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Myelodysplastic Syndrome with Deletion 5q Abnormality and Its Treatment
Comprehensive Review Myelodysplastic Syndrome with Deletion 5q Abnormality and Its Treatment Wendy Ingram, Ghulam J. Mufti Abstract The clinical features of myelodysplastic syndrome (MDS) associated with an interstitial deletion of chromosome 5q(31-33) are well characterized; however, the underlying molecular events of pathogenesis remain elusive. This class of immunomodulatory drugs has gained much interest after the report of high hematologic and cytogenetic response rates in patients with MDS and 5q abnormalities treated with lenalidomide. Lenalidomide demonstrates potent immunomodulatory properties through several pathways, including enhanced T-cell activation and augmentation of natural killer cell activity. Recent studies also report potential selectivity of lenalidomide on cell lines with 5q abnormality. This review outlines the mechanism of action of lenalidomide and the key trials in MDS associated with 5q deletion syndrome. Potential candidate genes mapped to the critically deleted region of chromosome 5q are described, and the roles of gene expression profiling and single-nucleotide polymorphism analysis are summarized herein. Clinical Leukemia, Vol. 1, No. 1, 28-33, 2006 Key words: Acute myeloid leukemia, Karyotype, Lenalidomide
Introduction Karyotypic abnormalities are a common feature of myelodysplastic syndromes (MDSs) identified in 60% of cases of primary MDS.1,2 An interstitial deletion of the long arm of chromosome 5 is a relatively common cytogenetic abnormality. Between 10% and 15% of patients with MDS present with an isolated 5q deletion (del[5q]) or del(5q) in combination with additional karyotypic changes.1,3 The del(5q) syndrome is a unique subtype of MDS characterized by macrocytic anemia, normal to increased platelet count, hypolobated megakaryocytes, and an interstitial deletion of the long arm of chromosome 5 usually involving q31-q33.4 The clinical features of del(5q) syndrome were first described in the mid-1970s by Van den Berghe et al5,6; however, del(5q) syndrome was only classified as a distinct entity in the World Health Organization classification of MDS.4 The syndrome typically affects women of middle to older age and confers a favorable prognosis compared with other subtypes of MDS.7-10 Prognostic scoring systems have been devised in an attempt to identify factors that adversely affect outcome in MDS,11 the most widely used being the international prognostic scoring system (IPSS).7 According to the IPSS, the percentage of bone marrow blasts, the presence or absence of karyotypic abnormalities, and peripheral blood cytopenias are the 3 most significant factors affecting outcome. Good-risk cytogenetic features include a normal karyotype, -Y, del(5q), or del(20q). The prognosis of patients with low-, intermediate-, and high-risk MDS is outlined in Table 1. A cohort of 76 patients with MDS and del(5q) involving band q31 were analyzed by Giagounidis et al.8 A projected median survival of 146 months at a median follow-up of 67 months in cases of isolated del(5q) was reported. However, the presence of 1 additional cytogenetic abnormality or > 5% bone marrow blasts conferred a significantly worse outcome. Despite prolonged survival and a low risk of leukemic transformation, the disease is typically characterized by a protracted history of regular blood transfusions. The resulting iron overload culminates in organ damage and can lead Department of Haematological Medicine, King’s College London, London, UK Submitted: July 11, 2006; Revised: August 4, 2006; Accepted: August 7, 2006 Address for correspondence: Ghulam J. Mufti, DM, Department of Haematological Medicine, King’s College London, Rayne Institute, 123 Coldharbour Ln, London, SE5 9NU, United Kingdom Fax: 207-346-3514; e-mail: [email protected] Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1931-6925, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.
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Clinical Leukemia • September 2006
Table 1 IPSS
Prognosis of Patient with MDS According to International Prognostic Scoring System
Risk Category
Regions Deleted on Chromosome 5q in MDS/AML Compared with del(5q) Syndrome20-22,25,26,32
Median Risk of 25% of Cases Undergoing Survival Transformation to AML (Years) (Years) 9.4
5.7
0.5-1 Intermediate-1
3.3
3.5
1.5-2 Intermediate-2
1.1
1.2
2.5
0.2
0.4
Low
0
Figure 1
High
DNA Marker Gene IL-3 CSF2 IRF-1 IL-5 IL-13 IL-4
p
MDS/AML Le Beau et al20
CDC25C D5S500
Zhao et al21 Horrigan et al22
HSPA9 EGR1 D5S594 HDAC3
1 q31
D5S166
q
CD14 FGF1 ADR2
2
D5S413 CSF1R PDGFR
Pathogenesis of del(5q) Syndrome
Table 2
Willman et al32
D5S479 D5S414
3
The molecular events underlying the pathogenesis of MDS remain elusive. The multi-hit hypothesis of leukemia is supported by mouse models of leukemogenesis, which demonstrate the need for mutations in > 1 gene in order for disease progression to occur. A proposal by Gilliland et al suggests the mutations predisposing one to leukemogenesis might be broadly classified into those that confer a proliferative advantage to the hematopoietic stem cell and/ or those that result in blockage of differentiation.13 The latter of these mechanisms might explain, in part, the cytopenias associated with the del(5q) syndrome. Further gene mutations, an example of which is the JAK2 (V617F) mutation, might subsequently confer a proliferative advantage to the cell. The JAK2 somatic mutation is
MDS/AML
IL-9 TGF1 SPOCK
Chromosome 5
to significant morbidity. Improving quality of life has increasingly been highlighted as an important objective of therapeutic modalities in MDS and myeloid leukemia, particularly in the elderly.12 Maximizing hemoglobin levels and, hence, decreasing the need for transfusions is at the forefront of the management of del(5q) syndrome and low-risk MDS. This review summarizes the advances in the pathogenesis of del(5q) syndrome, key genes mapped to the critically deleted segment, and outlines the clinical trials performed with lenalidomide and MDS with del(5q).
