Genetic Testing in the Myelodysplastic …williams.medicine.wisc.edu/mdsgenetics.pdfGENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES

Mayo Clin Proc. • May 2005;80(5):681-698 • www.mayoclinicproceedings.com 681

GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMESGENETICS IN CLINICAL PRACTICE

From the Department of Internal Medicine and Division of Hematology, MayoClinic College of Medicine, Rochester, Minn (D.P.S.); and H. Lee MoffittCancer 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 onGenetics in Clinical Practice will be available for purchase from the Proceed-ings Editorial Office at a later date.

© 2005 Mayo Foundation for Medical Education and Research

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 adiverse set of acquired somatic genetic abnormalities. Bone mar-row karyotyping provides important diagnostic and prognosticinformation and should be attempted in all patients who aresuspected of having MDS. Fluorescent in situ hybridization (FISH)studies on blood or marrow may also be valuable in selectedcases, such as patients who may have 5q– syndrome or those whohave undergone hematopoietic stem cell transplantation. TheMDS-associated cytogenetic abnormalities that have been de-fined by karyotyping and FISH studies have already contributedsubstantially to our current understanding of the biology of malig-nant myeloid disorders, but the pathobiological meaning of com-mon, recurrent chromosomal lesions such as del(5q), del(20q),and monosomy 7 is still unknown. The great diversity of thecytogenetic findings described in MDS highlights the molecularheterogeneity of this cluster of diseases. We review the commonand pathophysiologically interesting genetic abnormalities associ-ated with MDS, focusing on the clinical utility of conventionalcytogenetic assays and selected FISH studies. In addition, wediscuss a series of well-defined MDS-associated point mutationsand outline the potential for further insights from newer tech-niques such as global gene expression profiling and array-basedcomparative 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 myeloidleukemia; CMML = chronic myelomonocytic leukemia; CMML-Eos =CMML with eosinophilia; FAB = French-American-British; FISH = fluores-cent in situ hybridization; gDNA = genomic DNA; GTPase = guanosinetriphosphate hydrolases; IPSS = International Prognostic Scoring Sys-tem; MDS = myelodysplastic syndromes; M-FISH = multicolor FISH;PDGFR-βββββ = βββββ subunit of the platelet-derived growth factor receptor;SKY = spectral karyotyping; WHO = World Health Organization

DISEASE SYNOPSIS

The term myelodysplastic syndromes (MDS) describes adiverse group of acquired hematopoietic stem cell malig-nancies 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

with functional defects in neutrophils and platelets, whichcontribute to infection and bleeding complications, respec-tively, even in the absence of substantial deficits in bloodcell production. The variable risk of progression to acutemyeloid leukemia (AML) that is characteristic of MDSappears to be driven by somatic genomic instability andepigenetic genomic modification. Evidence for this in-cludes allelic imbalance arising from structural or nu-merical chromosome aberrations, defects in DNA repair,3,4

and linkage to genotoxic exposures including alkylatingagents, topoisomerase II inhibitors, environmental poisons,or radiation.1,2

This overview highlights the genetic and molecular ab-normalities in adults with MDS, methods to detect theseabnormalities, and their potential importance for diagnosis,prognosis, and therapy (Table 1). The inherited and ac-quired MDS in pediatric patients display genetic and clini-cal features distinct from those in adults5 and will not beaddressed in this review.

Most adult patients with MDS present to their physi-cians with signs and symptoms related to anemia orpancytopenia, or their ill ness is discovered incidentallywhen a blood cell count is performed for another purpose.The diagnosis of MDS is established from the character-istic appearance of a bone marrow aspirate, but in somecases cytologic features may be subtle, creating chal-lenges with respect to excluding nonneoplastic causesof cellular dysplasia. In such cases, genetic testing pro-vides an alternative means of diagnosis confirmation andoffers further insight into prognosis and managementconsiderations.

The 1976 French-American-British (FAB) Co-opera-tive Group classification scheme for MDS, revised andexpanded in 1982, provided the first widely used tool fordisease classification and prognostication.6 The FAB clas-sification is based on the proportion of immature blast cellsin the blood and marrow and on the presence or absence ofringed sideroblasts or peripheral blood monocytosis. In1999, a group of hematopathologists and clinicians operat-ing under the auspices of the World Health Organization(WHO) created a new classification system for MDS basedon the FAB framework but incorporated newer morpho-logic insights and, to a more limited extent, cytogeneticfindings (Table 2).7,8

For personal use. Mass reproduce only with permission from Mayo Clinic Proceedings.For personal use. Mass reproduce only with permission from Mayo Clinic Proceedings.

Mayo Clin Proc. • May 2005;80(5):681-698 • www.mayoclinicproceedings.com682

GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES

TABLE 1. Genetic Abnormalities Characteristic of MDS and Their Consequences*

Genetic ormolecular abnormality Functional consequence Importance in MDS

Karyotypic findings

Del(20q), isolated del(5q), Unknown IPSS good-risk markers; del(5q31.1) is associated with–Y, normal karyotype specific 5q– syndrome (female predominance,

dysplastic micromegakaryocytes, thrombocytosis)Complex karyotype, Unknown IPSS poor-risk markers

abnormalities ofchromosome 7

Other karyotypes not listed Unknown IPSS intermediate-risk markers, likely to be subdividedabove in the future as IPSS is revised

Trisomy 8 Unknown Increased risk of leukemic transformation (single-institution studies); male predominance; oralulceration (rare)

Abnormalities of 3q21 translocations are associated with overexpression Decreased survival (single-institution studies)chromosome 1q, of MDS1/EVI1 gene; 11q23 translocations rearrange3q21, and 11q23 MLL gene

Del(12p) Unknown; ETV6 gene at 12p13 is frequently involved Relatively benign prognosis (single-institution studies)in translocations in this region, which may be crypticor associated with deletions

t(8;21)(q22;q22), Leukemogenic rearrangements of core-binding factors WHO classifies cases with these translocations asinv(16)(p13q22) or involved in transcriptional regulation AML by definition, even if they would otherwiset(16;16)(p13;q22), and be consistent with MDSt(15;17)(q22;q12)

Isodicentric X Unknown; ABCB7 at Xq12-13 plays a critical role in Iron accumulation in elderly womeniron homeostasis

t(3;3)(q21;q26) or Rearranges MDS1/EVI1 gene at 3q26 in many cases; Thrombocytosis with megakaryocyte dysplasiainv(3)(q21q26.2) GATA2 at 3q21 is overexpressed in some cases

Isolated isochromosome (17q) Unknown Occasional myeloproliferative features; rapid trans-formation to leukemia and poor response to therapy

