanezaki et al., 2010; Klusmann et al., 2007; Langebrake et al., 2006; Malinge et al., 2009).
8. Treatment outcome
DS children with AMKL have an excellent prognostic, with an approximately 80% cure rate, in relation to children without DS who develop AML (Arico et al.; 2008; Creutzig et al., 2005; Gamis et al., 2003; Rao et al., 2006; Taub et al., 1996). This outcome is possible on contemporary AML protocols which based in reducing treatment intensity regimens has considerably reduced the mortality rates in children with DS (Creutzig et al., 2005; Gamis et al., 2003; Whitlock et al., 2005; Zeller et al., 2005).
AMKL blasts have shown hypersensitivity to varied chemotherapeutic drugs (Zwaan et al., 2002). Probably the hypersensibility of the blasts to cytarabine (ARA-C) is due of the effect of
Researchs in prospective clinical trials are trying to demonstrate whether treatment of TMD by low-dose cytarabine could prevent the arise of AMKL. Another related question to be clarified is whether treatment of clinically silent disease, identiﬁed by molecular detection of
In conclusion, many questions remain unanswered concerning the factors that contribute to the progression of TMD and AMKL in DS-patients. Progress in research to unravel these questions will improve diagnosis and treatment. Furthermore, ensuring the diagnosis of
Review supported by CAPES – Project CEP-FM 34/2008 and SES-DF 339/08.