Study Group
q34
del(5q) Syndrome
CD74
Jaju et al26
MEGF1 SPARC ATOX1
Boultwood et al25
GLR1
Diagrammatic representation of the regions deleted on chromosome 5q in MDS/AML compared with del(5q) syndrome. A summary of the DNA markers and genes of interest are shown. Abbreviations: ADR2 = adrenergic -2-, receptor, surface; ATOX1 = ATX1 antioxidant protein 1 homologue; CDC25C = cell division cycle 25C; CSF2 = colony-stimulating factor 2; CSF1R = colony-stimulating factor 1 receptor; EGR1 = early growth response 1; FGF1 = fibroblast growth factor 1; GLR1 = glycine receptor 1; HDAC3 = histone deacetylase 3; HSPA9 = human mortalin; MEGF1 = protein 1 with multiple EGF-like domains; PDGFR = platelet-derived growth factor receptor– polypeptide; SPARC = secreted protein acidic and richin cystein; SPOCK = sparc/osteonectin, cwcv and kazal-like domains proteoglycan; TGF1 = transforming growth factor–1
Summary of Reported Deleted Regions on Chromosome 5q31-3420-26 Disease Group Studied
Size of Deleted Region
Flanking Genes/ DNA Markers
Genes of Interest Mapped Within the Region
Le Beau et al20
MDS/AML
2.8 Mb
IL-9 D5S166*
CSF2, IL-3, IL-4, IL-5, IL-9
Zhao et al21
MDS/AML
1-1.5 Mb
D5S479* D5S500*
EGR1, CDC25C
AML
700 kb
D5S500* D5S594*
CDC25C, HSPA9, EGR1, CTNN1
Boultwood et al24
del(5q) Syndrome
5.6 Mb
FGFA NKSF1
GLR, ADR2, CSF1R, SPARC
Jaju et al26
del(5q) Syndrome
3 Mb
ADR2 IL-12b
CSF1R, SPARC, GR11
Boultwood et al25
del(5q) Syndrome
1.5 Mb
D5S413* GLR1
MEGF1, G3BP, SPARC, CSF1R
Study
Horrigan et al22
*Represent DNA markers. Abbreviations: ADR2 = adrenergic -2-, receptor, surface; CDC25C = cell division cycle 25C; CSF2 = colony-stimulating factor 2; CSF1R = colony-stimulating factor 1 receptor; CTNN1 = catenin (cadherin-associated protein), 1, 102 kDa; EGR1 = early growth response 1; FGFA = fibroblast growth factor acidic; G3BP = Ras-GAP SH3 domain binding protein; GLR = glycine receptor; GLR1 = glycine receptor 1; GR11 = glutamate receptor; HSPA9 = human mortalin; kb = kilobase; MEGF1 = protein 1 with multiple EGF-like domains; NKSF1 = natural killer cell stimulatory factor–1; SPARC = secreted protein acidic and richin cystein
Clinical Leukemia • September 2006
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MDS and Deletion 5q Abnormality
Figure 2
Single-Nucleotide Polymorphisms in a Patient with Myelodysplastic Syndrome Copy Number
Loss of Heterozygosity
A B C D E
A B C D E
5p15.33 5p15.32 5p15.31 5p15.2 5p15.1 5p14.3 5p14.2 5p14.1
Deleted Segment on 5q
5p13.3 5p13.2 5p13.1 5p12 5p11 5q11.2 5q12.1 5q12.3 5q13.1 5q13.2 5q13.3 5q14.1 5q14.2 5q14.3 5q15
5q22.3 5q23.1 5q23.2 5q23.3 5q31.1
Deleted Region
5q21.1 5q21.2 5q21.3 5q22.1
5q31.2 5q31.3 5q32 5q33.1 5q33.2 5q33.3 5q34 5q35.1 5q35.2 5q35.3
Single-nucleotide polymorphism analysis identifying a large region of LOH on chromosome 5q in a patient with MDS with no history of 5q deletion. The copy number is shown in pink, highlighting a fall in copy number. The yellow figure relates to retention of heterozygosity and outlines a region of LOH on the long arm of chromosome 5.
