GENETICS IN CLINICAL PRACTICE
GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
Genetic Testing in the Myelodysplastic Syndromes:
Molecular Insights Into Hematologic Diversity
DAVID P. STEENSMA, MD, AND ALAN F. LIST, MD
The myelodysplastic syndromes (MDS) are associated with a
diverse set of acquired somatic genetic abnormalities. Bone marrow karyotyping provides important diagnostic and prognostic
information and should be attempted in all patients who are
suspected of having MDS. Fluorescent in situ hybridization (FISH)
studies on blood or marrow may also be valuable in selected
cases, such as patients who may have 5q– syndrome or those who
have undergone hematopoietic stem cell transplantation. The
MDS-associated cytogenetic abnormalities that have been defined by karyotyping and FISH studies have already contributed
substantially to our current understanding of the biology of malignant myeloid disorders, but the pathobiological meaning of common, recurrent chromosomal lesions such as del(5q), del(20q),
and monosomy 7 is still unknown. The great diversity of the
cytogenetic findings described in MDS highlights the molecular
heterogeneity of this cluster of diseases. We review the common
and pathophysiologically interesting genetic abnormalities associated with MDS, focusing on the clinical utility of conventional
cytogenetic assays and selected FISH studies. In addition, we
discuss a series of well-defined MDS-associated point mutations
and outline the potential for further insights from newer techniques such as global gene expression profiling and array-based
comparative genomic hybridization.
Mayo Clin Proc. 2005;80(5):681-698
AA = all normal; AML = acute myeloid leukemia; AN = abnormal-normal;
APL = acute promyelocytic leukemia; CDR = commonly deleted region;
CGH = comparative genomic hybridization; CML = chronic myeloid
leukemia; CMML = chronic myelomonocytic leukemia; CMML-Eos =
CMML with eosinophilia; FAB = French-American-British; FISH = fluorescent in situ hybridization; gDNA = genomic DNA; GTPase = guanosine
triphosphate hydrolases; IPSS = International Prognostic Scoring System; MDS = myelodysplastic syndromes; M-FISH = multicolor FISH;
β = β subunit of the platelet-derived growth factor receptor;
PDGFR-β
SKY = spectral karyotyping; WHO = World Health Organization
DISEASE SYNOPSIS
The term myelodysplastic syndromes (MDS) describes a
diverse group of acquired hematopoietic stem cell malignancies unified by a common functional consequence—
ineffective production of mature blood cells.1,2 In MDS,
cytologic dysplasia of blood and marrow cells is associated
From the Department of Internal Medicine and Division of Hematology, Mayo
Clinic College of Medicine, Rochester, Minn (D.P.S.); and H. Lee Moffitt
Cancer Center & Research Institute at the University of South Florida, Tampa,
Fla (A.F.L.).
Individual reprints of this article are not available. The entire series on
Genetics in Clinical Practice will be available for purchase from the Proceedings Editorial Office at a later date.
© 2005 Mayo Foundation for Medical Education and Research
Mayo Clin Proc.
•
with functional defects in neutrophils and platelets, which
contribute to infection and bleeding complications, respectively, even in the absence of substantial deficits in blood
cell production. The variable risk of progression to acute
myeloid leukemia (AML) that is characteristic of MDS
appears to be driven by somatic genomic instability and
epigenetic genomic modification. Evidence for this includes allelic imbalance arising from structural or numerical chromosome aberrations, defects in DNA repair,3,4
and linkage to genotoxic exposures including alkylating
agents, topoisomerase II inhibitors, environmental poisons,
or radiation.1,2
This overview highlights the genetic and molecular abnormalities in adults with MDS, methods to detect these
abnormalities, and their potential importance for diagnosis,
prognosis, and therapy (Table 1). The inherited and acquired MDS in pediatric patients display genetic and clinical features distinct from those in adults5 and will not be
addressed in this review.
Most adult patients with MDS present to their physicians with signs and symptoms related to anemia or
pancytopenia, or their illness is discovered incidentally
when a blood cell count is performed for another purpose.
The diagnosis of MDS is established from the characteristic appearance of a bone marrow aspirate, but in some
cases cytologic features may be subtle, creating challenges with respect to excluding nonneoplastic causes
of cellular dysplasia. In such cases, genetic testing provides an alternative means of diagnosis confirmation and
offers further insight into prognosis and management
considerations.
The 1976 French-American-British (FAB) Co-operative Group classification scheme for MDS, revised and
expanded in 1982, provided the first widely used tool for
disease classification and prognostication.6 The FAB classification is based on the proportion of immature blast cells
in the blood and marrow and on the presence or absence of
ringed sideroblasts or peripheral blood monocytosis. In
1999, a group of hematopathologists and clinicians operating under the auspices of the World Health Organization
(WHO) created a new classification system for MDS based
on the FAB framework but incorporated newer morphologic insights and, to a more limited extent, cytogenetic
findings (Table 2).7,8
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
TABLE 1. Genetic Abnormalities Characteristic of MDS and Their Consequences*
Genetic or
molecular abnormality
Functional consequence
Importance in MDS
Karyotypic findings
Del(20q), isolated del(5q),
–Y, normal karyotype
Unknown
Complex karyotype,
abnormalities of
chromosome 7
Other karyotypes not listed
above
Trisomy 8
Unknown
Abnormalities of
chromosome 1q,
3q21, and 11q23
Del(12p)
t(8;21)(q22;q22),
inv(16)(p13q22) or
t(16;16)(p13;q22), and
t(15;17)(q22;q12)
Isodicentric X
t(3;3)(q21;q26) or
inv(3)(q21q26.2)
Isolated isochromosome (17q)
IPSS good-risk markers; del(5q31.1) is associated with
specific 5q– syndrome (female predominance,
dysplastic micromegakaryocytes, thrombocytosis)
IPSS poor-risk markers
Unknown
Unknown
3q21 translocations are associated with overexpression
of MDS1/EVI1 gene; 11q23 translocations rearrange
MLL gene
Unknown; ETV6 gene at 12p13 is frequently involved
in translocations in this region, which may be cryptic
or associated with deletions
Leukemogenic rearrangements of core-binding factors
involved in transcriptional regulation
Unknown; ABCB7 at Xq12-13 plays a critical role in
iron homeostasis
Rearranges MDS1/EVI1 gene at 3q26 in many cases;
GATA2 at 3q21 is overexpressed in some cases