Rearrangements of 5q33 PDGFR-β subunit constitutive activation; TEL at CMML with eosinophilia; frequent clinical responsessuch as t(5;12)(q33;p13) 12p13 is a common fusion partner, but many other to imatinib or other PDGF inhibitors; other molecularly

partners have been described defined myelodysplastic-myeloproliferative overlapsyndromes exist, including rearrangements ofPDGFR-α subunit

All metaphases abnormal Marker of clonality Poorer prognosis than patients with mixture of abnormaland normal metaphases

Point mutations

TP53 Mutations result in loss of function of tumor Mutated in 5%-10% of patients with MDS, moresuppressor p53, “the guardian of the genome”— commonly mutated in secondary MDS, may bean essential regulator of cell cycle, specifically associated with progression to AMLtransition from G0 to G1

NRAS Constitutively activates oncogene by preventing Codon 12 mutation is most common in MDS andnormal GTP recycling, resulting in uncontrolled found in 10%-15% of cases; acquisition of mutationgrowth signalling commonly associated with progression to AML

FLT3 Internal tandem duplications of juxtamembrane domain Found in up to 10% of MDS cases; acquisition ofor point mutations result in constitutive activation mutation commonly associated with progression toand uncontrolled growth signalling AML

RUNX1/AML1 Point mutations result in loss of normal trans-activating Most common point mutation described to date inpotential of the key RUNX1 core-binding factor MDS—in up to 25% of cases; associated with highsubunit and consequent changes in gene expression risk of progression to AML, previous radiation

exposure, and chromosome 7 abnormalitiesc-FMS Mutations constitutively activate the receptor for Occasionally associated with CMML

colony-stimulating factor 1, a cytokine that controlsthe production, differentiation, and function ofmonocyte-macrophages

PTPN11 Mutations alter function of the SHP2 that has diverse Rarely associated with CMML or other MDS,roles in cell signalling, including modulation of commonly associated with JMMLPDGFR phosphorylation and STAT activity

ATRX Point mutations result in loss of function of ATRX Acquired α-thalassemia; no apparent effect onmultiprotein complex with consequent changes in proliferative potential of thalassemic clonegene expression, including down-regulation of αglobin cluster, probably by epigenetic mechanisms

*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 = WorldHealth Organization.




TABLE 2. World Health Organization Classification of the Myelodysplastic Syndromes*

Disease subtype Blood findings Bone marrow findings

Refractory anemia Anemia Erythroid dysplasia only–ie, notNo blasts or rare (<1%) blasts myeloid or megakaryocytic dysplasiaNo Auer rods <5% blasts<1 × 109/L monocytes <15% ringed sideroblasts

Refractory anemia with Anemia Erythroid dysplasia onlyringed sideroblasts No blasts ≥15% ringed sideroblasts

No Auer rods <5% blasts<1 × 109/L monocytes

Refractory cytopenia with Cytopenias (bicytopenia or Dysplasia in ≥10% of cells in 2 ormultilineage dysplasia pancytopenia) more myeloid cell lines

No or rare (<1%) blasts <5% blasts in marrowNo Auer rods <15% ringed sideroblasts<1 × 109/L monocytes

Refractory cytopenia with Cytopenias (bicytopenia or Dysplasia in ≥10% of cells in 2 ormultilineage dysplasia pancytopenia) more myeloid cell linesand ringed sideroblasts No or rare (<1%) blasts ≥15% ringed sideroblasts

No Auer rods <5% blasts<1 × 109/L monocytes

Refractory anemia with Cytopenias Unilineage or multilineage dysplasiaexcess blasts 1 <5% blasts 5% to 9% blasts

No Auer rods No Auer rods<1 × 109/L monocytes

Refractory anemia with Cytopenias Unilineage or multilineage dysplasiaexcess blasts 2 5%-19% blasts 10%-19% blasts

Auer rods may be present Auer rods may be present<1 × 109/L monocytes

Myelodysplastic syndrome, Cytopenias Unilineage dysplasia in granulocytesunclassified No or rare blasts or megakaryocytes but no erythroid

No Auer rods dysplasia<5% blastsNo Auer rods

Myelodysplastic syndrome Anemia; other cytopenias Normal to increased megakaryocytesassociated with isolated possible with hypolobated nucleidel(5q) <5% blasts <5% blasts

Platelets normal or increased No Auer rodsIsolated del(5q)

*Chronic myelomonocytic leukemia and juvenile myelomonocytic leukemia are classified as myelodysplastic-myeloproliferative 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 develop-ment offer promise.9 For most patients, management isprimarily supportive, relying on transfusions and antimi-crobials as dictated by symptoms and infectious complica-tions. Select patients will experience hematologic improve-ment with the use of recombinant hematopoietic growthfactors, including erythropoietin and granulocyte colony-stimulating factor.10,11

With the advent of new therapeutic alternatives, themanagement strategy for many patients has shifted fromsole reliance on supportive measures to more active inter-vention. The Food and Drug Administration recently ap-proved the first agent with an indication for MDS, 5-

azacitidine (Vidaza, Pharmion Corporation, Boulder,Colo), an injectable nucleoside analogue that covalentlyincorporates into DNA and depletes DNA methyltrans-ferases to promote genome hypomethylation. This maycontribute to reactivation of methylation-silenced tumorsuppressor genes. In a national cooperative group open-label randomized trial of MDS, 5-azacitidine treatmentdelayed leukemic transformation and reduced the risk ofAML progression or death.12 Most other noninvestigationalagents offer limited benefit for the general population withMDS but are reasonable considerations for select individu-als. For example, thalidomide, when administered at lowdoses for an extended schedule, may restore erythropoiesisin some patients; however, neurotoxicity and other adverseeffects limit dose escalation and prolonged drug adminis-tration, particularly in elderly individuals.13 Antithymocyte




globulin and other immunosuppressants may be effectivein patients with hematopoietic inhibitory immune effec-tors; such patients may be identified through predictivemodelling.14-16 Older agents such as androgens and pyri-doxine are usually ineffective.17,18 Aggressive chemother-apy similar to that used for acute leukemia should be re-served for clinical trials; it is rarely curative.19

Younger patients are often candidates for curative strat-egies such as allogeneic hematopoietic stem cell transplan-tation; however, mortality and morbidity remain high, evenin experienced and specialized centers.20 Autologous trans-plantation may benefit selected patients without an alloge-neic donor, but the potential benefit of this approachappears limited to younger patients.21 Unfortunately, thevast majority of patients with MDS are not candidates forstem cell transplantation with conventional ablative con-ditioning regimens because the median age at the time ofdiagnosis is in the seventh decade of life, and comor-bidities are common. Reduced-intensity conditioning ornonmyeloablative approaches promise to expand the up-per age limit of candidates for stem cell therapy.20,22 Theoptimal timing of transplantation is a subject of activeinvestigation.23