identified in 6% of cases of patients with MDS associated with del(5q) and in 5% of other subtypes of MDS, except in refractory anemia with ring sideroblasts with thrombocytosis, in which this mutation occurs in > 60% of patients. JAK2 mutation in del(5q) syndrome is associated with proliferative bone marrow.14-17 The presence of additional gene mutations can, therefore, result in a growth advantage to the cell, culminating in disease progression. The hypothesis is supported by 2 studies comparing the incidence of additional gene mutations in isolated del(5q) or del(5q) in the presence of a complex karyotype. Crescenzi et al performed interphase fluorescent in situ hybridization (FISH) on 30 cases of MDS and acute myeloid leukemia (AML) and found no additional deletions in all 19 cases of isolated del(5q), whereas abnormalities, including p53, AML1, and NF1 mutations, were identified in a high proportion of those with a complex karyotype.18 In a study by Fidler et al, no mutations of NRAS, TP53, or FLT3 were identified in cases of del(5q) syndrome.19 The absence of additional gene mutations in del(5q) syndrome results in stable disease with a favorable prognosis and low risk of leukemic transformation. Identifying novel candidate genes central to the pathogenesis of del(5q) syndrome remains a matter of much research. The uniform
30
nature of the disease and the consistently deleted segment on chromosome 5q pointed toward the loss of a key tumor suppressor gene from this region. However, as outlined later, studies to date have failed to identify a gene on 5q31-q33 that is responsible for the 5q phenotype. Haploinsufficiency is a state arising from the deletion of a gene on 1 allele, leading to a decrease of the gene product by 50%, which is subsequently incapable of providing normal function. Haploinsufficiency is thought to play a key role in the pathogenesis of MDS and might also provide a mechanism for good responses to lenalidomide.
More than 1 region on chromosome 5q is reported to be deleted in myeloid disorders with del(5q). The critically deleted segment in del(5q) syndrome is shown to flank band 5q32, whereas in other subtypes of MDS and AML, the deleted region localizes to band 5q31.20-26 Table 2 summarizes the reports of the critically deleted segments on chromosome 5q, and Figure 1 shows a diagram representing the regions deleted in AML and MDS compared with del(5q) syndrome. Early studies of the critically deleted segment in del(5q) syndrome identified a region of 5 megabase (Mb); however, a more recent report describes a region of 1.5 Mb flanked by the genes D5S413 and glycine receptor 1, both distinct and telomeric to the deleted regions identified in MDS and AML.24-26 Chromosome 5q harbors several genes encoding for growth factors, cytokines, transcriptional regulators, and tumor suppressor genes.27-31 Examples include c-FMS, colony-stimulating factor 2, the early growth response gene, and the interleukins (ILs) IL-3, IL-4, IL-5, and IL-9. Initial reports of the tumor suppressor gene interferon regulatory factor (IRF-1) stimulated interest as this gene mapped to 5q31.1 and was shown, in a study of MDS and AML, to be the gene consistently deleted.32 However, a study of IRF-1 in del(5q) syndrome showed no homozygous loss of the IRF-1 gene, and further studies demonstrated the genes for IRF-1, colony-stimulating factor 2, IL-3, IL-4, and IL-5, all in fact mapped outside the critically deleted segment for del(5q) syndrome (Figure 1).25,26 Other tumor suppressor genes mapped to the long arm of chromosome 5 include the mutation in colorectal cancer and familial adenomatous polyposis genes; however, neither have been shown to be deleted in del(5q) syndrome.33-35 The most recent report by Boultwood et al, in which the 1.5 Mb region was described, reports 40 genes that map within the commonly deleted region in del(5q) syndrome, of which 33 are shown to be expressed in CD34+ cells and, therefore, represent potential candidate genes.25 Genes of interest include the putative tumor suppressor genes protein 1 with multiple EGF-like domains and secreted protein acidic and richin cystein. Protein 1 with multiple EGF-like domains represents a member of the cadherin gene family responsible for cell adhesion, and secreted protein acidic and richin cystein is a protein involved in cell-matrix interactions; however, to date, no mutations have been identified in these genes.
Gene Expression Potential benefits of gene expression profiling (GEP) include the identification of novel candidate genes involved in the pathogenesis of MDS and providing unique profiles of different subtypes of MDS, thereby aiding the classification and prognosis of disease.
Clinical Leukemia • September 2006
Wendy Ingram, Ghulam J. Mufti The ability to distinguish between normal controls, low-risk and high-risk MDS, and AML on the basis of differences in patterns of gene expression of CD34+ cells is reported by 2 separate groups.36,37 Hofmann et al identified 11 genes that predicted the risk of the disease and propose that GEP could provide a useful tool in risk evaluation at the time of diagnosis.36 The use of GEP in del(5q) syndrome has been reported in 2 studies. Pellagatti et al analyzed 21 cases of MDS, 7 of which had a diagnosis of del(5q) syndrome.38 Gene expression profiling of the neutrophils using complementary DNA technology was performed, and the results of the 7 cases were compared, with 6 patients having refractory anemia and normal karyotype. A significant difference in the expression of 71 genes was identified, with a difference in expression profile of 5 genes. However, only 1 of the 5 genes, the ATX1 antioxidant protein 1 homologue gene, was mapped to the critically deleted segment of chromosome 5q. A further study by the same group recently described the results of GEP of CD34+ cells in 55 cases of MDS, 20 of which had del(5q) syndrome.39 Expression profiles obtained from the del(5q) syndrome cohort were quite distinct from other subtypes of MDS. Approximately 40% of genes found to be downregulated were mapped to chromosome 5q, most likely reflecting the loss of 1 allele and supporting the hypothesis of haploinsufficiency as the pivotal mechanism in the pathogenesis of the disease. Of particular interest was the overexpression of histone genes, genes encoding for proteins in the actin cytoskeleton, ARPC2, CORO1C, CAPZA2, as well as megakaryocyte and platelet genes PF4V1, PPBP, and CD61.39 In addition, interferon-stimulated genes, IFIT1 and IFITM1 were significantly upregulated in all cases of MDS, which might provide a potential diagnostic marker of MDS and might contribute to the clinical features of MDS, such as peripheral blood cytopenias. The abnormal expression of genes identified by GEP provides further insight into the molecular pathogenesis of MDS, in particular to disease evolution; however, to date, it has failed to identify a critical novel candidate gene or the mechanism behind the selective deletion in del(5q) syndrome. The introduction of a high resolution, genome-wide, array-based technology utilizing single-nucleotide polymorphisms (SNPs) has been applied to solid tumors and leukemias.40-47 The data generated, expressed as a change in copy number, is interpreted as a deletion of a region of the chromosome, loss of heterozygosity (LOH), or uniparental disomy. A report of 10K SNP analysis in AML demonstrated LOH as a result of uniparental disomy in 20% of cases.47 A further report by the same group identified 4 homozygous mutations in AML at distinct loci and hypotheses that the mutation precedes mitotic recombination, which subsequently acts as a second “hit” responsible for the removal of the wild-type allele.48 Single-nucleotide polymorphism analysis in MDS is currently being explored. The use of 500K SNP analysis in cases of MDS with normal cytogenetics is being analyzed by our group. Of interest is the ability to identify significant deletions, including del(5q), or regions of LOH that are not detected on routine cytogenetic analysis using G-banding technique (Figure 2).