Unknown
Rearrangements of 5q33
such as t(5;12)(q33;p13)
PDGFR-β subunit constitutive activation; TEL at
12p13 is a common fusion partner, but many other
partners have been described
All metaphases abnormal
Marker of clonality
TP53
NRAS
FLT3
RUNX1/AML1
c-FMS
PTPN11
ATRX
Point mutations
Mutations result in loss of function of tumor
suppressor p53, “the guardian of the genome”—
an essential regulator of cell cycle, specifically
transition from G0 to G1
Constitutively activates oncogene by preventing
normal GTP recycling, resulting in uncontrolled
growth signalling
Internal tandem duplications of juxtamembrane domain
or point mutations result in constitutive activation
and uncontrolled growth signalling
Point mutations result in loss of normal trans-activating
potential of the key RUNX1 core-binding factor
subunit and consequent changes in gene expression
Mutations constitutively activate the receptor for
colony-stimulating factor 1, a cytokine that controls
the production, differentiation, and function of
monocyte-macrophages
Mutations alter function of the SHP2 that has diverse
roles in cell signalling, including modulation of
PDGFR phosphorylation and STAT activity
Point mutations result in loss of function of ATRX
multiprotein complex with consequent changes in
gene expression, including down-regulation of α
globin cluster, probably by epigenetic mechanisms
IPSS intermediate-risk markers, likely to be subdivided
in the future as IPSS is revised
Increased risk of leukemic transformation (singleinstitution studies); male predominance; oral
ulceration (rare)
Decreased survival (single-institution studies)
Relatively benign prognosis (single-institution studies)
WHO classifies cases with these translocations as
AML by definition, even if they would otherwise
be consistent with MDS
Iron accumulation in elderly women
Thrombocytosis with megakaryocyte dysplasia
Occasional myeloproliferative features; rapid transformation to leukemia and poor response to therapy
CMML with eosinophilia; frequent clinical responses
to imatinib or other PDGF inhibitors; other molecularly
defined myelodysplastic-myeloproliferative overlap
syndromes exist, including rearrangements of
PDGFR-α subunit
Poorer prognosis than patients with mixture of abnormal
and normal metaphases
Mutated in 5%-10% of patients with MDS, more
commonly mutated in secondary MDS, may be
associated with progression to AML
Codon 12 mutation is most common in MDS and
found in 10%-15% of cases; acquisition of mutation
commonly associated with progression to AML
Found in up to 10% of MDS cases; acquisition of
mutation commonly associated with progression to
AML
Most common point mutation described to date in
MDS—in up to 25% of cases; associated with high
risk of progression to AML, previous radiation
exposure, and chromosome 7 abnormalities
Occasionally associated with CMML
Rarely associated with CMML or other MDS,
commonly associated with JMML
Acquired α-thalassemia; no apparent effect on
proliferative potential of thalassemic clone
*Gene expression changes that have been documented via microarray analyses are not included because their relevance has not yet been established. AML =
acute myeloid leukemia; ATRX = α-thalassemia mental retardation X-linked; c-FMS = McDonough feline sarcoma virus oncogene homolog; CMML =
chronic myelomonocytic leukemia; FLT3 = fms-related tyrosine kinase 3; GTP = guanosine triphosphate; IPSS = International Prognostic Scoring System;
JMML = juvenile myelomonocytic leukemia; MDS = myelodysplastic syndromes; NRAS = neuroblastoma viral rat sarcoma oncogene homolog; PDGFR =
platelet-derived growth factor receptor; PTPN11 = protein tyrosine phosphatase, nonreceptor type 11; RUNX1 = runt-related transcription factor 1; SHP2 =
SH2 domain-containing protein tyrosine phosphatase 2; STAT = signal transducer and activator of transcription; TP53 = tumor protein p53; WHO = World
Health Organization.
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
TABLE 2. World Health Organization Classification of the Myelodysplastic Syndromes*
Disease subtype
Blood findings
Refractory anemia
Bone marrow findings
Anemia
No blasts or rare (<1%) blasts
No Auer rods
<1 × 109/L monocytes
Anemia
No blasts
No Auer rods
<1 × 109/L monocytes
Cytopenias (bicytopenia or
pancytopenia)
No or rare (<1%) blasts
No Auer rods
<1 × 109/L monocytes
Cytopenias (bicytopenia or
pancytopenia)
No or rare (<1%) blasts
No Auer rods
<1 × 109/L monocytes
Cytopenias
<5% blasts
No Auer rods
<1 × 109/L monocytes
Cytopenias
5%-19% blasts
Auer rods may be present
<1 × 109/L monocytes
Cytopenias
No or rare blasts
No Auer rods
Refractory anemia with
ringed sideroblasts
Refractory cytopenia with
multilineage dysplasia
Refractory cytopenia with
multilineage dysplasia
and ringed sideroblasts
Refractory anemia with
excess blasts 1
Refractory anemia with
excess blasts 2
Myelodysplastic syndrome,
unclassified
Myelodysplastic syndrome
associated with isolated
del(5q)
Anemia; other cytopenias
possible
<5% blasts
Platelets normal or increased
Erythroid dysplasia only–ie, not
myeloid or megakaryocytic dysplasia
<5% blasts
<15% ringed sideroblasts
Erythroid dysplasia only
≥15% ringed sideroblasts
<5% blasts
Dysplasia in ≥10% of cells in 2 or
more myeloid cell lines
<5% blasts in marrow
<15% ringed sideroblasts
Dysplasia in ≥10% of cells in 2 or
more myeloid cell lines
≥15% ringed sideroblasts
<5% blasts
Unilineage or multilineage dysplasia
5% to 9% blasts
No Auer rods
Unilineage or multilineage dysplasia
10%-19% blasts
Auer rods may be present
Unilineage dysplasia in granulocytes
or megakaryocytes but no erythroid
dysplasia
<5% blasts
No Auer rods
Normal to increased megakaryocytes
with hypolobated nuclei
<5% blasts
No Auer rods
Isolated del(5q)
*Chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia are classified as myelodysplasticmyeloproliferative overlap syndromes by the World Health Organization.
Modified from Blood.7
TREATMENT OVERVIEW
The current spectrum of treatments for MDS is limited;
however, encouraging results with novel agents in development offer promise.9 For most patients, management is
primarily supportive, relying on transfusions and antimicrobials as dictated by symptoms and infectious complications. Select patients will experience hematologic improvement with the use of recombinant hematopoietic growth
factors, including erythropoietin and granulocyte colonystimulating factor.10,11
With the advent of new therapeutic alternatives, the
management strategy for many patients has shifted from
sole reliance on supportive measures to more active intervention. The Food and Drug Administration recently approved the first agent with an indication for MDS, 5Mayo Clin Proc.