Iron overload remains a clinical challenge, especially inpatients who require frequent red blood cell transfusions,emphasizing the need for iron chelation therapy for patientswith otherwise favorable prognostic features in whom ex-tended survival is expected. Given the paucity of disease-altering therapies for MDS, clinical trial enrollment is para-mount to advance the field. At least 2 agents in clinicaldevelopment, the immunomodulatory drug lenalidomide(CC-5013, Revlimid; Celgene Corporation, Warren, NJ)and the demethylating agent decitabine (Dacogen, Super-gen, Inc, Dublin, Calif), have shown promising preliminaryresults, and Food and Drug Administration review shouldoccur in the months ahead.24,25

STANDARD CYTOGENETIC STUDIES ANDTEST RATIONALE

At present, the most widely available genetic test applied inthe evaluation of patients with MDS is cytogenetic analysisby conventional metaphase karyotyping. Karyotyping hasbeen applied widely since the 1970s, and the method forperforming these assays and their limitations have beenreviewed recently.26,27 Briefly, standard cytogenetic assaysinvolve light-microscopic visualization of metaphase chro-mosomes prepared by (1) inducing the cells of interest todivide, (2) arresting their division at the appropriate mitoticstage with colchicine, (3) lysing the cells, and (4) Giemsastaining of the chromosome spread to resolve the chromo-some size and banding pattern (Figure 1).

In diagnostically ambiguous cases, an abnormal kar-yotype can provide important evidence to support thepresence of a clonal, neoplastic process.28 Additionally, cy-togenetic studies provide important prognostication dis-crimination. The 1997 International Prognostic ScoringSystem (IPSS) for MDS, based on a review of 816 patientswith “primary” MDS (ie, no history of treatment with che-motherapy or radiotherapy), incorporates 3 elements withindependent 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) thetype of chromosomal abnormality present.29 Using these 3elements, a score generated by the IPSS algorithm repro-ducibly classifies patients with MDS into a highest-riskgroup with a median survival of only 0.4 years, a pair ofintermediate-risk groups, and a lowest-risk group with amedian survival of 5.7 years overall, 11.8 years for patients60 years of age or younger. The IPSS algorithm has beenvalidated independently.30,31

The IPSS clusters the varied chromosomal abnormali-ties observed in MDS into 3 risk groups: poor risk (includ-ing abnormalities of chromosome 7 or complex karyotype[≥3 clonal abnormalities]), good risk (including a normalkaryotype, isolated interstitial deletion of the long arm ofchromosome 5 [del(5q)], chromosome 20q interstitial dele-tions [del(20q)], or clonal loss of the Y chromosome, seenin 7.7% of otherwise healthy elderly men32), and intermedi-ate risk (including all other abnormalities not defined asgood or poor).29 The corresponding median survival bycytogenetic 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 maintainsprognostic validity for both standard therapy and alloge-neic stem cell transplantation. In one analysis, patients witha poor-risk cytogenetic pattern had a 3.5-fold greater risk ofrelapse after stem cell transplantation than did patients witha good-risk karyotype.33 Similar findings have been re-ported for patients treated with intensive antileukemic che-motherapy, with or without subsequent transplantation.19,34

Not surprisingly, acquisition of higher-risk IPSS cytoge-netic abnormalities during the course of disease adverselyimpacts survival because of leukemic evolution or cyto-penic complications.35

COMMON CYTOGENETIC ABNORMALITIES IN PRIMARY MDSAlthough not specific for MDS, the recurrent chromosomalabnormalities defined by the IPSS (ie, –7/del(7q), del(5q),del(20q), and –Y) are among the most common detected inMDS. Overall, 40% to 50% of patients with primary MDSpresent with 1 or more clonal karyotypic abnormalities,whereas in those with secondary MDS (ie, after therapeuticchemotherapy or radiotherapy for an unrelated condition),




FIGURE 1. Conventional cytogenetic study of bone marrow cells in a patient with amyelodysplastic syndrome (MDS). This abnormal karyotype includes several abnormalitiesassociated with MDS, including numerical abnormalities (trisomy 8 and monosomy 17[arrowheads]), and 2 common deletions (deletions of the long arm of chromosome 20 andof the long arm of chromosome 5 between bands q13 and q33 [arrows]). Because morethan 3 abnormalities are present, this karyotype is considered complex. Photographcourtesy of Jack L. Spurbeck, Cytogenetics Laboratory, Mayo Clinic, Rochester, Minn.

the proportion of patients with an abnormal karyotype ex-ceeds 80%.2,28,31,36-38 The relative frequency of specific cyto-genetic abnormalities in primary MDS ranges from as highas 30% for del(5q)—either as an isolated anomaly (10%) oras part of a more complex karyotype (20%)—to 20% fortrisomy 8 and 15% for monosomy 7.39 Numerous otherrecurrent abnormalities have been observed, but each ac-counts for less than 10% of cases of primary MDS.

CYTOGENETIC ABNORMALITIES IN SECONDARY MDSThe distribution of chromosome abnormalities is skewed intreatment-related MDS, in which structural or numericaldeletions of chromosome 7 (40%) and chromosome 5(40%) predominate.36,39 Together, abnormalities of chro-mosome 5 and 7 account for approximately 75% of karyo-typic abnormalities in secondary MDS,36 whereas all otherkaryotypic abnormalities occur with a frequency of 10% orless.39 In addition to the higher frequency of abnormalkaryotypes, secondary MDS displays a disproportionatepercentage of IPSS poor-risk abnormalities, and as withprimary MDS these are associated with an unfavorableprognosis. This cytogenetic pattern is frequently linked toand believed to be induced by prior treatment with alkylat-ing antineoplastics.36

The IPSS good-risk abnormalities occur with sufficientrarity in secondary MDS to raise suspicion that such cases

instead represent de novo and treatment-unrelated MDS.Nevertheless, single-institution studies indicate that patientswith such karyotypes fare better than their counterparts withsecondary disease who have poor-risk karyotype abnormali-ties.36 When the spectrum of abnormalities associated withprimary and secondary MDS is compared, secondary formsmore commonly exhibit monosomy 5, monosomy 7, andder(17p) aberrations, whereas in primary MDS, isolateddel(5q) and normal karyotype predominate.39 Treatmentwith topoisomerase II inhibitors has been linked to chromo-some 3q inversions or translocations and to balanced translo-cations involving 11q23 deregulating the MLL gene.40,41