Mechanism of Action of Lenalidomide Lenalidomide is an orally active thalidomide analogue belonging to the class of immunomodulatory drugs. The development
of immunomodulatory drugs has stemmed from previous reports describing clinical responses after the use of thalidomide in MDS. In particular, erythroid responses are observed with thalidomide, with up to a third of patients responding and transfusion independence seen in a minority of cases.49-52 The mechanism by which thalidomide exerts its activity is not well understood. Groups have analyzed a variety of factors, including levels of tumor necrosis factor–, basic fibroblast growth factor, vascular endothelial growth factor, and bone marrow apoptosis; however, studies have failed to clarify the precise mechanism of action.49,53 In addition, thalidomide is generally poorly tolerated in patients with MDS, hence the need for novel agents. Immunomodulatory drugs are a group of drugs with potent immunomodulatory and antiangiogenic properties that lack the teratogenic and neurotoxic side effects of thalidomide. Most of the preclinical studies of lenalidomide are reported in myeloma cell lines. In vitro evidence of decreased IL-6, IL-12, and tumor necrosis factor– production; enhanced T-cell activation through increased production of IL-2 and IFN- augmentation of natural killer cell cytotoxicity; activation of caspase 8–mediated apoptosis; and an inhibitory effect on growth factor–induced Akt phosphorylation highlight the wideranging mechanisms of action of these compounds.54-56 Of particular relevance to del(5q) syndrome is the recent literature on the in vitro activity of lenalidomide on tumor cell lines. Cell lines with chromosome 5 deletion, including T-cell, B-cell, and myeloid lineage, all showed inhibition with lenalidomide in vitro. A G0/G1–induced cell cycle arrest with inhibition of Akt and Grb2-associated binder 1 (Gab1) phosphorylation and prevention of the binding of Gab1 to its erythropoietin (Epo) receptor was observed with Namalwa CSN.70.57 Namalwa CSN.70, a Burkitt’s lymphoma cell line, was found to be the most sensitive cell line to lenalidomide. The use of a sensitive cell line potentially enables the mechanism of action of lenalidomide to be explored in more detail. No effect of lenalidomide on Gab1 in UT-7 cells was observed, whereas data by List et al report enhanced STAT phosphorylation and increased Epo sensitivity in the presence of lenalidomide in UT-7 cells.58 The differences in observations between the 2 studies do not show conflicting data, because Gab1 is not involved in Epoinduced STAT5 activation. Preliminary data presented by Boultwood et al demonstrate upregulation of the SPARC gene in erythroblasts after treatment with lenalidomide.59 The SPARC gene is known to be mapped to the commonly deleted region of chromosome 5q identified in del(5q) syndrome and might explain the enhanced STAT5 phosphorylation and Epo sensitivity seen with lenalidomide. An additional gene of interest is CDC25C, which localizes to chromosome 5q. Its role in relation to response to treatment with lenalidomide is currently being explored by List et al. In a further study by Jädersten et al published only in abstract form, the effect of lenalidomide on CD34+ cells from healthy donors was compared with patients with MDS with deletion 5q31.60 A selective inhibition of the del(5q) clone was observed, compared with no effect on normal CD34+ cells. The potential for selectivity of the abnormal clone by lenalidomide might provide valuable insight into the mechanism of action of the drug and further our knowledge of critical genes central to the pathogenesis of del(5q) syndrome.
Clinical Leukemia • September 2006
31
MDS and Deletion 5q Abnormality
Table 3
Summary of Trials of Lenalidomide in MDS del(5q)62,64 Study
List et al62 (MDS-001) List et al64 (MDS-003)
Number Erythroid Response (%) of Patients
Cytogenetic Response (%)
Complete Cytogenetic Response (%)
All cases
43
56
55 (11 of 20)
50 (10 of 20)
del(5q) Cases
12
83
83
75
All cases
148
64*
76
55
Isolated del(5q)
111
69*
Not applicable
Not applicable
Complex karyotype
37
49*
Not applicable
Not applicable
*Denotes percentage of cases with transfusion independence.