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azacitidine (Vidaza, Pharmion Corporation, Boulder,
Colo), an injectable nucleoside analogue that covalently
incorporates into DNA and depletes DNA methyltransferases to promote genome hypomethylation. This may
contribute to reactivation of methylation-silenced tumor
suppressor genes. In a national cooperative group openlabel randomized trial of MDS, 5-azacitidine treatment
delayed leukemic transformation and reduced the risk of
AML progression or death.12 Most other noninvestigational
agents offer limited benefit for the general population with
MDS but are reasonable considerations for select individuals. For example, thalidomide, when administered at low
doses for an extended schedule, may restore erythropoiesis
in some patients; however, neurotoxicity and other adverse
effects limit dose escalation and prolonged drug administration, particularly in elderly individuals.13 Antithymocyte
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683
GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
globulin and other immunosuppressants may be effective
in patients with hematopoietic inhibitory immune effectors; such patients may be identified through predictive
modelling.14-16 Older agents such as androgens and pyridoxine are usually ineffective.17,18 Aggressive chemotherapy similar to that used for acute leukemia should be reserved for clinical trials; it is rarely curative.19
Younger patients are often candidates for curative strategies such as allogeneic hematopoietic stem cell transplantation; however, mortality and morbidity remain high, even
in experienced and specialized centers.20 Autologous transplantation may benefit selected patients without an allogeneic donor, but the potential benefit of this approach
appears limited to younger patients.21 Unfortunately, the
vast majority of patients with MDS are not candidates for
stem cell transplantation with conventional ablative conditioning regimens because the median age at the time of
diagnosis is in the seventh decade of life, and comorbidities are common. Reduced-intensity conditioning or
nonmyeloablative approaches promise to expand the upper age limit of candidates for stem cell therapy.20,22 The
optimal timing of transplantation is a subject of active
investigation.23
Iron overload remains a clinical challenge, especially in
patients who require frequent red blood cell transfusions,
emphasizing the need for iron chelation therapy for patients
with otherwise favorable prognostic features in whom extended survival is expected. Given the paucity of diseasealtering therapies for MDS, clinical trial enrollment is paramount to advance the field. At least 2 agents in clinical
development, the immunomodulatory drug lenalidomide
(CC-5013, Revlimid; Celgene Corporation, Warren, NJ)
and the demethylating agent decitabine (Dacogen, Supergen, Inc, Dublin, Calif), have shown promising preliminary
results, and Food and Drug Administration review should
occur in the months ahead.24,25
STANDARD CYTOGENETIC STUDIES AND
TEST RATIONALE
At present, the most widely available genetic test applied in
the evaluation of patients with MDS is cytogenetic analysis
by conventional metaphase karyotyping. Karyotyping has
been applied widely since the 1970s, and the method for
performing these assays and their limitations have been
reviewed recently.26,27 Briefly, standard cytogenetic assays
involve light-microscopic visualization of metaphase chromosomes prepared by (1) inducing the cells of interest to
divide, (2) arresting their division at the appropriate mitotic
stage with colchicine, (3) lysing the cells, and (4) Giemsa
staining of the chromosome spread to resolve the chromosome size and banding pattern (Figure 1).
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In diagnostically ambiguous cases, an abnormal karyotype can provide important evidence to support the
presence of a clonal, neoplastic process.28 Additionally, cytogenetic studies provide important prognostication discrimination. The 1997 International Prognostic Scoring
System (IPSS) for MDS, based on a review of 816 patients
with “primary” MDS (ie, no history of treatment with chemotherapy or radiotherapy), incorporates 3 elements with
independent prognostic power into the staging algorithm:
(1) the proportion of myeloblasts in the patient’s marrow,
(2) the number of blood cell lineage deficits, and (3) the
type of chromosomal abnormality present.29 Using these 3
elements, a score generated by the IPSS algorithm reproducibly classifies patients with MDS into a highest-risk
group with a median survival of only 0.4 years, a pair of
intermediate-risk groups, and a lowest-risk group with a
median survival of 5.7 years overall, 11.8 years for patients
60 years of age or younger. The IPSS algorithm has been
validated independently.30,31
The IPSS clusters the varied chromosomal abnormalities observed in MDS into 3 risk groups: poor risk (including abnormalities of chromosome 7 or complex karyotype
[≥3 clonal abnormalities]), good risk (including a normal
karyotype, isolated interstitial deletion of the long arm of
chromosome 5 [del(5q)], chromosome 20q interstitial deletions [del(20q)], or clonal loss of the Y chromosome, seen
in 7.7% of otherwise healthy elderly men32), and intermediate risk (including all other abnormalities not defined as
good or poor).29 The corresponding median survival by
cytogenetic risk group was 0.8 years (poor risk), 2.4 years
(intermediate risk), and 3.8 years (good risk) (Figure 2).29
The IPSS is an important clinical tool and maintains
prognostic validity for both standard therapy and allogeneic stem cell transplantation. In one analysis, patients with
a poor-risk cytogenetic pattern had a 3.5-fold greater risk of
relapse after stem cell transplantation than did patients with
a good-risk karyotype.33 Similar findings have been reported for patients treated with intensive antileukemic chemotherapy, with or without subsequent transplantation.19,34
Not surprisingly, acquisition of higher-risk IPSS cytogenetic abnormalities during the course of disease adversely
impacts survival because of leukemic evolution or cytopenic complications.35
COMMON CYTOGENETIC ABNORMALITIES IN PRIMARY MDS
Although not specific for MDS, the recurrent chromosomal
abnormalities defined by the IPSS (ie, –7/del(7q), del(5q),
del(20q), and –Y) are among the most common detected in
MDS. Overall, 40% to 50% of patients with primary MDS
present with 1 or more clonal karyotypic abnormalities,
whereas in those with secondary MDS (ie, after therapeutic
chemotherapy or radiotherapy for an unrelated condition),
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
FIGURE 1. Conventional cytogenetic study of bone marrow cells in a patient with a
myelodysplastic syndrome (MDS). This abnormal karyotype includes several abnormalities
associated with MDS, including numerical abnormalities (trisomy 8 and monosomy 17
[arrowheads]), and 2 common deletions (deletions of the long arm of chromosome 20 and
of the long arm of chromosome 5 between bands q13 and q33 [arrows]). Because more
than 3 abnormalities are present, this karyotype is considered complex. Photograph
courtesy of Jack L. Spurbeck, Cytogenetics Laboratory, Mayo Clinic, Rochester, Minn.
the proportion of patients with an abnormal karyotype exceeds 80%.2,28,31,36-38 The relative frequency of specific cytogenetic abnormalities in primary MDS ranges from as high
as 30% for del(5q)—either as an isolated anomaly (10%) or
as part of a more complex karyotype (20%)—to 20% for
trisomy 8 and 15% for monosomy 7.39 Numerous other
recurrent abnormalities have been observed, but each accounts for less than 10% of cases of primary MDS.
CYTOGENETIC ABNORMALITIES IN SECONDARY MDS
The distribution of chromosome abnormalities is skewed in
treatment-related MDS, in which structural or numerical
deletions of chromosome 7 (40%) and chromosome 5
(40%) predominate.36,39 Together, abnormalities of chromosome 5 and 7 account for approximately 75% of karyotypic abnormalities in secondary MDS,36 whereas all other
karyotypic abnormalities occur with a frequency of 10% or
less.39 In addition to the higher frequency of abnormal
karyotypes, secondary MDS displays a disproportionate
percentage of IPSS poor-risk abnormalities, and as with
primary MDS these are associated with an unfavorable
prognosis. This cytogenetic pattern is frequently linked to
and believed to be induced by prior treatment with alkylating antineoplastics.36
The IPSS good-risk abnormalities occur with sufficient
rarity in secondary MDS to raise suspicion that such cases
Mayo Clin Proc.
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instead represent de novo and treatment-unrelated MDS.
