RICERCA ITALIANA     

New insights into the mutational load and into the possibilities of cytogenetic and molecular monitoring of myeloid dysplasia/neoplasia

Abstract

The purpose of this research programme is to analyse some of the gene and chromosome mutations, both constitutional and acquired, which cause myeloid dysplasia/neoplasia. The aims are both speculative, in order to identify nosological subgroups characterized by specific mutations and by peculiar pathogenetic mechanisms, and practical, to optimize the follow-up of the disease course. The methods used include chromosome analyses, fluorescent in situ hybridization (FISH) and molecular techniques: in particular an original quantitative real-time PCR (RQ-PCR) technique on genomic DNA set up by one of our Research Unit (RU) will give precise and accurate quantitative results applied to patients with specific chromosome anomalies.
The main goals regard the following points.
Shwachman Syndrome, a Mendelian disease predisposing to Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukaemia (AML) will be investigated as to the SBDS gene mutations to confirm the diagnosis and to search for genotype correlations with the phenotype or with acquired chromosome anomalies. In the patients with the most frequent chromosome anomalies in the bone marrow, as structural rearrangements of n. 7 or deletion of the long arms of n. 20, the follow-up of the abnormal clone will be done by the mentioned techniques and the results will be compared among them. The parental origin of the abnormal chromosome will be investigated in these patients as well.
The hereditary thrombocytopenia FPD/AML (OMIM #601399), another disorder predisposing to MDS/AML, will be studied as to RUNX1 gene mutations, to define the diagnosis. All the patients will be tested by standard and molecular cytogenetic techniques to look in the bone marrow for monosomy 7 and trisomy 8, specifically correlated to MDS/AML development. The RQ-PCR technique already mentioned will be applied to follow the state of the abnormal clone during the course.
The same techniques will be done to investigate other Mendelian disorders predisposing to MDS/AML, like familial MDS/AML with monosomy 7 or trisomy 8 as clonal chromosome anomalies.
Acquired mutations of genes PTPN11, NRAS and KRAS will be studied in cases of MDS, and, in particular, in juvenile myelomonocytic leukaemia (JMML), where these mutations are known to have a specific role. SBDS and RUNX1 gene mutations will be studied too, because these mutations might have important implications, if acquired. The mutations found will be investigated on DNA from different cell tissues to exclude their constitutional presence.
In myeloid diseases associated to monosomy 7, the monitoring of the abnormal clone will be done on genomic DNA from about 126,000 cells by the RQ-PCR technique set up by the RU 1. The results obtained will be compared to those of chromosome and FISH analyses.
The same investigation criteria will be used to monitor the trisomy 8 in myeloid dysplasia/neoplasia. In this case, this will be useful also to define the clonal marrow expansion in patients with constitutional trisomy 8 mosaicism.
In MDS and myeloproliferative disorders cases with a deletion of the long arms of chromosome 20, all the monitoring techniques already mentioned will be used, with similar criteria and goals.
In chronic myeloid leukaemia (CML), the presence of Ph-positive cells will be quantified by a new molecular approach, again by RQ-PCR on genomic marrow DNA. This modified RQ-PCR technique is efficient to quantify directly the Ph-positive cells, as it is based on the ratio between the amplified BCR/ABL fusion product and the BCR one. The higher sensibility and specificity of this technique will allow to control precisely the amount of leukaemic marrow cells in the disease course, but also to evaluate the consistence of the leukaemic stem cell compartment. The results will be compared to those obtained by cytogenetic and molecular methods. <<<