OTHER RECURRENT CYTOGENETIC ABNORMALITIES IN MDSNumerous chromosomal abnormalities occur with moder-ate frequency (ie, 1%-10%) in MDS; however, because ofoverall low prevalence, their prognostic relevance remainsundefined. Limited case series have linked trisomy 8 withan increased risk of AML transformation, and abnormali-ties of chromosome 1q, 3q21, and 11q23 with an overallinferior survival.30,31,40,42 In contrast, del(12p) as a sole ab-normality has been associated with an indolent diseasecourse.31 The prognostic relevance of less common (ie,<1% of cases) yet recurrent chromosomal abnormalitiesremains uncertain.43-47 Although multiple independent andkaryotypically discordant clones may be identified in se-




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’sbone marrow karyotype. These Kaplan-Meier curves show that the highest risk of death andleukemia is in patients with MDS with chromosome 7 abnormalities (Chrom 7 abn) or a complexkaryotype; the lowest risk is associated with loss of the Y chromosome, isolated interstitialdeletion of the long arm of chromosome 5, a normal karyotype, or chromosome 20q interstitialdeletions [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 tobe influenced by the highest-risk karyotype. The MitelmanDatabase of Chromosome Aberrations in Cancer, part of theCancer Genome Anatomy Project of the National Cancer

Institute, includes a comprehensive catalogue of chromo-some abnormalities in patients with MDS that can be ac-cessed at: http://cgap.nci.nih.gov/Chromosomes/Mitelman(accessibility verified March 29, 2005).

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del(5q)Normaldel(20q)Misc. single+8DoubleMisc. doubleChrom 7 abnMisc. complexComplex

–Y 17 (2)48 (6)

489 (60)16 (2)74 (9)38 (5)

14 (2)10 (1)15 (2)66 (8)

29 (3)

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The karyotypic diversity included in the Mitelman data-base illustrates that the diagnostic category of MDS con-tains disorders that phenotypically overlap yet have a mo-lecularly distinct pathobiology. Although the IPSS hasproved its clinical usefulness, the IPSS intermediate-riskcategory includes potentially hundreds of chromosomalabnormalities, suggesting that further refinement of thealgorithm will be possible. As therapy for MDS improves,the prognosis associated with particular karyotypes maychange. By analogy, before the introduction of retinoic acidtherapy for acute promyelocytic leukemia (APL) in the1980s, the classic t(15;17) APL-associated chromosomaltranslocation was regarded as an intermediate-risk leuke-mia karyotype, but with modern drug therapy APL isamong the most curable AML subtypes.50

Although balanced favorable translocations such ast(15;17) and t(8;21) are relatively common in AML, they arerare in MDS in which deletions and numerical chromosomalabnormalities predominate.2,29,51 Given the responsiveness toconventional leukemia chemotherapy, the WHO categorizes4 specific rearrangements involving core-binding factor tran-scriptional repressors [ie, t(8;21)(q22;q22), inv(16)(p13q22)or t(16;16)(p13;q22), and t(15;17)(q22;q12)] as AML, re-gardless of the bone marrow myeloblast percentage.7,8 Forthose exceptional cases that harbor such karyotypic abnor-malities with a low myeloblast burden, it remains unclearwhether AML therapy impacts survival as it does in moretypical AML. 52,53

CLONAL CYTOGENETIC ABNORMALITIES IN THE ABSENCE OF

MORPHOLOGIC EVIDENCE OF MYELODYSPLASIA

In selected cases in which marrow aspiration is performedfor evaluation of cytopenias, routine staging, or follow-upof another malignancy, clonal karyotypic abnormalities maybe identified in the absence of cytologic dysplasia that wouldsupport a diagnosis of MDS.54 The constitutional karyotypein nonhematopoietic tissue in such cases has been normal,confirming an acquired somatic cytogenetic abnormality.This constellation of findings may represent a forme frusteof MDS,54 evidenced by an increased risk of leukemic trans-formation and subsequent cytopenic mortality.54 These un-common cases emphasize the importance of cytogeneticevaluation 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 conven-tional cytogenetics, the abnormality may be present in allexamined metaphases (all abnormal [AA]) or in only afraction of the metaphases (sometimes termed abnormal-normal [AN]).28,35 The AA pattern correlates with higher-

risk MDS subtypes identified by the FAB MDS pathologicclassification,6 and patients with AA have a poorer overallsurvival than those with AN.28

The favorable modifying effect of increasing propor-tions of normal metaphases has precedent in other hemato-logic malignancies. The clinical usefulness of the reduc-tion in the number of abnormal metaphases with therapy isperhaps best illustrated by chronic myeloid leukemia(CML). In CML, a “major cytogenetic response” repre-sents an important therapeutic goal because patients whoachieve a complete cytogenetic remission experience asuperior progression-free survival compared with thosepatients with persistence of any proportion of metaphasesbearing the Philadelphia chromosome.55-57 By analogy, aninternational working group that created standardized re-sponse criteria for clinical trials of MDS included reduc-tions in the number of abnormal metaphases as one of theseresponse criteria, although the clinical importance of thismeasurement had not yet been formally established inMDS.58 Evidence from recent clinical trials testing newpotentially disease-modifying agents supports this notion.In a large phase 2 study of intravenous decitabine inhigher-risk MDS, 31% of patients achieved a completecytogenetic remission, with a corresponding reduction inthe relative risk of death (0.38 compared with those inwhom the abnormal clone persisted).59 These preliminarydata suggest that a cytogenetic response may also be animportant disease-modifying marker in MDS.

BONE MARROW VS PERIPHERAL BLOOD CYTOGENETIC STUDIES

IN MDSMost chromosomal studies in patients with MDS are per-formed on bone marrow cells. Peripheral blood karyotyp-ing has a higher failure rate than marrow studies and rarelyadds useful information beyond that available from themarrow study.37 Unlike CML, in which tracking the pro-portion of cells containing the Philadelphia chromosomeor its molecular equivalent is critical for monitoring ther-apy and the results may prompt management changes inthe absence of the need for an additional marrow aspira-tion, there is currently little compelling clinical indicationfor frequent assessment of chromosomal status in MDS. Inspecial situations in which chromosomal information isdesirable but a marrow examination is not required, such asmeasuring donor/host chimerism after stem cell transplan-tation, fluorescent in situ hybridization (FISH) assays (dis-cussed subsequently) may be preferable.