Nevertheless, single-institution studies indicate that patients
with such karyotypes fare better than their counterparts with
secondary disease who have poor-risk karyotype abnormalities.36 When the spectrum of abnormalities associated with
primary and secondary MDS is compared, secondary forms
more commonly exhibit monosomy 5, monosomy 7, and
der(17p) aberrations, whereas in primary MDS, isolated
del(5q) and normal karyotype predominate.39 Treatment
with topoisomerase II inhibitors has been linked to chromosome 3q inversions or translocations and to balanced translocations involving 11q23 deregulating the MLL gene.40,41
OTHER RECURRENT CYTOGENETIC ABNORMALITIES IN MDS
Numerous chromosomal abnormalities occur with moderate frequency (ie, 1%-10%) in MDS; however, because of
overall low prevalence, their prognostic relevance remains
undefined. Limited case series have linked trisomy 8 with
an increased risk of AML transformation, and abnormalities of chromosome 1q, 3q21, and 11q23 with an overall
inferior survival.30,31,40,42 In contrast, del(12p) as a sole abnormality has been associated with an indolent disease
course.31 The prognostic relevance of less common (ie,
<1% of cases) yet recurrent chromosomal abnormalities
remains uncertain.43-47 Although multiple independent and
karyotypically discordant clones may be identified in se-
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
100
90
Survival
80
No. (%)
of patients
–Y
del(5q)
Normal
del(20q)
Misc. single
+8
Double
Misc. double
Chrom 7 abn
Misc. complex
Complex
Percentage
70
60
50
40
30
17 (2)
48 (6)
489 (60)
16 (2)
74 (9)
38 (5)
29 (3)
14 (2)
10 (1)
15 (2)
66 (8)
20
10
0
0
1 2
3 4 5 6
7
8 9 10 11 12 13 14 15 16 17 18
Year
AML evolution
100
90
80
Percentage
70
60
50
40
30
20
10
0
0
1 2
3 4 5 6
7
8 9 10 11 12 13 14 15 16 17 18
Year
FIGURE 2. The International Prognostic Scoring System for myelodysplastic syndromes (MDS)
predicts survival time (top) and time to leukemic transformation (bottom) based on the patient’s
bone marrow karyotype. These Kaplan-Meier curves show that the highest risk of death and
leukemia is in patients with MDS with chromosome 7 abnormalities (Chrom 7 abn) or a complex
karyotype; the lowest risk is associated with loss of the Y chromosome, isolated interstitial
deletion of the long arm of chromosome 5, a normal karyotype, or chromosome 20q interstitial
deletions [del(20q)]. Data were derived from a review of 816 patients with primary MDS. AML =
acute myeloid leukemia; Misc = miscellaneous. From Blood,29 with permission.
lected cases,48,49 their prognostic importance is believed to
be influenced by the highest-risk karyotype. The Mitelman
Database of Chromosome Aberrations in Cancer, part of the
Cancer Genome Anatomy Project of the National Cancer
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Institute, includes a comprehensive catalogue of chromosome abnormalities in patients with MDS that can be accessed at: http://cgap.nci.nih.gov/Chromosomes/Mitelman
(accessibility verified March 29, 2005).
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
The karyotypic diversity included in the Mitelman database illustrates that the diagnostic category of MDS contains disorders that phenotypically overlap yet have a molecularly distinct pathobiology. Although the IPSS has
proved its clinical usefulness, the IPSS intermediate-risk
category includes potentially hundreds of chromosomal
abnormalities, suggesting that further refinement of the
algorithm will be possible. As therapy for MDS improves,
the prognosis associated with particular karyotypes may
change. By analogy, before the introduction of retinoic acid
therapy for acute promyelocytic leukemia (APL) in the
1980s, the classic t(15;17) APL-associated chromosomal
translocation was regarded as an intermediate-risk leukemia karyotype, but with modern drug therapy APL is
among the most curable AML subtypes.50
Although balanced favorable translocations such as
t(15;17) and t(8;21) are relatively common in AML, they are
rare in MDS in which deletions and numerical chromosomal
abnormalities predominate.2,29,51 Given the responsiveness to
conventional leukemia chemotherapy, the WHO categorizes
4 specific rearrangements involving core-binding factor transcriptional repressors [ie, t(8;21)(q22;q22), inv(16)(p13q22)
or t(16;16)(p13;q22), and t(15;17)(q22;q12)] as AML, regardless of the bone marrow myeloblast percentage.7,8 For
those exceptional cases that harbor such karyotypic abnormalities with a low myeloblast burden, it remains unclear
whether AML therapy impacts survival as it does in more
typical AML.52,53
CLONAL CYTOGENETIC ABNORMALITIES IN THE ABSENCE OF
MORPHOLOGIC EVIDENCE OF MYELODYSPLASIA
In selected cases in which marrow aspiration is performed
for evaluation of cytopenias, routine staging, or follow-up
of another malignancy, clonal karyotypic abnormalities may
be identified in the absence of cytologic dysplasia that would
support a diagnosis of MDS.54 The constitutional karyotype
in nonhematopoietic tissue in such cases has been normal,
confirming an acquired somatic cytogenetic abnormality.
This constellation of findings may represent a forme fruste
of MDS,54 evidenced by an increased risk of leukemic transformation and subsequent cytopenic mortality.54 These uncommon cases emphasize the importance of cytogenetic
evaluation in the investigation of unexplained cytopenias,
even in the absence of overt morphologic evidence of MDS.
RELEVANCE OF ALL ABNORMAL VS ABNORMAL-NORMAL
CYTOGENETIC PATTERN
When a chromosomal abnormality is detected by conventional cytogenetics, the abnormality may be present in all
examined metaphases (all abnormal [AA]) or in only a
fraction of the metaphases (sometimes termed abnormalnormal [AN]).28,35 The AA pattern correlates with higherMayo Clin Proc.
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risk MDS subtypes identified by the FAB MDS pathologic
classification,6 and patients with AA have a poorer overall
survival than those with AN.28
The favorable modifying effect of increasing proportions of normal metaphases has precedent in other hematologic malignancies. The clinical usefulness of the reduction in the number of abnormal metaphases with therapy is
perhaps best illustrated by chronic myeloid leukemia
(CML). In CML, a “major cytogenetic response” represents an important therapeutic goal because patients who
achieve a complete cytogenetic remission experience a
superior progression-free survival compared with those
patients with persistence of any proportion of metaphases
bearing the Philadelphia chromosome.55-57 By analogy, an
international working group that created standardized response criteria for clinical trials of MDS included reductions in the number of abnormal metaphases as one of these
response criteria, although the clinical importance of this
measurement had not yet been formally established in
MDS.58 Evidence from recent clinical trials testing new
potentially disease-modifying agents supports this notion.
In a large phase 2 study of intravenous decitabine in
higher-risk MDS, 31% of patients achieved a complete
cytogenetic remission, with a corresponding reduction in
the relative risk of death (0.38 compared with those in
whom the abnormal clone persisted).59 These preliminary
data suggest that a cytogenetic response may also be an
important disease-modifying marker in MDS.