Principal Investigator

Francesco Pasquali Università degli Studi INSUBRIA Varese-Como

Research Objectives

The mutational load at the origin of myeloid dysplasia/neoplasia includes a large spectrum of identified gene and chromosome mutations. These are gene constitutional mutations that cause Mendelian predisposing disorders, clonal gene mutations acquired in the bone marrow, constitutional chromosome anomalies (sometimes in mosaicism), and clonal chromosome anomalies acquired in the bone marrow.
This research programme is intended to analyse some of these mutations by molecular and cytogenetic techniques, in order to identify nosological subgroups, characterized by specific mutations and by peculiar pathogenetic mechanisms, and to optimize the follow-up of the disease course. This latter goal will be pursued, in particular, with a quantitative real-time PCR (RQ-PCR) technique on genomic DNA. The U. R. n. 1 has set up this original technique which is able to quantify precisely and accurately the amount of cells with a given chromosome anomaly.
The main goals are summarized in the following points.
1. Shwachman Syndrome (SS), a Mendelian disease predisposing to myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML), will be investigated as to the different mutations of the SBDS gene, to confirm the diagnosis, and to identify single mutations correlated to clinical features or to specific acquired chromosome anomalies. In the SS patients with the most frequent acquired chromosome changes in the bone marrow (a long-arm isochromosome 7, or a structural rearrangement of a chromosome 7, and a long-arm deletion of a chromosome 20), the follow-up of the abnormal clone will be done by the RQ-PCR technique on genomic DNA mentioned above. The results will be compared to those obtained with standard cytogenetic and fluorescent in situ hybridization (FISH) analyses. Moreover, the parental origin of the abnormal chromosome will be investigated in these patients.
2. The mutation analysis of the gene RUNX1 will be performed in cases of FPD/AML (OMIM #601399), an autosomal dominant form of thrombocytopenia, which is another disorder predisposing to MDS/AML, to confirm the diagnosis. All the patients will be tested by standard and molecular cytogenetic techniques to look in their bone marrow for monosomy 7 and trisomy 8, that are significantly associated to MDS/AML development. In these patients the abnormal clone will be monitored by the RQ-PCR technique too, and the results compared to those of cytogenetics and FISH.
3. A similar investigation protocol will be applied to other predisposing Mendelian disorders and also to familial MDS/AML cases in which a monosomy 7 or a trisomy 8 are present.
4. Acquired mutations of genes PTPN11, NRAS and KRAS will be studied in cases of MDS, and, in particular, in cases of juvenile myelomonocitic leukaemia (JMML), where these mutations are known to have a specific role. A search for SBDS and RUNX1 mutations will be done in these patients as well, because also these might play a specific role. These mutational analyses will be repeated on DNA from different tissues, to confirm the acquired origin of the mutation, and to obtain some informations as to their pathogenetic implications.
5. In MDS/AML with monosomy 7, the control of the size of the abnormal clone is followed usually by standard chromosome and FISH analyses on interphase nuclei with alphoid centromere-specific probes. We will use RQ-PCR on genomic DNA in subjects constitutionally heterozygous for a chromosome 7 polymorphism to obtain more precise evaluations. Every quantitative analysis will be repeated nine times to allow a statistical evaluation with 95% confidence interval on the DNA of around 126,000 bone marrow cells. The results obtained will be compared to those of chromosome and FISH analyses.
6. The same procedure of monitoring by RQ-PCR will be applied to cases characterized by trisomy 8 in the bone marrow. The quantitative results will be precise and accurate as well, and instrumental to control the disease course, which may be MDS/AML or a chronic myeloproliferative disease. The results may help, in the case of trisomy 8, also to identify the expansion of an abnormal bone marrow clone in subjects with constitutional trisomy 8 mosaicism, what might predict the development of MDS/AML. Also here the results obtained will be compared to those of chromosome and FISH analyses performed on the same material of RQ-PCR.
7. Cases with a deletion of the long arms of chromosome 20 as acquired anomaly will be followed with the techniques described, by RQ-PCR. The results obtained may be relevant to indicate the disease state: this may be a MDS or a chronic myeloproliferative disorder, with higher or lower risk of acute transformation.
8. In chronic myeloid leukaemia (CML) the presence of Ph-positive cells will be quantified by the mentioned molecular approach of RQ-PCR on genomic DNA from bone marrow. The standard RQ-PCR method actually used quantifies indirectly the leukaemic cells in relation to the amount of the BCR/ABL fusion product. This monitoring method, based on the m-RNA quantification, does not reveal the presence of Ph-positive cells not expressing the fusion product. Our new approach, on the contrary, quantifies directly the Ph-positive cells because the number of Ph-positive marrow cells is extrapolated by the ratio between the BCR/ABL amplification product and that of intact BCR. The high specificity of this technique on DNA will allow to control precisely the amount of leukaemic cells in the bone marrow, and, in addition, to evaluate the consistence of the leukaemic stem cell compartment responsible of relapses after imatinib therapy. All the RQ-PCR results will be compared to those of other monitoring techniques. <<<