INDICATIONS FOR CYTOGENETIC TESTING IN MDS

When should a karyotype be obtained for patients withMDS? Given the importance of the IPSS risk assessment, a




chromosomal study is an essential part of the initial evalua-tion (Table 3). Acquisition of new chromosome abnormali-ties (ie, cytogenetic evolution) portends disease progres-sion and therefore justifies cytogenetic evaluation in theroutine management of MDS. Some clinicians obtain acytogenetic study every time a marrow aspiration is per-formed during the care of patients with MDS, whereasothers obtain such studies only in relationship to plannedtherapeutic trials or when there are signs of a change in thepatient’s condition. No evidence base exists to help guidethis practice, and therefore such decisions are best left tothe discretion of the individual treating clinician. Aftertransplantation, karyotypic analysis may not only help inearly determination of relapse (ie, before morphologic evi-dence of recurrent disease is present) but also is useful inassessment of donor-host chimerism in the case of sexmismatches between marrow donor and host.

MOLECULAR CYTOGENETICS IN MDS

Delineating the pathogenetically relevant alterations inMDS at the gene level, even for recurrent cytogeneticabnormalities, has proved challenging. The so-called 5q–syndrome is perhaps the best example of the difficultiesfaced. First delineated in the early 1970s, 5q– syndrome isa form of refractory anemia characterized by female pre-dominance, atypical megakaryocytes, erythroid hypoplasiawith red blood cell transfusion dependence, and a low risk ofleukemic transformation with prolonged survival. Theseclinical and pathological features are consistently associatedwith an isolated interstitial deletion of the long arm of chro-mosome 5 that involves 5q31.1.60,61 Although earlier reportsvaried in the definition of the 5q– syndrome, general consen-sus is that this description should be limited to patients with

an isolated 5q31.1 interstitial deletion in the absence of othercytogenetic abnormalities, a low bone marrow blast percent-age, and no history of genotoxic therapy.62

The WHO’s most recent classification schema for ma-lignant hematopoietic diseases formally recognizes 5q–syndrome as a discrete entity within the general category ofMDS.7 Although no other specific MDS-associated chro-mosome aberration has been recognized by the WHO as adistinct pathologic and clinical syndrome, other recurrentchromosome abnormalities have been linked with specificdisease features. For example, isolated isochromosome 17qmay be associated with myeloproliferative features andpoor response to therapy,63 isodicentric X has been linkedto iron overload in older women,64 trisomy 8 displays amale predominance,65-67 and inv(3)(q21q26.2) is associatedwith thrombocytosis with megakaryocytic dysplasia andpoor therapeutic response.42,68

Boultwood and colleagues at Oxford have carefully de-fined the commonly deleted region (CDR) in patients with5q– syndrome using FISH and other molecular mappingtechniques, and they have cloned several novel genes in theprocess.69-72 The defined CDR spans 1.5 megabase pairs at5q31.1.71 This CDR is relatively gene rich, containing noless than 40 discrete genes, 33 of which are expressed inearly hematopoietic cells.71 Several of these genes aremembers of the interleukin family or have other potentialpromoting roles in hematopoiesis. A number of geneswithin the CDR are not well characterized, and their func-tion is as yet unknown. To date, systematic sequencing ofthese genes in patients with 5q– syndrome has not re-vealed pathogenetic mutations, and thus it is now be-lieved that the pathophysiology of 5q– syndrome mayrelate to gene dosage rather than mutational gene deregu-lation in the CDR.

TABLE 3. Summary of Genetic Test Indications in Myelodysplastic Syndromes*

Conventional cytogenetics FISH

Screening No NoAt diagnosis

Bone marrow Yes Testing for 5q deletions may predict response to lenalidomide,24

Blood Not necessary unless patient refuses and panel FISH testing is reasonable if cytogenetic study isbone marrow examination inadequate

During therapyBone marrow If the patient is enrolled in a clinical study, If FISH was performed at diagnosis and the patient is enrolled

otherwise no compelling indication in a clinical study, otherwise no compelling indicationAfter hematopoietic

stem cell transplantationBone marrow 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 abnormalityis present before transplantation

Blood No May be useful for minimally invasive assessment ofchimerism and early relapse

Leukemic transformationBone marrow Yes No clear role

*See text for test interpretation. FISH = fluorescent in situ hybridization.




Other groups are investigating additional recurrentMDS-associated chromosome abnormalities in an attemptto identify critical gene targets, including del(20q), trisomy8, del(7q), and del(9q). In selected cases, study of patientswith parallel inherited disorders may provide insight. Forexample, patients with constitutional trisomy 8 mosaicismappear to have a high risk of developing myeloid malignan-cies including MDS and leukemia.73 Chromosome 7 may beparticularly challenging because there are multiple CDRs,including 7q22 and several in the region 7q31-7q35.74-76

Perhaps the most important molecular cytogenetic dis-covery in recent years with immediate therapeutic implica-tion is the reciprocal chromosome translocation involvingchromosome 5q33, where the gene encoding the β subunitof the platelet-derived growth factor receptor (PDGFR-β)is located. Although a number of chromosomes and genesmay partner with 5q33, the clinical phenotype is distinctand is now recognized by the WHO classification aschronic myelomonocytic leukemia (CMML) with eosino-philia (CMML-Eos). (The WHO classification removed allCMML subtypes from the MDS group, where they hadbeen placed in the widely used 1982 FAB MDS classi-fication, and placed them in a separate myelodysplastic/myeloproliferative overlap category. This was done be-cause patients with CMML frequently exhibit prominentmyeloproliferative features, unlike patients with other sub-types of MDS.6,7,77) The CMML-Eos phenotype arises fromthe generation of novel fusion transcripts with constitutiveactivation of the PDGF-β receptor tyrosine kinase.78-82

Transgenic mouse models have shown that these novel re-ceptor tyrosine kinase fusion genes are singularly respon-sible for the generation of these myelodysplastic/myelopro-liferative disorders and are selectively responsive to PDGFRkinase inhibitors such as imatinib (Gleevec, Novartis, Basel,Switzerland).79,83,84 Several patients with CMML-Eos havebeen reported to achieve rapid hematologic control and sus-tained cytogenetic remission with imatinib monotherapy85 ortreatment with SU11657, an experimental agent that alsoinhibits PDGF signalling.86

