Galili Raza_EU Haematology 03/03/2010 15:34 Page 32
Myelodysplastic Syndromes
CD34
+
cell differentiation and also inhibited terminal monocyte and that the observed decreased expression in 5q- patients of the two
granulocyte differentiation in cell lines. Most recently, Hussein et al.
15
ribosomal genes in the CDR – RBM22 and RPS14 – may be important
looked at expression levels of these haematopoiesis-associated miRNAs in the development of 5q- syndrome, analogous to the causative role
(-150, -155, -221 and -222) in marrow cells of 52 MDS patients of all played by the ribosomal gene RPS19 in Blackfan-Diamond syndrome.
23
subtypes (eight with isolated del(5q)) compared with controls. Indeed, Ebert et al.
24
shortly thereafter used RNAi to inhibit each gene
Surprisingly, miRNA-150 increased expression was the only perturbation of the CDR individually in CD34
+
cells cultured to produce mature
detected and this was confined to patients with del(5q). The expression erythrocytes or mature megakaryocytes as the functional readout.
of MYB, one of the targets of miRNA-150, was found to be inversely Only inhibition of RPS14 resulted in blocked erythropoiesis in the
correlated with miRNA-150 expression, suggesting that decreased levels presence of normal megakaryocyte differentiation – a recapitulation
of MYB, a main component of erythropoiesis,
16
may contribute to the of the 5q- phenotype. Forced expression of RPS14 in CD34
+
marrow
anaemia found in 5q- syndrome patients. cells from 5q- patients re-established erythrpoietic differentiation,
showing that RPS14 loss on one allele was the causative event for the
DNA Microarrays 5q- syndrome. Further studies
25
showed that 55 genes associated with
The ability to simultaneously measure the expression of virtually all ribosome and protein synthesis were differentially expressed in RA
known genes in a limited number of cells has dramatically changed with normal karyotype and 5q- syndrome patients compared with
basic biology and medical research. In the field of oncology and controls. Confirming the importance of deregulated ribosomal genes,
haematology, numerous studies have established expression profiles Sohal et al.
26
used meta-analysis to compare the CD34
+
expression
that aid in the diagnosis and classification of tumour types. However, for profiles of 60 cases of non-del(5q) MDS patients with 52 normal
MDS the heterogeneity of the tumour cells together with the controls. This study showed that ribosomal protein genes were the
confounding influence of an abnormal bone marrow microenvironment most significantly deregulated class of genes in the MDS group, with
has somewhat limited the use of this technology. Initial studies focused decreased expression in many of those genes. Therefore, results of
on limiting heterogeneity by use of CD34
+
cells combined with selection expression arrays point to deregulated ribosomal biogenesis, in
of specific French–American–British (FAB) subtypes. While these studies addition to excessive apoptosis and proliferation, as a unifying
gave clues in terms of the biological pathways deregulated in MDS and mechanism in the evolution of MDS.
therefore most likely involved with the pathology of the disease, they
were of limited clinical benefit. Our group sought to approach the Single Nucleotide Polymorphism Arrays
problem in a different manner. The del(5q) MDS patients were shown in Sequencing results of the human genome show that there is
a phase II clinical trial to be highly responsive to therapy with great variation within individuals at the single nucleotide level. This
lenalidomide, a derivative of thalidomide.
17
Interestingly, in a second variation, known as SNPs, most typically has no deleterious effect and
phase II trial approximately 25% of patients without del(5q) were also therefore is highly conserved in different populations. Therefore, it is
responsive to this agent.
18
No apparent clinical parameters could predict possible to use SNPs as genetic markers that may be associated
which non-del(5q) patients were likely to respond. We postulated that with specific diseases, clinical presentation and response to therapy or
perhaps there was an expression profile that could identify these prognosis. SNP arrays are essentially DNA arrays that detect specific
responding patients. Using pre-therapy marrow mononuclear cells SNPs resulting in a ‘SNP map’ of individuals. Currently, >50 common
and the Affymetrix 2.0 DNA array, a unique 32-gene expression profile SNP variations have been found to be associated with a number of
was generated that separated responders, irrespective of karyotype, diverse diseases including type 2 diabetes, immune disorders and
from non-responders.
19
This profile comprised many erythroid genes cardiac disease.
27
More recently, copy number variation (CNV, a short
that were underexpressed in responders compared with the non- or long sequence of nucleotides) has been found to account for much
responders. Further in vitro studies demonstrated that these genes are of the variation seen in the genome and to be associated with disease
initially expressed at very low levels in CD34
+
cells, but increase phenotype.
28
CNV is now incorporated into the newer high-density
expression when differentiated along the erythroid lineage. Thus, versions of SNP arrays. An additional advantage of these arrays is that
patients presenting primarily with a defect in erythroid differentiation, it is possible to detect copy-neutral loss of heterozygosity (LOH). LOH
whether due to selective apoptosis or other mechanisms, responded to reflects the loss or gain of an allele. However, in some instances while
lenalidomide. This profile was also used to predict response with 80% two alleles are present, they are not from both parents; instead, one is
accuracy. The ability to predict lenalidomide response in non-del(5q) inherited from a single parent and duplicated. This is referred to as
patients would be of significant clinical value, as such patients are not uniparental disomy (UPD) or copy-neutral LOH. When this allele is
currently US Food and Drug Administration (FDA)-approved to receive abnormal, it may result in a disease phenotype. Several recent studies
this drug, which could alleviate their severe anaemia. A clinical of MDS have found that some patients with marrow cells that have
confirmatory trial is currently being conducted. cytogenetically normal karyotypes actually contain numerous CNV and
UPD.
29,30
These changes are clonal and are not found in the normal
Expression microarray studies in MDS have sought to identify tissues of the patients. While the percentage of patients with these
molecular pathways that could explain the diverse clinical abnormalities varies between studies, it appears to be a significant
phenotypes evolving from marrows with common features of subgroup within cytogenetically normal MDS patients. The clinical
dysplasia and increased proliferation with excessive apoptosis. Initial utility of such studies is clearly demonstrated by Heinrichs et al.,
31
who
studies by Boultwood et al.
13
showed that Wnt/B-catenin signalling examined the clinical course of two patients with low-risk International
and protein ubiquitination pathways were deregulated in 5q- Prognostic Scoring System (IPSS) scores and normal karyotype who
syndrome patients compared with RA with normal karyotype or were found to have UPDs on chromosome 7q. These patients had
healthy controls. Wnt/B-catenin signalling regulates stem cell fate and unexpectedly rapid disease progression, more suggestive of that
transformation events and both pathways have been implicated in normally seen in patients with karyotypic deletions of chromosome 7,
other myeloid malignancies.
20–22
Additionally, this group hypothesised for whom therapy is usually aggressive. Regions of UPD/CNV may
32
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