BONE MARROW VS PERIPHERAL BLOOD CYTOGENETIC STUDIES
MDS
Most chromosomal studies in patients with MDS are performed on bone marrow cells. Peripheral blood karyotyping has a higher failure rate than marrow studies and rarely
adds useful information beyond that available from the
marrow study.37 Unlike CML, in which tracking the proportion of cells containing the Philadelphia chromosome
or its molecular equivalent is critical for monitoring therapy and the results may prompt management changes in
the absence of the need for an additional marrow aspiration, there is currently little compelling clinical indication
for frequent assessment of chromosomal status in MDS. In
special situations in which chromosomal information is
desirable but a marrow examination is not required, such as
measuring donor/host chimerism after stem cell transplantation, fluorescent in situ hybridization (FISH) assays (discussed subsequently) may be preferable.
IN
INDICATIONS FOR CYTOGENETIC TESTING IN MDS
When should a karyotype be obtained for patients with
MDS? Given the importance of the IPSS risk assessment, a
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
TABLE 3. Summary of Genetic Test Indications in Myelodysplastic Syndromes*
Conventional cytogenetics
Screening
At diagnosis
Bone marrow
Blood
During therapy
Bone marrow
After hematopoietic
stem cell transplantation
Bone marrow
Blood
Leukemic transformation
Bone marrow
FISH
No
No
Yes
Not necessary unless patient refuses
bone marrow examination
Testing for 5q deletions may predict response to lenalidomide,24
and panel FISH testing is reasonable if cytogenetic study is
inadequate
If the patient is enrolled in a clinical study,
otherwise no compelling indication
If FISH was performed at diagnosis and the patient is enrolled
in a clinical study, otherwise no compelling indication
At 3-6 mo and thereafter as indicated
May be useful to assess chimerism in donor-recipient sex
mismatch or to assess for early relapse if FISH abnormality
is present before transplantation
May be useful for minimally invasive assessment of
chimerism and early relapse
No
Yes
No clear role
*See text for test interpretation. FISH = fluorescent in situ hybridization.
chromosomal study is an essential part of the initial evaluation (Table 3). Acquisition of new chromosome abnormalities (ie, cytogenetic evolution) portends disease progression and therefore justifies cytogenetic evaluation in the
routine management of MDS. Some clinicians obtain a
cytogenetic study every time a marrow aspiration is performed during the care of patients with MDS, whereas
others obtain such studies only in relationship to planned
therapeutic trials or when there are signs of a change in the
patient’s condition. No evidence base exists to help guide
this practice, and therefore such decisions are best left to
the discretion of the individual treating clinician. After
transplantation, karyotypic analysis may not only help in
early determination of relapse (ie, before morphologic evidence of recurrent disease is present) but also is useful in
assessment of donor-host chimerism in the case of sex
mismatches between marrow donor and host.
MOLECULAR CYTOGENETICS IN MDS
Delineating the pathogenetically relevant alterations in
MDS at the gene level, even for recurrent cytogenetic
abnormalities, has proved challenging. The so-called 5q–
syndrome is perhaps the best example of the difficulties
faced. First delineated in the early 1970s, 5q– syndrome is
a form of refractory anemia characterized by female predominance, atypical megakaryocytes, erythroid hypoplasia
with red blood cell transfusion dependence, and a low risk of
leukemic transformation with prolonged survival. These
clinical and pathological features are consistently associated
with an isolated interstitial deletion of the long arm of chromosome 5 that involves 5q31.1.60,61 Although earlier reports
varied in the definition of the 5q– syndrome, general consensus is that this description should be limited to patients with
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an isolated 5q31.1 interstitial deletion in the absence of other
cytogenetic abnormalities, a low bone marrow blast percentage, and no history of genotoxic therapy.62
The WHO’s most recent classification schema for malignant hematopoietic diseases formally recognizes 5q–
syndrome as a discrete entity within the general category of
MDS.7 Although no other specific MDS-associated chromosome aberration has been recognized by the WHO as a
distinct pathologic and clinical syndrome, other recurrent
chromosome abnormalities have been linked with specific
disease features. For example, isolated isochromosome 17q
may be associated with myeloproliferative features and
poor response to therapy,63 isodicentric X has been linked
to iron overload in older women,64 trisomy 8 displays a
male predominance,65-67 and inv(3)(q21q26.2) is associated
with thrombocytosis with megakaryocytic dysplasia and
poor therapeutic response.42,68
Boultwood and colleagues at Oxford have carefully defined the commonly deleted region (CDR) in patients with
5q– syndrome using FISH and other molecular mapping
techniques, and they have cloned several novel genes in the
process.69-72 The defined CDR spans 1.5 megabase pairs at
5q31.1.71 This CDR is relatively gene rich, containing no
less than 40 discrete genes, 33 of which are expressed in
early hematopoietic cells.71 Several of these genes are
members of the interleukin family or have other potential
promoting roles in hematopoiesis. A number of genes
within the CDR are not well characterized, and their function is as yet unknown. To date, systematic sequencing of
these genes in patients with 5q– syndrome has not revealed pathogenetic mutations, and thus it is now believed that the pathophysiology of 5q– syndrome may
relate to gene dosage rather than mutational gene deregulation in the CDR.