FLUORESCENT IN SITU HYBRIDIZATION

Although conventional cytogenetic analysis remains thestandard for purposes of diagnosis and prognosis in MDS,interest is increasing in the application of more sensitivetechniques such as FISH. Reviews of FISH applicationsin hematologic malignancies have been published re-cently.27,87 Briefly, FISH involves hybridization of patientgenetic material with a fluorescently labelled DNA probedesigned to anneal either to specific DNA sequences or tochromosomal features such as centromeres. The advan-tages of FISH include its applicability to either peripheralblood or bone marrow, the opportunity to analyze inter-

phase cells instead of only dividing cells, and the capacityto analyze substantially more cells than is possible byconventional cytogenetic analysis. When chromosomal rear-rangements are present in only a small subset of neoplasticcells, a common situation in MDS, FISH can offer increasedsensitivity over conventional cytogenetics. For purposes ofinvestigation of specific MDS-associated chromosomal de-letions, FISH probes are extremely useful for narrowing thecommon CDRs,71,74,76,88-91 and they can also identify the pre-cise types of cells involved in the neoplastic process.90,92-98

Several groups have developed FISH “panels” that aredesigned to detect the chromosome abnormalities mostcommonly identified in patients with MDS, such as thoseinvolving chromosomes 5, 7, 8, 11, 13, and 20.99,100 SuchFISH analyses in MDS occasionally reveal cryptic cytoge-netic abnormalities that are not recognized on metaphaseanalysis.101-103 However, conventional cytogenetics andFISH should be viewed as complementary because thereare also abnormalities better detected by conventional cy-togenetics than FISH.100,103-105

Many of the abnormalities detected by FISH that are notalways recognized by conventional cytogenetics are amongthose included in the IPSS (eg, monosomy 7106) or thosefound by other groups to have prognostic importance inMDS (eg, trisomy 8102,106 and 12p rearrangements101). Chro-mosome aberrations detected only by FISH have not yetbeen shown to have the same prognostic importance asthose revealed by conventional cytogenetics, and thus atpresent there is no compelling clinical reason to performFISH at the time of diagnosis of MDS if the karyotype issuccessful. However, because the critical molecular defectis likely to be the same regardless of how the chromosomalbreakpoint is detected, specific high-yield FISH assays willprobably be incorporated eventually into prognostic andtreatment-response algorithms,107 as in CML.26,108 For ex-ample, in the European clinical trial of decitabine for MDS,2 patients had chromosomal responses detected by FISHthat correlated with clinical and cytogenetic response.59

If standard karyotyping is unsuccessful because of afibrotic marrow, failure of aspirated cells to grow in cul-ture, or other technical problems, it is reasonable to resortto FISH using a panel designed to detect abnormalitieswith recognized prognostic relevance. Additionally, inview of the promising recent findings by List et al24 of ahigh rate of complete hematologic and cytogenetic re-sponse to lenalidomide therapy in patients with MDS with5q31.1 deletions, it may be especially valuable to performFISH directed at detection of loss of the CDR on chromo-some 5, particularly when the marrow morphologic fea-tures suggest 5q– syndrome. Patients with cryptic 5q dele-tions might then be referred for appropriate clinical trials.Over time, as the presence of other cytogenetic abnormali-




ties is correlated with response to novel therapies, the roleof 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 toanalyze all human autosomes and sex chromosomes at thesame time by labelling metaphase cells with band-selectivecolor probes (Figure 3).109,110 These methods offer greatersensitivity than conventional karyotyping and are useful forcharacterizing cryptic chromosomal rearrangements and theorigin of structurally ill-defined chromosomal material suchas “marker” chromosomes, in which the normal counterpartis not readily identified because of an ambiguous bandingpattern.

Despite the heightened power of discrimination that M-FISH and SKY technologies may offer,111 their clinicalusefulness in MDS has yet to be proved. For example, inpatients with MDS with a normal karyotype by conventionalcytogenetics, unrecognized cryptic abnormalities are rarelyidentified by M-FISH or SKY.99,100,112 In contrast, additionalrearrangements are often detected when chromosome paint-ing methods are performed in patients with MDS with abnor-mal karyotypes109,111,113-116; however, the clinical relevance ofthese abnormalities is as yet undefined.

COMPARATIVE GENOMIC HYBRIDIZATION

Comparative genomic hybridization (CGH) is a new tech-nique that applies fluorescent hybridization to detect differ-ences in DNA copy number between a patient sample and anormal control, giving insight into loss or gain of chromo-somal segments.117 The ratio of the signal strength from thepatient DNA to the reference DNA is measured in eachchromosomal region. Until recently, the resolution of CGHwas limited and only allowed detection of chromosomalgains or losses of 20 megabase pairs or greater. Despitethis, even first-generation CGH probes provided sufficientsensitivity to discover cryptic rearrangements in patientswith myeloid diseases including MDS, and soon the resolu-tion had improved to almost 1 megabase pair.118,119 Newerarray-based CGH methods, including a recently describedtiling array that maps the entire human genome with morethan 30,000 clones, promise to improve sensitivity suffi-cient to resolve even minute DNA alterations.117,120

POINT MUTATIONS IN MDS

Conventional cytogenetic studies and FISH probes detectlarge chromosomal rearrangements. However, isolatedbase pair changes or so-called point mutations may alsoinactivate or result in constitutive gain of function of theaffected gene, with important biological consequences.

Discovery of these mutations is one of the major openfrontiers in MDS molecular pathology, with initial investi-gations focusing on recognized tumor suppressor genes.

TP53The tumor suppressor gene TP53, a critical cell-cyclecheckpoint regulator, was the first gene evaluated in MDSbecause of its high frequency of inactivation in solid tu-mors. In myeloid malignancies such as MDS and AML,TP53 mutations are uncommon, detected in no more than15% of patients with primary MDS, with a higher fre-quency in atomic bomb survivors and patients treated pre-viously with chemotherapy or radiotherapy.121-130 Such mu-tations are generally demonstrable at the time of diagnosisand may have independent prognostic value.128,131

Even in tumor types in which TP53 mutations are com-mon, TP53 mutation testing is rarely performed in clinicalpractice, unless a germline mutation leading to a generalcancer susceptibility syndrome (the Li-Fraumeni syn-drome) is suspected. This is primarily because patientswith TP53-mutant neoplasias have not yet been shown tobenefit from differential clinical management. For both in-herited and acquired mutations, direct genetic analysis ofTP53 by sequencing is required because immunohis-tochemical results do not correlate well with mutation sta-tus.132,133 Newer high-throughput technology may change thecost-benefit analysis of TP53 mutation screening and makethis assay more accessible to clinicians (although its useful-ness remains to be determined). For instance, denaturinghigh-performance liquid chromatography, which separateswild-type and mutant DNA on the basis of the differentialaffinity of DNA heteroduplexes and homoduplexes onpolystyrene beads coated with a gradient of organic sol-vents, has shown promise as a more rapid TP53 screeningmethod in MDS and other tumors134 (D.P.S., unpublisheddata, 2004).