Clinical Trials of Lenalidomide in MDS Associated with del(5q) Hematologic improvement is reported in up to a third of patients who receive thalidomide.49-52 Dose increase is, however, limited by its toxicity. Lenalidomide is an amino-substituted thalidomide analogue that lacks teratogenicity in animal models and is associated with fewer side effects.61 In a pivotal study of lenalidomide in MDS reported by List et al, 3 dose regimens (10 mg per day, 25 mg per day, or 10 mg per day for 21 days repeated every 28 days) were analyzed in 43 patients, including 12 cases with deletion 5q31.1 (del[5q31.1]).62 Enrollment criteria included symptomatic anemia (hemogobin < 10 g/dL) or transfusion-dependent anemia ( 4 units of red blood cells within 8 weeks). The overall response rate was 56%, with 20 of 32 transfusion-dependent cases achieving transfusion independence. The erythroid response, assessed using the International Working Group criteria,63 in the del(5q) cohort was significantly better than other groups at 83% versus 57% in those with normal cytogenetics. In addition, a cytogenetic response was observed in 10 of 12 patients with del(5q31.1), with a complete cytogenetic response confirmed by interphase FISH in 5 of 12 patients. The drug was well tolerated; however, significant myelosuppression occurred at increased doses, requiring interruption in 77% of the cohort receiving 25 mg daily.62 A multicenter phase II study of lenalidomide was subsequently performed in 148 patients with MDS with del(5q31.1).64 Patients with low or intermediate-1 risk by IPSS, transfusion dependence (> 2 units in the past 8 weeks), and del(5q) with or without additional cytogenetic abnormalities were eligible. The drug was administered at 10 mg per day or 10 mg per day every 21 days. Preliminary data are available in abstract form. Of the 122 evaluable cases, 64% achieved transfusion independence, of which a cytogenetic response was noted in 76% and a complete cytogenetic response in 55%. Cytopenias were the most common adverse event recorded. The median response duration was not reached at a median follow up of 9.3 months. A summary of the clinical trials of lenalidomide is shown in Table 3.62,64 The studies of lenalidomide to date have shown it to be a highly effective compound in the treatment of MDS, in particular, when associated with the 5q abnormality. Reports of its use in the presence of a complex karyotype involving del(5q) also show promising results. The phase II study (MDS-003) outlined earlier, included 37 cases of complex karyotype, with transfusion independence seen in 49% of cases compared with 69% in isolated del(5q).64 A report of the use of
32
lenalidomide in a patient with a complex karyotype and < 5% bone marrow blasts is described by Giagounidis et al.65 The patient achieved complete cytogenetic remission at 6 months of treatment confirmed by interphase FISH, normalization of bone marrow megakaryocyte morphology, and transfusion independence. A 3-arm multicenter study of lenalidomide is currently under way in Europe, with randomization among 5 mg, 10 mg, or best supportive care. A survival advantage will, however, require long-term follow-up because of the prolonged survival in this cohort. In addition, quality of life studies will provide crucial information in evaluating the benefits of lenalidomide.
Conclusion To date, no consensus has been reached regarding potential candidate genes involved in the pathogenesis of del(5q) syndrome. It is clear that secondary hits are important in the progression of the disease; however, in the setting of del(5q) syndrome, haploinsufficiency might account for the characteristic clinical features recognized in this disease. In addition, in vitro studies of lenalidomide on tumor cell lines or bone marrow from patients with del(5q) syndrome could provide crucial information to key genes involved in the pathogenesis of the disease, while also furthering our knowledge of the mechanism of action of the drug. Of particular interest is the recent finding of the upregulation of the SPARC gene after treatment with lenalidomide. The use of gene expression profiling and SNP analysis has enabled the investigation of vast numbers of genes and might provide a useful tool in aiding the classification of MDS, predicting prognosis, and in particular through the analysis of changes in gene expression prelenalidomide and postlenalidomide therapy, could identify genes worthy of further study.
Acknowledgements We thank Alan List for his critical review of the manuscript and Natalie Twine for help in preparing the figures for illustration. Wendy Ingram is sponsored by the Leukaemia Research Fund.