Timescale

24 months

National and international background

The present research programme concerns:
- Mutational analysis of patients with a myelodysplastic syndrome (MDS), acquired or associated with Mendelian predisposing disorders;
- A new monitoring method of MDS, acute myeloid leukaemia (AML) and chronic myeloproliferative diseases characterized by chromosome changes.
These two types of investigations are linked by the fact that the Mendelian disorders at risk of myeloid dysplasia/neoplasia are characterized by the presence of specific chromosome anomalies, and their monitoring is of primary importance during the disease course; on the other hand, the mutation analysis is needed to obtain a diagnostic confirmation of these predisposing disorders, and may also identify subtypes of MDS/AML in which specific chromosome anomalies may be significantly acquired.
1. MDS
MDS are usually sporadic, but rare families are known with MDS occurring in sibs, and monogenic disorders as well in which the patients have an increased risk of developing MDS. These will be discussed more precisely at the following point, but include, for instance, Shwachman Syndrome (SS), Fanconi Anaemia (FA), Kostman Disease (KD), Cyclic Neutropenia (CN). These observations indicate that mutations in several different genes may cause or contribute to the development of the MDS phenotype.
The definition of different MDS subtypes related to specific mutations of identified genes may lead to a better classification, and will eventually provide basic biological data to develop specific therapies.
Monosomy 7 (-7) is one of the most common chromosomal anomalies observed in MDS, along with trisomy 8, deletions of the long arms of chromosomes 5 and 20, and different structural alterations of chromosome 7: both numerical and structural anomalies are thus present in MDS, with different mechanisms of origin. The possibility that the two types of anomaly may recognize a common origin mechanism has been discussed, as was the case of some constitutional anomalies (Schinzel, 1997).
In particular, as to total or partial -7, this is one of the most frequent anomalies in MDS/AML in general, and is particularly frequent in secondary post-therapy forms (Vardiman et al, 2002; Steensma e List, 2005). It is typically found in some of the constitutional syndromes, already mentioned, in evolution into leukaemia or dysplasia, as in Fanconi anaemia (Thurston et al, 1999), in other congenital neutropenias, and in familial myelodysplasia cases (Minelli et al, 2001; Scheres e Green, 2004; Maserati et al, 2004). The pathogenetic role of monosomy 7 is still unknown, in spite of recent data on the levels of expression of some chromosome 7 loci (Schoch et al, 2005). Monosomy 7 is certainly associated with a poor prognosis both in MDS and in AML (Greenberg et al, 1997; Vardiman et al, 2002; Steensma e List, 2005).
According to Luna-Fineman et al (1995), -7 is a secondary event, which follows the development of MDS. It was discussed if -7 (total or partial), may cause MDS because of the presence on chromosome 7 of an oncosuppressor gene (Shannon et al, 1992). Shannon et al (1992) and Minelli et al (2001), while studying familial cases of MDS associated to -7 demonstrated that, in the three families studied, this hypothesis does not hold true, while it remains to demonstrate the existence of a mutator gene which induces the chromosomal anomaly first and, subsequently, the MDS.
It is well known that chromosome 7 harbours imprinted regions and that maternal uniparental disomy (UPD) for this chromosome results in a phenotype which includes also a growth deficiency (Spence et al 1988; Kotzot et al, 2000), whereas paternal UPD may be seen in subjects with normal growth and development (Hoglund et al, 1994). Thus, at least in theory, also the acquired loss of the paternal or maternal chromosome 7 may result in different biological effects.
Epidemiological studies in patients with MDS not associated with monogenic disorders demonstrated that -7 in cases of infantile MDS is more frequent in males, and unpublished data from our group indicates that the age of onset in the male is lower if the lost chromosome is the maternal one.
The frequency of trisomy 8 in MDS/AML is similar to that of monosomy 7. Its pathogenetic role is not clear (Greenberg et al, 1997), but recent data show the importance of the dosage effect for some involved loci (Schoch et al, 2005). A specific prognostic value of trisomy 8 is not recognized (Greenberg et al, 1997). We showed that in some cases the trisomy 8 is in fact constitutional, with a mosaicism confined to some tissues, and not acquired: this would be the case of 15-20% of the patients (Seghezzi et al, 1996; Maserati et al, 2002).
The deletion of the long arms of a chromosome 20 is frequently found in MDS/AML and in chronic myeloproliferative disorders, and a minimal commonly deleted region involved has been defined (Bench et al, 2000; Wang et al, 2000). MDS with a del(20)(q11) have a good prognosis (Greenberg et al, 1997; Steensma e List, 2005),