NRAS AND FLT3The RAS proto-oncogene superfamily encodes guanosinetriphosphate hydrolases (GTPase) that are critical regula-tors of cellular growth-related signals. Three RAS proto-oncogenes (H, N, and K) encode four 21-kd G proteins,including 2 alternatively spliced K-Ras products that areposttranslationally modified before incorporation into theinner leaflet of the plasma membrane. RAS mutations occurat critical regulatory sites (eg, codons 12, 13, and 61) thatinactivate the GTPase response normally stimulated by thebinding of GTPase-activating proteins, thereby extendingthe half-life and signalling activity of the Ras-GTP boundmutant. In myeloid malignancies, NRAS mutations pre-dominate, especially a common mutation in codon 12;nonetheless, the frequency of activating RAS mutations in




unselected cases of MDS is relatively low, ranging from10% to 15% of MDS. In contrast, in CMML, RAS muta-tions are found in 40% to 60% of patients and are believedto contribute to growth factor hypersensitivity of myelo-monocytic progenitors.130,135-139

Internal tandem duplications and other constitutively ac-tivating mutations of the receptor tyrosine kinase FLT3 arecommon in AML but are relatively rare in MDS.130,138,140-143

Acquisition of NRAS and/or FLT3 mutations has been asso-ciated with progression of MDS to AML.137,138,140

RUNX1/AML1The most common point mutations detected in MDS todate are those involving the core-binding–factor subunitRUNX1/AML1.144-151 In leukemia, this transcription factorand oncogene is involved in common balanced transloca-tions such as t(8;21), which leads to formation of aberrantlyactive chimeric fusion proteins and uncontrolled cellgrowth. Although early reports estimated that RUNX1/AML1 mutations were rare in MDS,148 more recent studieshave described these mutations in as many as 25% ofpatients,150 and single-institution studies have associatedRUNX1/AML1 mutation status with chromosome 7q abnor-malities,149 more advanced forms of MDS,150,151 and a highrisk of progression to overt leukemia.150,151

ATRXSpecific gene mutations may be associated with uniqueMDS phenotypes. For instance, the rare complication of

acquired α-thalassemia (hemoglobin H disease) arising inthe context of MDS was recently linked to mutations in theATRX gene at Xq13.1.152,153 Inherited mutations in ATRXcause a mild form of α-thalassemia and severe syndromicX-linked mental retardation,154 whereas acquired somaticATRX mutations create a severe imbalance in globin syn-thesis, a large proportion of hemoglobin H-containingcells, and the unusual finding of microcytic, hypochromicred cell indices.152,153,155 The distinct behavior of mutationslike ATRX when they are acquired in the context of MDS,rather than inherited in the germline, may offer importantinsights into MDS pathobiology.155

MUTATIONS FOUND PREDOMINANTLY IN CMMLIn addition to having unique clinical features and uniquechromosomal translocations [such as the t(5;12) PDGFRβ-TEL fusion], CMML has several point mutations thatappear with great frequency but are uncommon in otherMDS subtypes. Patients with CMML have a high fre-quency of activating RAS mutations and a disproportion-ately higher frequency of c-FMS point mutations, althoughthe latter are rare overall in myeloid disorders.156,157 Like-wise, mutations in the protein tyrosine phosphatase genePTPN11 (which cause a form of Noonan syndrome wheninherited) are extremely uncommon in MDS in general,more common in CMML, and especially common (ie,34% frequency) in the pediatric syndrome juvenile myelo-monocytic leukemia, which shares some features withCMML.158,159

FIGURE 3. Multiplex fluorescent in situ hybridization (M-FISH) study of a patient with a malignant myeloid disorder. Conventional G-bandedcytogenetic 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 analysisconfirmed 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 rearrange-ment. Arrows designate the abnormal chromosomes. Photograph courtesy of Rhett P. Ketterling, MD, Division of Laboratory Medicine and Pathol-ogy, Mayo Clinic, Rochester, Minn. From Br J Haematol,109 with permission.




PROSPECTS FOR A MOLECULAR CLASSIFICATION OF MDSAlthough the association of unique disease subtypes withspecific point mutations promises molecular clarity for theperplexing heterogeneity of MDS, the prospect of a mo-lecular classification to replace the current morphologic-and cytogenetics-based WHO scheme threatens to be un-wieldy. For example, one of us (D.P.S.) recently detectedacquired, clonally restricted point mutations in the GATA1gene (chromosome Xp11.23) and RUNX1/AML1 gene(chromosome 21q22.3) as well as a cancer-associatedgermline polymorphism in TP53 (chromosome 17p13.1) ina single patient with MDS with acquired thalassemia butwithout a demonstrable ATRX mutation—a phenotypeseemingly unrelated to the patient’s karyotype of trisomy 8and del(20q) (Figure 4).

The frequencies of specific point mutations describedpreviously are likely to be underestimates. Unlike AML, inwhich the marrow is almost completely replaced by neo-plastic cells, blood and marrow in MDS include a heteroge-neous mix of neoplastic and residual normal cells, anddirect sequencing may be sensitive only to mutant DNAencompassing at least 20% to 30% of the tested sample.Techniques such as denaturing high-performance liquidchromatography, denaturing gradient gel electrophoresis,and polymerase chain reaction–single-strand conforma-tional polymorphism are more sensitive to mosaicism thandirect sequencing.153,160-162 In addition, for a more accurateestimate of mutation frequency, it is important not only toassay genes of interest at the genomic DNA (gDNA) levelbut also the gene message since messenger RNA splicingvariants are common causes of human disease.163

Mutations that affect splicing can be missed if sequenc-ing of gDNA is limited only to the coding region (exons)because splicing abnormalities frequently arise from muta-tions residing in introns, both within the consensus splicedonor and acceptor sites and outside of these areas (ie, inintronic splice enhancer or inhibitor sites). In the case ofATRX, patients with MDS with clonally restricted splicingabnormalities predicted to result in loss of critical ATRXprotein domains have been described, but in all of thesecases the causative mutation has not yet been identified atthe gDNA level.155,164 A mutation in a cryptic splice en-hancer or inhibitor element at some distance from the siteof the splicing abnormality is likely responsible. The ex-pensive and time-consuming search for splicing mutationsand missense mutations as well as the need to use a tech-nique sensitive to mosaicism illustrates why point mutationdetection in MDS has not yet become mainstream.