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
Other groups are investigating additional recurrent
MDS-associated chromosome abnormalities in an attempt
to identify critical gene targets, including del(20q), trisomy
8, del(7q), and del(9q). In selected cases, study of patients
with parallel inherited disorders may provide insight. For
example, patients with constitutional trisomy 8 mosaicism
appear to have a high risk of developing myeloid malignancies including MDS and leukemia.73 Chromosome 7 may be
particularly challenging because there are multiple CDRs,
including 7q22 and several in the region 7q31-7q35.74-76
Perhaps the most important molecular cytogenetic discovery in recent years with immediate therapeutic implication is the reciprocal chromosome translocation involving
chromosome 5q33, where the gene encoding the β subunit
of the platelet-derived growth factor receptor (PDGFR-β)
is located. Although a number of chromosomes and genes
may partner with 5q33, the clinical phenotype is distinct
and is now recognized by the WHO classification as
chronic myelomonocytic leukemia (CMML) with eosinophilia (CMML-Eos). (The WHO classification removed all
CMML subtypes from the MDS group, where they had
been placed in the widely used 1982 FAB MDS classification, and placed them in a separate myelodysplastic/
myeloproliferative overlap category. This was done because patients with CMML frequently exhibit prominent
myeloproliferative features, unlike patients with other subtypes of MDS.6,7,77) The CMML-Eos phenotype arises from
the generation of novel fusion transcripts with constitutive
activation of the PDGF-β receptor tyrosine kinase.78-82
Transgenic mouse models have shown that these novel receptor tyrosine kinase fusion genes are singularly responsible for the generation of these myelodysplastic/myeloproliferative disorders and are selectively responsive to PDGFR
kinase inhibitors such as imatinib (Gleevec, Novartis, Basel,
Switzerland).79,83,84 Several patients with CMML-Eos have
been reported to achieve rapid hematologic control and sustained cytogenetic remission with imatinib monotherapy85 or
treatment with SU11657, an experimental agent that also
inhibits PDGF signalling.86
FLUORESCENT IN SITU HYBRIDIZATION
Although conventional cytogenetic analysis remains the
standard for purposes of diagnosis and prognosis in MDS,
interest is increasing in the application of more sensitive
techniques such as FISH. Reviews of FISH applications
in hematologic malignancies have been published recently.27,87 Briefly, FISH involves hybridization of patient
genetic material with a fluorescently labelled DNA probe
designed to anneal either to specific DNA sequences or to
chromosomal features such as centromeres. The advantages of FISH include its applicability to either peripheral
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phase cells instead of only dividing cells, and the capacity
to analyze substantially more cells than is possible by
conventional cytogenetic analysis. When chromosomal rearrangements are present in only a small subset of neoplastic
cells, a common situation in MDS, FISH can offer increased
sensitivity over conventional cytogenetics. For purposes of
investigation of specific MDS-associated chromosomal deletions, FISH probes are extremely useful for narrowing the
common CDRs,71,74,76,88-91 and they can also identify the precise types of cells involved in the neoplastic process.90,92-98
Several groups have developed FISH “panels” that are
designed to detect the chromosome abnormalities most
commonly identified in patients with MDS, such as those
involving chromosomes 5, 7, 8, 11, 13, and 20.99,100 Such
FISH analyses in MDS occasionally reveal cryptic cytogenetic abnormalities that are not recognized on metaphase
analysis.101-103 However, conventional cytogenetics and
FISH should be viewed as complementary because there
are also abnormalities better detected by conventional cytogenetics than FISH.100,103-105
Many of the abnormalities detected by FISH that are not
always recognized by conventional cytogenetics are among
those included in the IPSS (eg, monosomy 7106) or those
found by other groups to have prognostic importance in
MDS (eg, trisomy 8102,106 and 12p rearrangements101). Chromosome aberrations detected only by FISH have not yet
been shown to have the same prognostic importance as
those revealed by conventional cytogenetics, and thus at
present there is no compelling clinical reason to perform
FISH at the time of diagnosis of MDS if the karyotype is
successful. However, because the critical molecular defect
is likely to be the same regardless of how the chromosomal
breakpoint is detected, specific high-yield FISH assays will
probably be incorporated eventually into prognostic and
treatment-response algorithms,107 as in CML.26,108 For example, in the European clinical trial of decitabine for MDS,
2 patients had chromosomal responses detected by FISH
that correlated with clinical and cytogenetic response.59
If standard karyotyping is unsuccessful because of a
fibrotic marrow, failure of aspirated cells to grow in culture, or other technical problems, it is reasonable to resort
to FISH using a panel designed to detect abnormalities
with recognized prognostic relevance. Additionally, in
view of the promising recent findings by List et al24 of a
high rate of complete hematologic and cytogenetic response to lenalidomide therapy in patients with MDS with
5q31.1 deletions, it may be especially valuable to perform
FISH directed at detection of loss of the CDR on chromosome 5, particularly when the marrow morphologic features suggest 5q– syndrome. Patients with cryptic 5q deletions might then be referred for appropriate clinical trials.
Over time, as the presence of other cytogenetic abnormali-
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
ties is correlated with response to novel therapies, the role
of specific FISH tests in MDS is likely to expand.
MULTICOLOR FISH AND SPECTRAL KARYOTYPING
“Chromosome painting” methods such as multicolor FISH
(M-FISH) or spectral karyotyping (SKY) offer the ability to
analyze all human autosomes and sex chromosomes at the
same time by labelling metaphase cells with band-selective
color probes (Figure 3).109,110 These methods offer greater
sensitivity than conventional karyotyping and are useful for
characterizing cryptic chromosomal rearrangements and the
origin of structurally ill-defined chromosomal material such
as “marker” chromosomes, in which the normal counterpart
is not readily identified because of an ambiguous banding
pattern.
Despite the heightened power of discrimination that MFISH and SKY technologies may offer,111 their clinical
usefulness in MDS has yet to be proved. For example, in
patients with MDS with a normal karyotype by conventional
cytogenetics, unrecognized cryptic abnormalities are rarely
identified by M-FISH or SKY.99,100,112 In contrast, additional
rearrangements are often detected when chromosome painting methods are performed in patients with MDS with abnormal karyotypes109,111,113-116; however, the clinical relevance of
these abnormalities is as yet undefined.
COMPARATIVE GENOMIC HYBRIDIZATION
Comparative genomic hybridization (CGH) is a new technique that applies fluorescent hybridization to detect differences in DNA copy number between a patient sample and a
normal control, giving insight into loss or gain of chromosomal segments.117 The ratio of the signal strength from the
patient DNA to the reference DNA is measured in each
chromosomal region. Until recently, the resolution of CGH
was limited and only allowed detection of chromosomal
gains or losses of 20 megabase pairs or greater. Despite
this, even first-generation CGH probes provided sufficient
sensitivity to discover cryptic rearrangements in patients
with myeloid diseases including MDS, and soon the resolution had improved to almost 1 megabase pair.118,119 Newer
array-based CGH methods, including a recently described
tiling array that maps the entire human genome with more
than 30,000 clones, promise to improve sensitivity sufficient to resolve even minute DNA alterations.117,120
POINT MUTATIONS IN MDS
Conventional cytogenetic studies and FISH probes detect
large chromosomal rearrangements. However, isolated
base pair changes or so-called point mutations may also
inactivate or result in constitutive gain of function of the
affected gene, with important biological consequences.
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Discovery of these mutations is one of the major open
frontiers in MDS molecular pathology, with initial investigations focusing on recognized tumor suppressor genes.
TP53
The tumor suppressor gene TP53, a critical cell-cycle
checkpoint regulator, was the first gene evaluated in MDS
because of its high frequency of inactivation in solid tumors. In myeloid malignancies such as MDS and AML,
TP53 mutations are uncommon, detected in no more than
15% of patients with primary MDS, with a higher frequency in atomic bomb survivors and patients treated previously with chemotherapy or radiotherapy.121-130 Such mutations are generally demonstrable at the time of diagnosis
and may have independent prognostic value.128,131
Even in tumor types in which TP53 mutations are common, TP53 mutation testing is rarely performed in clinical
practice, unless a germline mutation leading to a general
cancer susceptibility syndrome (the Li-Fraumeni syndrome) is suspected. This is primarily because patients
with TP53-mutant neoplasias have not yet been shown to
benefit from differential clinical management. For both inherited and acquired mutations, direct genetic analysis of
TP53 by sequencing is required because immunohistochemical results do not correlate well with mutation status.132,133 Newer high-throughput technology may change the
cost-benefit analysis of TP53 mutation screening and make
this assay more accessible to clinicians (although its usefulness remains to be determined). For instance, denaturing
high-performance liquid chromatography, which separates
wild-type and mutant DNA on the basis of the differential
affinity of DNA heteroduplexes and homoduplexes on
polystyrene beads coated with a gradient of organic solvents, has shown promise as a more rapid TP53 screening
method in MDS and other tumors134 (D.P.S., unpublished
data, 2004).