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Refractory anemia with ringed sideroblasts associated with marked thrombocytosis (RARS-T), another myeloproliferative condition characterized by JAK2 V617F mutation. Blood 2006. [Epub ahead of print]. 17. Remacha AF, Nomdedeu JF, Puget G, et al. Occurrence of the JAK2 V617F mutation in the WHO provisional entity: myelodysplastic/myeloproliferative disease, unclassifiable-refractory anemia with ringed sideroblasts associated with marked thrombocytosis. Haematologica 2006; 91:719-720. 18. Crescenzi B, La Starza R, Romoli S, et al. Submicroscopic deletions in 5q- associated malignancies. Haematologica 2004; 89:281-285. 19. Fidler C, Watkins F, Bowen DT, et al. NRAS, FLT3 and TP53 mutations in patients with myelodysplastic syndrome and a del(5q). Haematologica 2004; 89:865-866. 20. Le Beau MM, Espinosa R III, Neuman WL, et al. Cytogenetic and molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases. Proc Natl Acad Sci U S A 1993; 90:5484-5488. 21. Zhao N, Stoffel A, Wang PW, et al. Molecular delineation of the smallest commonly deleted region of chromosome 5 in malignant myeloid diseases to 1-1.5 Mb and preparation of a PAC-based physical map. Proc Natl Acad Sci U S A 1997; 94:6948-6953. 22. Horrigan SK, Arbieva ZH, Xie HY, et al. Delineation of a minimal interval and identification of 9 candidates for a tumor suppressor gene in malignant myeloid disorders on 5q31. Blood 2000; 95:2372-2377. 23. Horrigan SK, Bartoloni L, Speer MC, et al. A radiation hybrid breakpoint map of the acute myeloid leukemia (AML) and limb-girdle muscular dystrophy 1A (LGMD1A) regions of chromosome 5q31 localizing 122 expressed sequences. Genomics 1999; 57:24-35. 24. Boultwood J, Fidler C, Lewis S, et al. Molecular mapping of uncharacteristically small 5q deletions in two patients with the 5q- syndrome: delineation of the critical region on 5q and identification of a 5q- breakpoint. Genomics 1994; 19:425-432. 25. Boultwood J, Fidler C, Strickson AJ, et al. Narrowing and genomic annotation of the commonly deleted region of the 5q- syndrome. Blood 2002; 99:4638-4641. 26. Jaju RJ, Boultwood J, Oliver FJ, et al. Molecular cytogenetic delineation of the critical deleted region in the 5q- syndrome. Genes Chromosomes Cancer 1998; 22:251-256. 27. Huebner K, Isobe M, Croce CM, et al. The human gene encoding GM-CSF is at 5q21-q32, the chromosome region deleted in the 5q- anomaly. Science 1985; 230:1282-1285. 28. Le Beau MM, Westbrook CA, Diaz MO, et al. Evidence for the involvement of GM-CSF and FMS in the deletion (5q) in myeloid disorders. Science 1986; 231:984-987. 29. Nienhuis AW, Bunn HF, Turner PH, et al. Expression of the human c-fms protooncogene in hematopoietic cells and its deletion in the 5q- syndrome. Cell 1985; 42:421-428. 30. Boultwood J, Rack K, Kelly S, et al. Loss of both CSF1R (FMS) alleles in patients with myelodysplasia and a chromosome 5 deletion. Proc Natl Acad Sci U S A 1991; 88:6176-6180. 31. van Leeuwen BH, Martinson ME, Webb GC, et al. Molecular organization of the cytokine gene cluster, involving the human IL-3, IL-4, IL-5, and GM-CSF genes, on human chromosome 5. Blood 1989; 73:1142-1148. 32. Willman CL, Sever CE, Pallavicini MG, et al. Deletion of IRF-1, mapping to chromosome 5q31.1, in human leukemia and preleukemic myelodysplasia. Science 1993; 259:968-971. 33. Nishisho I, Nakamura Y, Miyoshi Y, et al. Mutations of chromosome 5q21 genes in FAP and colorectal cancer patients. Science 1991; 253:665-669. 34. Joslyn G, Carlson M, Thliveris A, et al. Identification of deletion mutations and three new genes at the familial polyposis locus. Cell 1991; 66:601-613. 35. Kinzler KW, Nilbert MC, Vogelstein B, et al. Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers. Science 1991; 251:1366-1370. 36. Hofmann WK, de Vos S, Komor M, et al. Characterization of gene expression of CD34+ cells from normal and myelodysplastic bone marrow. Blood 2002;
100:3553-3560. 37. Miyazato A, Ueno S, Ohmine K, et al. Identification of myelodysplastic syndromespecific genes by DNA microarray analysis with purified hematopoietic stem cell fraction. Blood 2001; 98:422-427. 38. Pellagatti A, Esoof N, Watkins F, et al. Gene expression profiling in the myelodysplastic syndromes using cDNA microarray technology. Br J Haematol 2004; 125:576-583. 39. Pellagatti A, Cazzola M, Giagounidis AA, et al. Gene expression profiles of CD34+ cells in myelodysplastic syndromes: involvement of interferon stimulated genes and correlation to FAB subtype and karyotype. Blood 2006; 108:337-345. 40. Lieberfarb ME, Lin M, Lechpammer M, et al. Genome-wide loss of heterozygosity analysis from laser capture microdissected prostate cancer using single nucleotide polymorphic allele (SNP) arrays and a novel bioinformatics platform dChipSNP. Cancer Res 2003; 63:4781-4785. 