2. Myelodysplastic/myeloproliferative diseases
Juvenile myelomonocytic leukaemia (JMML) is a disease occurring mosty in children, it has a clonal origin, and concurrently shows both myeloproliferative and myelodysplastic features, and was therefore included in a group distinct from MDS in myeloid neoplasms classification (Vardiman et al, 2002). The response to standard chemotherapy is usually poor and stem cell transplantation is the only curative approach; however relapses are frequent. JMML develops because of a dysregulation of signal transduction of the the Ras pathway. The JMML cells become selectively hypersensitive to in vitro granulocyte macrophage colony-stimulating factor (GM-CSF). Acquired mutations of three genes (RAS, NF1, and PTPN11), all relevant in the GM-CSF/Ras signal transduction pathway, account for up to 70% of cases of JMML (Goemans et al, 2005; Tartaglia et al, 2006). Mutations in the three genes resulted to be mutually exclusive. PTPN11 constitutional mutations are observed as causal in patients with Noonan and Leopard syndromes, who often develop JMML. Understanding the pathogenesis of the disease is instrumental to the development of therapies aimed to correct the GM-CSF/Ras signal transduction pathway (Tartaglia e Gelb, 2006).
3. Mendelian disorders predisposing to MDS/AML
Shwachman et al in 1964 described a syndrome consisting of pancreatic insufficiency, normal sweat electrolytes, without lung involvement but with pancytopenia, metaphyseal dysostosis (Pringle et al, 1968) and autosomal recessive inheritance. The disease-gene has been identified by Boocock et al (2003), who also found some frequent mutations. No genotype-phenotype correlations have been established, in particular with respect to the risk of developing MDS/AML. Woods et al (1981) showed that in SS there is an increased risk of developing onco-haematological disorders and Smith et al (1996) described the frequency of occurrence of clonal marrow chromosome anomalies with specific involvement of chromosome 7 associated with MDS/AML evolution. Cunningham et al (2002) described some children affected by SS showing clonal chromosomal abnormalities, among which an isochromosome for the long arm of chromosome 7 was especially frequent; they also suggest that this i(7)(q10) may be associated to a different type of disease with no indication to a bone marrow transplantation only based on the identification of the clonal chromosomal abnormality; bone marrow transplantation should be limited only to cases with overt progression to MDS.
Maserati et al (2000) and Sokolic et al (1999) reported patients affected with SS in which an i(7)(q10) or a partial monosomy 7 were associated to non- dysplastic bone marrow cytomorphology. The fact that these chromosome anomalies may be present without MDS/AML suggests that, in addition to the association to a marrow pattern different from other SS cases, they should be regarded as a primary event in MDS/AML development. It was shown that the so called i(7)(q10) of the literature often is a more complex rearrangement, and that the structural 7 anomalies derive from an instability of this chromosome specific to SS (Maserati et al, 2006).
In other Mendelian neutropenias, as in Kostmann disease (severe congenital agranulocytosis) and in cyclic neutropenia, it has been discussed whether -7 associated with MDS has a primary causative role (Naparstek et al, 1995; Kalra et al, 1995). In Fanconi anaemia, it has been described a mutator effect of the BRCA2 gene, which in presence of biallelic mutations causes the typical chromosomal instability (Howlett et al, 2002). Also in FA numerical and structural alterations of chromosome 7 are associated with MDS, even if the steps and the links between chromosomal instability, chromosome 7 anomalies, and MDS are not fully understood.
The "Familial platelet disorder with predisposition to acute myelogenous leukemia" (FPD/AML) is an autosomal dominant disorder with qualitative and quantitative alterations of platelets and increased risk of developing AML.
Song et al (1999) identified mutations in the CBFA2 (or RUNX1, or AML1) gene, localized on chromosome 21, in 6 FPD/AML families. Bone marrow analysis in FPD/AML patients also showed a reduction in megakaryocytic colonies demonstrating that haploinsufficiency of CBFA2/RUNX1 affects platelet production and may help in acquiring other mutations which, in turn, will cause the leukaemia. Preudhomme et al (2000) identified mutations of the RUNX1 gene in a group of patients with various forms of MDS and AML, also showing several numerical chromosome anomalies, among which -7 and acquired tri- or tetra-somy chromosome 21 were present. Osato et al (2001) showed that the mutant RUNX1 protein acts with a dominant negative effect, creating a higher propensity for leukaemia development. They also stated that, as these RUNX1 mutations are per se insufficient for leukaemogenesis, other cooperating genetic and cytogenetic alterations should be sought to fully explain the mechanisms of leukaemogenesis in these patients.