MITOCHONDRIAL GENE MUTATIONS IN MDSA recent report of mutations in the mitochondrial genomein patients with MDS, including frequent mutations in the

cytochrome-c oxidase genes, generated some excitementbecause it offered an alternative genomic source poten-tially contributing to the MDS phenotype.165 Mitochondriaare a logical target for MDS mutation analysis, given thepresence of ringed sideroblasts—pathological iron-ladenmitochondria—in many patients, which suggests a possiblecommon metabolic disturbance in mitochondrial enzymessuch as cytochrome.166 Mitochondrial enzymes are en-coded by both the 16.6 kilobase pair mitochondrial ge-nome and the nuclear genome. However, another group ofinvestigators sequenced the entire mitochondrial genomein 10 patients with MDS and were unable to reproduce thefindings.167 Several amino acid changes were found inpatients but not controls, but the investigators suggestedthat this might be a reflection of limited clonality amonghematopoietic stem cells rather than an indication of mi-tochondrial genome instability.167 Subsequently, a novelsomatic mutation of mitochondrial transfer RNA was de-tected in a patient with MDS,168 and work continues in thisarea.

FUNCTIONAL IMPORTANCE OF POINT MUTATIONS IN MDSIn each of the genetic mutations described previously,functional consequences arise from modification of theprotein encoded. For instance, RUNX1/AML1 point muta-tions alter DNA binding and transactivating potential of themutant RUNX1/AML1.147 In light of the inherent genomicinstability in MDS, “innocent bystander” mutations, ie,those with no known functional consequence, may occurwith equal or greater frequency. Patients with MDS some-times have clonally restricted gene mutations that do notresult in amino acid substitutions and have no obviouseffect on transcript splicing (Figure 4). If there is no detri-mental effect on cell survival, darwinian pressure in themarrow will not select against hematopoietic clones bear-ing this class of mutation. ATRX mutations may be anexample of a neutral mutation with a readily discerniblephenotype (ie, defective α globin production in redcells).153,155 A vast number of point mutations likely awaitdiscovery in MDS. The task of determining which of theseare important in the pathobiology of the disease will bechallenging.

GENE EXPRESSION PROFILING

A gene need not be altered structurally to modify its ex-pression and affect normal cellular homeostasis. One of theprincipal ways in which gene expression can be altered isthrough epigenetic modification, including changes in theway DNA and its associated histones are packaged intochromatin, and, as a consequence, the accessibility of thatDNA for transcription. The term epigenetic refers to heri-




Patient’sblood

Patient-derivedlymphoblastoid

cell line

Wild-typeblood

FIGURE 4. Analysis of exon 6 of GATA1 in archival blood and marrow samples from a groupof patients with myelodysplastic syndromes. Top, Chromatogram from denaturing high-performance liquid chromatography Transgenomic WAVE mutation detection system. Mostpatient 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 wild-type and mutant DNA. Bottom, Sequencing of the patient’s GATA1 gene revealed anacquired 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–transformedlymphoblastoid 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 anamino acid change. RNA was unavailable to determine whether the mutation might affectsplicing.

table alterations in the pattern of gene expression that arenot a consequence of changes in the nucleotide sequence ofa gene.169 Such changes are of interest in cancer, in partbecause abnormal epigenetic silencing of genes has beenshown to be associated with neoplasia development andprogression.170 For instance, 5′ methylation of cytosinenucleotides in the promoter region of tumor suppressorgenes can lead to silencing of those genes.171 In MDS, thefirst gene found to be commonly hypermethylated andsilenced was the P15INK4B gene that encodes a cyclin-dependent kinase inhibitor.172-177 Methylation silencing ofp15 is rare in patients with low leukemic cell burden;

however, it is detected in more than 75% of cases withexcess blasts and occurs uniformly with AML transforma-tion, suggesting a pathogenetic role in disease progression.Epigenetic gene silencing in human neoplasia has swiftlygained importance as a therapeutic target because of itspotential reversibility with pharmacological agents.169,178

The clinically beneficial nucleoside analogue 5-azacitidinehas dual therapeutic effects resulting from direct cytotoxic-ity as a nucleoside analogue and from inhibition of ge-nomic methylation.12,179 Treatment with decitabine, the ac-tive metabolite of 5-azacitidine, has been associated withdemethylation of hypermethylated P15INK4B in MDS, al-

Abs

orba

nce

Column retention time




though not consistently.179 The addition of histone de-acetylase inhibitors and other epigenetic modifiers todemethylating agents holds considerable promise for pow-erful chromatin remodelling strategies.180

Until recently, molecular investigations focused on ex-pression of 1 or more genes of interest at a time. Forexample, in a few patients with MDS with intact TP53genes (ie, no coding mutation), expression of the gene hasbeen found to be decreased.181 This down-regulation mayresult in loss of p53 function with biological consequencesequivalent to those resulting from mutational inactivation.The advent of complementary DNA microarray technologyoffers the ability to survey the expression pattern of thou-sands of genes concurrently.182 The results of MDSmicroarray studies have not yet yielded many pathophysi-ological insights, although some leads have been gener-ated.183-186 For instance, the first ATRX mutation was identi-fied after a microarray study of purified neutrophilsshowed dramatic down-regulation of the gene comparedwith normal controls (ultimately found to be the conse-quence 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 withhomology to epidermal growth factor that was first identi-fied as a regulator of adipocyte differentiation, in patientswith MDS but not in patients with AML.183 If microarraystudies finally fulfil their promise, it is hoped that they willlead to more accurate MDS diagnosis, classification, andprognostic discrimination and provide more insight into thedisease pathobiology. Recent microarray-generated ad-vancements in the understanding of diffuse large B-celllymphoma187 and breast cancer188 illustrate the tremendouspower of this technology.

CONCLUSION

Genetic testing in MDS is currently an essential compo-nent of clinical care of patients, and testing optionsare likely to evolve in the near future. At present, con-ventional cytogenetic studies remain a critical part ofthe diagnostic evaluation of MDS, and the results offervaluable prognostic information. Detection of specificMDS-associated point mutations and measurement ofgene expression patterns are not yet part of routine clini-cal care, but they promise to expand clinicians’ ability todiagnose, prognosticate, and ultimately treat patients withMDS.

We thank Drs. Richard J. Gibbons (University of Oxford, Ox-ford, England) and Ayalew Tefferi (Mayo Clinic College ofMedicine, Rochester, Minn) for helpful comments on the submit-ted manuscript.

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The Genetics in Clinical Practice series will continue in the July issue.


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Genetic Testing in the Myelodysplastic …williams.medicine.wisc.edu/mdsgenetics.pdfGENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES GENETIC TESTING IN THE MYELODYSPLASTIC SYNDROMES