41. Zhao X, Weir BA, LaFramboise T, et al. Homozygous deletions and chromosome amplifications in human lung carcinomas revealed by single nucleotide polymorphism array analysis. Cancer Res 2005; 65:5561-5570. 42. Janne PA, Li C, Zhao X, et al. High-resolution single-nucleotide polymorphism array and clustering analysis of loss of heterozygosity in human lung cancer cell lines. Oncogene 2004; 23:2716-2726. 43. Dumur CI, Dechsukhum C, Ware JL, et al. Genome-wide detection of LOH in prostate cancer using human SNP microarray technology. Genomics 2003; 81:260-269. 44. Shearer PD, Valentine MB, Grundy P, et al. Hemizygous deletions of chromosome band 16q24 in Wilms tumor: detection by fluorescence in situ hybridization. Cancer Genet Cytogenet 1999; 115:100-105. 45. Murthy SK, DiFrancesco LM, Ogilvie RT, et al. Loss of heterozygosity associated with uniparental disomy in breast carcinoma. Mod Pathol 2002; 15:1241-1250. 46. Henry I, Bonaiti-Pellie C, Chehensse V, et al. Uniparental paternal disomy in a genetic cancer-predisposing syndrome. Nature 1991; 351:665-667. 47. Raghavan M, Lillington DM, Skoulakis S, et al. Genome-wide single nucleotide polymorphism analysis reveals frequent partial uniparental disomy due to somatic recombination in acute myeloid leukemias. Cancer Res 2005; 65:375-378. 48. Fitzgibbon J, Smith LL, Raghavan M, et al. Association between acquired uniparental disomy and homozygous gene mutation in acute myeloid leukemias. Cancer Res 2005; 65:9152-9154. 49. Zorat F, Shetty V, Dutt D, et al. The clinical and biological effects of thalidomide in patients with myelodysplastic syndromes. Br J Haematol 2001; 115:881-894. 50. Musto P, Falcone A, Sanpaolo G, et al. Thalidomide abolishes transfusion-dependence in selected patients with myelodysplastic syndromes. Haematologica 2002; 87:884-886. 51. Strupp C, Germing U, Aivado M, et al. Thalidomide for the treatment of patients with myelodysplastic syndromes. Leukemia 2002; 16:1-6. 52. Raza A, Meyer P, Dutt D, et al. Thalidomide produces transfusion independence in long-standing refractory anemias of patients with myelodysplastic syndromes. Blood 2001; 98:958-965. 53. Invernizzi R, Travaglino E, De Amici M, et al. Thalidomide treatment reduces apoptosis levels in bone marrow cells from patients with myelodysplastic syndromes. Leuk Res 2005; 29:641-647. 54. Corral LG, Haslett PA, Muller GW, et al. Differential cytokine modulation and T cell activation by two distinct classes of thalidomide analogues that are potent inhibitors of TNF-alpha. J Immunol 1999; 163:380-386. 55. Davies FE, Raje N, Hideshima T, et al. Thalidomide and immunomodulatory derivatives augment natural killer cell cytotoxicity in multiple myeloma. Blood 2001; 98:210-216. 56. Dredge K, Horsfall R, Robinson SP, et al. Orally administered lenalidomide (CC5013) is anti-angiogenic in vivo and inhibits endothelial cell migration and Akt phosphorylation in vitro. Microvasc Res 2005; 69:56-63. 57. Gandhi AK, Kang J, Naziruddin S, et al. Lenalidomide inhibits proliferation of Namalwa CSN.70 cells and interferes with Gab1 phosphorylation and adaptor protein complex assembly. Leuk Res 2006; 30:849-858. 58. List AF, Dewald G, Bennett JM, et al. Hematologic and cytogenetic (CTG) response to lenalidomide (CC-5013) in patients with transfusion-dependent (TD) myelodysplastic syndrome (MDS) and chromosome 5q31.1 deletion: results of a multicenter MDS003 study. J Clin Oncol 2005; 23(16 suppl):2s (Abstract #5). 59. Boultwood J, Wainscoat J. Molecular pathogenesis of the myelodysplastic syndromes, including the 5q- syndrome. Hematology (European Hematology Association) 2006; 2:69-74. 60. Jadersten M, Pellagatti A, Forsblom A, et al. Lenalidomide selectively inhibits in vitro growth of the malignant clone in myelodysplastic syndrome (MDS) patients with 5q deletion. Blood 2005; 106:960a (Abstract #3438). 61. Bartlett JB, Dredge K, Dalgleish AG. The evolution of thalidomide and its IMiD derivatives as anticancer agents. Nat Rev Cancer 2004; 4:314-322. 62. List A, Kurtin S, Roe DJ, et al. Efficacy of lenalidomide in myelodysplastic syndromes. N Engl J Med 2005; 352:549-557. 63. Cheson BD, Bennett JM, Kantarjian H, et al. Report of an international working group to standardize response criteria for myelodysplastic syndromes. Blood 2000; 96:3671-3674. 64. List AF, Dewald G, Bennett JM, et al. Hematologic and cytogenetic (CTG) response to lenalidomide (CC-5013) in patients with transfusion-dependent (TD) myelodysplastic syndrome (MDS) and chromosome 5q31.1 deletion: results of the multicenter MDS-003 study. J Clin Oncol 2005; 23:2s (Abstract #5). 65. Giagounidis AA, Germing U, Strupp C, et al. Prognosis of patients with del(5q) MDS and complex karyotype and the possible role of lenalidomide in this patient subgroup. Ann Hematol 2005; 84:569-571.