NRAS AND FLT3
The RAS proto-oncogene superfamily encodes guanosine
triphosphate hydrolases (GTPase) that are critical regulators of cellular growth-related signals. Three RAS protooncogenes (H, N, and K) encode four 21-kd G proteins,
including 2 alternatively spliced K-Ras products that are
posttranslationally modified before incorporation into the
inner leaflet of the plasma membrane. RAS mutations occur
at critical regulatory sites (eg, codons 12, 13, and 61) that
inactivate the GTPase response normally stimulated by the
binding of GTPase-activating proteins, thereby extending
the half-life and signalling activity of the Ras-GTP bound
mutant. In myeloid malignancies, NRAS mutations predominate, especially a common mutation in codon 12;
nonetheless, the frequency of activating RAS mutations in
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
FIGURE 3. Multiplex fluorescent in situ hybridization (M-FISH) study of a patient with a malignant myeloid disorder. Conventional G-banded
cytogenetic analysis (left) was karyotyped as 46,X,t(X;1)(q24;q25),t(6;8)(q25;q24.1),11,13,add(15)(q26),12mar[19]/46,XX[1].32 M-FISH analysis
confirmed the t(X;1) and t(6;8), identified the 2 markers as der(13)t(11;13), and identified the add(15) as a der(15)t(13;15). In addition,
t(9;11)(p21;q23) was detected, involving chromosome 9 and one of the marker chromosomes, and was confirmed as an MLL gene rearrangement. Arrows designate the abnormal chromosomes. Photograph courtesy of Rhett P. Ketterling, MD, Division of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minn. From Br J Haematol,109 with permission.
unselected cases of MDS is relatively low, ranging from
10% to 15% of MDS. In contrast, in CMML, RAS mutations are found in 40% to 60% of patients and are believed
to contribute to growth factor hypersensitivity of myelomonocytic progenitors.130,135-139
Internal tandem duplications and other constitutively activating mutations of the receptor tyrosine kinase FLT3 are
common in AML but are relatively rare in MDS.130,138,140-143
Acquisition of NRAS and/or FLT3 mutations has been associated with progression of MDS to AML.137,138,140
RUNX1/AML1
The most common point mutations detected in MDS to
date are those involving the core-binding–factor subunit
RUNX1/AML1.144-151 In leukemia, this transcription factor
and oncogene is involved in common balanced translocations such as t(8;21), which leads to formation of aberrantly
active chimeric fusion proteins and uncontrolled cell
growth. Although early reports estimated that RUNX1/
AML1 mutations were rare in MDS,148 more recent studies
have described these mutations in as many as 25% of
patients,150 and single-institution studies have associated
RUNX1/AML1 mutation status with chromosome 7q abnormalities,149 more advanced forms of MDS,150,151 and a high
risk of progression to overt leukemia.150,151
ATRX
Specific gene mutations may be associated with unique
MDS phenotypes. For instance, the rare complication of
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acquired α-thalassemia (hemoglobin H disease) arising in
the context of MDS was recently linked to mutations in the
ATRX gene at Xq13.1.152,153 Inherited mutations in ATRX
cause a mild form of α-thalassemia and severe syndromic
X-linked mental retardation,154 whereas acquired somatic
ATRX mutations create a severe imbalance in globin synthesis, a large proportion of hemoglobin H-containing
cells, and the unusual finding of microcytic, hypochromic
red cell indices.152,153,155 The distinct behavior of mutations
like ATRX when they are acquired in the context of MDS,
rather than inherited in the germline, may offer important
insights into MDS pathobiology.155
MUTATIONS FOUND PREDOMINANTLY IN CMML
In addition to having unique clinical features and unique
chromosomal translocations [such as the t(5;12) PDGFRβTEL fusion], CMML has several point mutations that
appear with great frequency but are uncommon in other
MDS subtypes. Patients with CMML have a high frequency of activating RAS mutations and a disproportionately higher frequency of c-FMS point mutations, although
the latter are rare overall in myeloid disorders.156,157 Likewise, mutations in the protein tyrosine phosphatase gene
PTPN11 (which cause a form of Noonan syndrome when
inherited) are extremely uncommon in MDS in general,
more common in CMML, and especially common (ie,
34% frequency) in the pediatric syndrome juvenile myelomonocytic leukemia, which shares some features with
CMML.158,159
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
PROSPECTS FOR A MOLECULAR CLASSIFICATION OF MDS
Although the association of unique disease subtypes with
specific point mutations promises molecular clarity for the
perplexing heterogeneity of MDS, the prospect of a molecular classification to replace the current morphologicand cytogenetics-based WHO scheme threatens to be unwieldy. For example, one of us (D.P.S.) recently detected
acquired, clonally restricted point mutations in the GATA1
gene (chromosome Xp11.23) and RUNX1/AML1 gene
(chromosome 21q22.3) as well as a cancer-associated
germline polymorphism in TP53 (chromosome 17p13.1) in
a single patient with MDS with acquired thalassemia but
without a demonstrable ATRX mutation—a phenotype
seemingly unrelated to the patient’s karyotype of trisomy 8
and del(20q) (Figure 4).
The frequencies of specific point mutations described
previously are likely to be underestimates. Unlike AML, in
which the marrow is almost completely replaced by neoplastic cells, blood and marrow in MDS include a heterogeneous mix of neoplastic and residual normal cells, and
direct sequencing may be sensitive only to mutant DNA
encompassing at least 20% to 30% of the tested sample.
Techniques such as denaturing high-performance liquid
chromatography, denaturing gradient gel electrophoresis,
and polymerase chain reaction–single-strand conformational polymorphism are more sensitive to mosaicism than
direct sequencing.153,160-162 In addition, for a more accurate
estimate of mutation frequency, it is important not only to
assay genes of interest at the genomic DNA (gDNA) level
but also the gene message since messenger RNA splicing
variants are common causes of human disease.163
Mutations that affect splicing can be missed if sequencing of gDNA is limited only to the coding region (exons)
because splicing abnormalities frequently arise from mutations residing in introns, both within the consensus splice
donor and acceptor sites and outside of these areas (ie, in
intronic splice enhancer or inhibitor sites). In the case of
ATRX, patients with MDS with clonally restricted splicing
abnormalities predicted to result in loss of critical ATRX
protein domains have been described, but in all of these
cases the causative mutation has not yet been identified at
the gDNA level.155,164 A mutation in a cryptic splice enhancer or inhibitor element at some distance from the site
of the splicing abnormality is likely responsible. The expensive and time-consuming search for splicing mutations
and missense mutations as well as the need to use a technique sensitive to mosaicism illustrates why point mutation
detection in MDS has not yet become mainstream.