4. Myeloid displasia/neoplasia course monitoring
Most cases of myelodysplastic syndromes and acute and chronic myeloproliferative disorders are characterized, as was stated for MDS, by clonal chromosome anomalies. These changes often have a specific pathogenetic role, which is well defined in many cases and lead to efficient targeted therapies, and they are also used in the clinical routine to control the disease course. On the other hand, several predisposing disorders are known: these are Mendelian diseases (some already mentioned) or constitutional chromosome mosaicisms, in which the risk to develop a myeloid dysplasia/neoplasia is characterized by specific chromosome abnormalities. In these conditions the cytogenetic follow-up during the course of the disease has an important predictive value. In both the type of conditions, the follow-up is done by standard chromosome analyses and by molecular cytogenetic techniques on metaphases and interphase nuclei, whereas also molecular genetic techniques are used when the chromosome anomalies imply rearrangements of specific DNA sequences.
We take into consideration here some of the most frequent and significant diseases in which the cytogenetic follow-up is important and usually done.
- Monosomy 7 in MDS/AML: the poor prognosis of these cases is well established (Greenberg et al, 1997; Vardiman et al, 2002; Steensma e List, 2005), although the pathogenetic role of monosomy 7 is not yet established, notwithstanding recent data on gene/dosage effect (Schoch et al, 2005). The importance to carry out a routine cytogenetic follow-up is obviously relevant.
- Trisomy 8 in MDS/AML: Relevant is the fact that the 15-20% of MDS/AML with trisomy 8 originate from a constitutional mosaicism (Maserati et al, 2002): thus, the control of the trisomic clone is necessary in these subjects to predict the expansion of the trisomic clone that would herald the evolution. The control of the trisomic clone in MDS/AML is in any case relevant to monitor the course of the disease.
- Deletion del(20)(q11) in MDS and chronic myeloproliferative diseases: the monitoring of the abnormal clone is essential for the disease control.
- Philadelphia chromosome (Ph) in chronic myeloid leukaemia (CML): The presence of Ph-positive cells is monitored during the disease course by standard and molecular cytogenetic techniques, as well as molecular methods, that evaluate the expression of the chimeric protein derived from the BCR/ABL rearrangement (Gabert et al, 2003). A problem still open is the persistence of a Ph-positive clone not expressing the chimeric BCR/ABL protein (Goldman, 2004). These quiescent cells, leukaemic stem cells, are thought to be responsible of relapses and/or resistance to the drug (Michor et al, 2005; Huntly e Gilliland, 2005).
- Mendelian conditions predisposing to MDS/AML: It has been suggested that the mutations causing the already mentioned Mendelian disorders predisposing to MDS/AML include in their pleiotropy a mutator effect, inducing specific and recurrent chromosome anomalies. It is the case of Shwachman syndrome (Maserati et al, 2000; Maserati et al, 2006), of FPD/AML associated with monosomy 7 and trisomy 8 (Minelli et al, 2004), of Rothmund-Thomson syndrome again with anomalies of chromosomes 7 and 8, of other hereditary neutropenias with monosomy 7. The same model was recently suggested for the genesis of MDS/AML in other Mendelian disorders, always with the involvement of chromosome 7 (Haimi et al, 2006).
Moreover, the hypothesis of the action of a mutator gene has been suggested in familial MDS/AML cases with entire or partial monosomy 7 (Minelli et al, 2001), and also in familial MDS/AML cases with both anomalies of chromosomes 7 and 8 (Maserati et al, 2004).
The control of the evolution of clones with the chromosome anomalies mentioned above is routinely performed in some more frequent predisposing disorders, as in Shwachman syndrome (Dror et al, 2002). Its importance is both theoretical, as to the understanding of the natural history of the disease, and practical, as to the possibility to predict a MDS/AML development. Chromosome changes, in fact, may be found in the bone marrow long before MDS/AML development (Maserati et al, 2006).
The research unit n. 1 of this programme has developed recently a quantitative real-time PCR technique on genomic DNA, based in the case of unbalanced chromosome anomalies on the use of polymorphisms, which is able to quantify the proportion of cells bearing the chromosome change with high levels of precision and accuracy (Mattarucchi et al, 2005). The same technique, with some methodological differences, may be applied also to the precise quantification of cell clones characterized by a balanced chromosome anomaly. <<<