MITOCHONDRIAL GENE MUTATIONS IN MDS
A recent report of mutations in the mitochondrial genome
in patients with MDS, including frequent mutations in the
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cytochrome-c oxidase genes, generated some excitement
because it offered an alternative genomic source potentially contributing to the MDS phenotype.165 Mitochondria
are a logical target for MDS mutation analysis, given the
presence of ringed sideroblasts—pathological iron-laden
mitochondria—in many patients, which suggests a possible
common metabolic disturbance in mitochondrial enzymes
such as cytochrome.166 Mitochondrial enzymes are encoded by both the 16.6 kilobase pair mitochondrial genome and the nuclear genome. However, another group of
investigators sequenced the entire mitochondrial genome
in 10 patients with MDS and were unable to reproduce the
findings.167 Several amino acid changes were found in
patients but not controls, but the investigators suggested
that this might be a reflection of limited clonality among
hematopoietic stem cells rather than an indication of mitochondrial genome instability.167 Subsequently, a novel
somatic mutation of mitochondrial transfer RNA was detected in a patient with MDS,168 and work continues in this
area.
FUNCTIONAL IMPORTANCE OF POINT MUTATIONS IN MDS
In each of the genetic mutations described previously,
functional consequences arise from modification of the
protein encoded. For instance, RUNX1/AML1 point mutations alter DNA binding and transactivating potential of the
mutant RUNX1/AML1.147 In light of the inherent genomic
instability in MDS, “innocent bystander” mutations, ie,
those with no known functional consequence, may occur
with equal or greater frequency. Patients with MDS sometimes have clonally restricted gene mutations that do not
result in amino acid substitutions and have no obvious
effect on transcript splicing (Figure 4). If there is no detrimental effect on cell survival, darwinian pressure in the
marrow will not select against hematopoietic clones bearing this class of mutation. ATRX mutations may be an
example of a neutral mutation with a readily discernible
phenotype (ie, defective α globin production in red
cells).153,155 A vast number of point mutations likely await
discovery in MDS. The task of determining which of these
are important in the pathobiology of the disease will be
challenging.
GENE EXPRESSION PROFILING
A gene need not be altered structurally to modify its expression and affect normal cellular homeostasis. One of the
principal ways in which gene expression can be altered is
through epigenetic modification, including changes in the
way DNA and its associated histones are packaged into
chromatin, and, as a consequence, the accessibility of that
DNA for transcription. The term epigenetic refers to heri-
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Absorbance
GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
Column retention time
Patient’s
blood
Patient-derived
lymphoblastoid
cell line
Wild-type
blood
FIGURE 4. Analysis of exon 6 of GATA1 in archival blood and marrow samples from a group
of patients with myelodysplastic syndromes. Top, Chromatogram from denaturing highperformance liquid chromatography Transgenomic WAVE mutation detection system. Most
patient samples exhibit only a single peak from homoduplexed wild-type DNA (right peak),
but DNA derived from a peripheral blood buffy coat from one patient exhibits a second peak
(left peak, arrow), indicating the presence of a DNA heteroduplex formed from a mix of wildtype and mutant DNA. Bottom, Sequencing of the patient’s GATA1 gene revealed an
acquired c.1066C>T mutation (described in terms of Genbank Accession NM_002049.2)
(arrow), which was not present in DNA derived from an Epstein-Barr virus–transformed
lymphoblastoid cell line from the patient or from DNA derived from a wild-type (normal)
individual. Surprisingly, this clonally restricted mutation did not obviously result in an
amino acid change. RNA was unavailable to determine whether the mutation might affect
splicing.
table alterations in the pattern of gene expression that are
not a consequence of changes in the nucleotide sequence of
a gene.169 Such changes are of interest in cancer, in part
because abnormal epigenetic silencing of genes has been
shown to be associated with neoplasia development and
progression.170 For instance, 5′ methylation of cytosine
nucleotides in the promoter region of tumor suppressor
genes can lead to silencing of those genes.171 In MDS, the
first gene found to be commonly hypermethylated and
silenced was the P15INK4B gene that encodes a cyclindependent kinase inhibitor.172-177 Methylation silencing of
p15 is rare in patients with low leukemic cell burden;
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however, it is detected in more than 75% of cases with
excess blasts and occurs uniformly with AML transformation, suggesting a pathogenetic role in disease progression.
Epigenetic gene silencing in human neoplasia has swiftly
gained importance as a therapeutic target because of its
potential reversibility with pharmacological agents.169,178
The clinically beneficial nucleoside analogue 5-azacitidine
has dual therapeutic effects resulting from direct cytotoxicity as a nucleoside analogue and from inhibition of genomic methylation.12,179 Treatment with decitabine, the active metabolite of 5-azacitidine, has been associated with
demethylation of hypermethylated P15INK4B in MDS, al-
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GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES
though not consistently.179 The addition of histone deacetylase inhibitors and other epigenetic modifiers to
demethylating agents holds considerable promise for powerful chromatin remodelling strategies.180
Until recently, molecular investigations focused on expression of 1 or more genes of interest at a time. For
example, in a few patients with MDS with intact TP53
genes (ie, no coding mutation), expression of the gene has
been found to be decreased.181 This down-regulation may
result in loss of p53 function with biological consequences
equivalent to those resulting from mutational inactivation.
The advent of complementary DNA microarray technology
offers the ability to survey the expression pattern of thousands of genes concurrently.182 The results of MDS
microarray studies have not yet yielded many pathophysiological insights, although some leads have been generated.183-186 For instance, the first ATRX mutation was identified after a microarray study of purified neutrophils
showed dramatic down-regulation of the gene compared
with normal controls (ultimately found to be the consequence of a point mutation in a consensus splice site).152
Another group has detected overexpression of DLK1
(Delta-like homolog), a gene of unknown function with
homology to epidermal growth factor that was first identified as a regulator of adipocyte differentiation, in patients
with MDS but not in patients with AML.183 If microarray
studies finally fulfil their promise, it is hoped that they will
lead to more accurate MDS diagnosis, classification, and
prognostic discrimination and provide more insight into the
disease pathobiology. Recent microarray-generated advancements in the understanding of diffuse large B-cell
lymphoma187 and breast cancer188 illustrate the tremendous
power of this technology.
CONCLUSION
Genetic testing in MDS is currently an essential component of clinical care of patients, and testing options
are likely to evolve in the near future. At present, conventional cytogenetic studies remain a critical part of
the diagnostic evaluation of MDS, and the results offer
valuable prognostic information. Detection of specific
MDS-associated point mutations and measurement of
gene expression patterns are not yet part of routine clinical care, but they promise to expand clinicians’ ability to
diagnose, prognosticate, and ultimately treat patients with
MDS.
We thank Drs. Richard J. Gibbons (University of Oxford, Oxford, England) and Ayalew Tefferi (Mayo Clinic College of
Medicine, Rochester, Minn) for helpful comments on the submitted manuscript.
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The Genetics in Clinical Practice series will continue in the July issue.
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