Clinical Leukemia • September 2006
33
Introduction Karyotypic abnormalities are a common feature of myelodysplastic syndromes (MDSs) identified in 60% of cases of primary MDS.1,2 An interstitial deletion of the long arm of chromosome 5 is a relatively common cytogenetic abnormality. Between 10% and 15% of patients with MDS present with an isolated 5q deletion (del[5q]) or del(5q) in combination with additional karyotypic changes.1,3 The del(5q) syndrome is a unique subtype of MDS characterized by macrocytic anemia, normal to increased platelet count, hypolobated megakaryocytes, and an interstitial deletion of the long arm of chromosome 5 usually involving q31-q33.4 The clinical features of del(5q) syndrome were first described in the mid-1970s by Van den Berghe et al5,6; however, del(5q) syndrome was only classified as a distinct entity in the World Health Organization classification of MDS.4 The syndrome typically affects women of middle to older age and confers a favorable prognosis compared with other subtypes of MDS.7-10 Prognostic scoring systems have been devised in an attempt to identify factors that adversely affect outcome in MDS,11 the most widely used being the international prognostic scoring system (IPSS).7 According to the IPSS, the percentage of bone marrow blasts, the presence or absence of karyotypic abnormalities, and peripheral blood cytopenias are the 3 most significant factors affecting outcome. Good-risk cytogenetic features include a normal karyotype, -Y, del(5q), or del(20q). The prognosis of patients with low-, intermediate-, and high-risk MDS is outlined in Table 1. A cohort of 76 patients with MDS and del(5q) involving band q31 were analyzed by Giagounidis et al.8 A projected median survival of 146 months at a median follow-up of 67 months in cases of isolated del(5q) was reported. However, the presence of 1 additional cytogenetic abnormality or > 5% bone marrow blasts conferred a significantly worse outcome. Despite prolonged survival and a low risk of leukemic transformation, the disease is typically characterized by a protracted history of regular blood transfusions. The resulting iron overload culminates in organ damage and can lead Department of Haematological Medicine, King’s College London, London, UK Submitted: July 11, 2006; Revised: August 4, 2006; Accepted: August 7, 2006 Address for correspondence: Ghulam J. Mufti, DM, Department of Haematological Medicine, King’s College London, Rayne Institute, 123 Coldharbour Ln, London, SE5 9NU, United Kingdom Fax: 207-346-3514; e-mail: [email protected] Electronic forwarding or copying is a violation of US and International Copyright Laws. Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by CIG Media Group, LP, ISSN #1931-6925, provided the appropriate fee is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers, MA 01923 USA 978-750-8400.
28
Clinical Leukemia • September 2006
Table 1 IPSS
Prognosis of Patient with MDS According to International Prognostic Scoring System
Risk Category
Regions Deleted on Chromosome 5q in MDS/AML Compared with del(5q) Syndrome20-22,25,26,32
Median Risk of 25% of Cases Undergoing Survival Transformation to AML (Years) (Years) 9.4
5.7
0.5-1 Intermediate-1
3.3
3.5
1.5-2 Intermediate-2
1.1
1.2
2.5
0.2
0.4
Low
0
Figure 1
High
DNA Marker Gene IL-3 CSF2 IRF-1 IL-5 IL-13 IL-4
p
MDS/AML Le Beau et al20
CDC25C D5S500
Zhao et al21 Horrigan et al22
HSPA9 EGR1 D5S594 HDAC3
1 q31
D5S166
q
CD14 FGF1 ADR2
2
D5S413 CSF1R PDGFR
Pathogenesis of del(5q) Syndrome
Table 2
Willman et al32
D5S479 D5S414
3
The molecular events underlying the pathogenesis of MDS remain elusive. The multi-hit hypothesis of leukemia is supported by mouse models of leukemogenesis, which demonstrate the need for mutations in > 1 gene in order for disease progression to occur. A proposal by Gilliland et al suggests the mutations predisposing one to leukemogenesis might be broadly classified into those that confer a proliferative advantage to the hematopoietic stem cell and/ or those that result in blockage of differentiation.13 The latter of these mechanisms might explain, in part, the cytopenias associated with the del(5q) syndrome. Further gene mutations, an example of which is the JAK2 (V617F) mutation, might subsequently confer a proliferative advantage to the cell. The JAK2 somatic mutation is
MDS/AML
IL-9 TGF1 SPOCK
Chromosome 5
to significant morbidity. Improving quality of life has increasingly been highlighted as an important objective of therapeutic modalities in MDS and myeloid leukemia, particularly in the elderly.12 Maximizing hemoglobin levels and, hence, decreasing the need for transfusions is at the forefront of the management of del(5q) syndrome and low-risk MDS. This review summarizes the advances in the pathogenesis of del(5q) syndrome, key genes mapped to the critically deleted segment, and outlines the clinical trials performed with lenalidomide and MDS with del(5q).
Study Group
q34
del(5q) Syndrome
CD74
Jaju et al26
MEGF1 SPARC ATOX1
Boultwood et al25
GLR1
Diagrammatic representation of the regions deleted on chromosome 5q in MDS/AML compared with del(5q) syndrome. A summary of the DNA markers and genes of interest are shown. Abbreviations: ADR2 = adrenergic -2-, receptor, surface; ATOX1 = ATX1 antioxidant protein 1 homologue; CDC25C = cell division cycle 25C; CSF2 = colony-stimulating factor 2; CSF1R = colony-stimulating factor 1 receptor; EGR1 = early growth response 1; FGF1 = fibroblast growth factor 1; GLR1 = glycine receptor 1; HDAC3 = histone deacetylase 3; HSPA9 = human mortalin; MEGF1 = protein 1 with multiple EGF-like domains; PDGFR = platelet-derived growth factor receptor– polypeptide; SPARC = secreted protein acidic and richin cystein; SPOCK = sparc/osteonectin, cwcv and kazal-like domains proteoglycan; TGF1 = transforming growth factor–1
Summary of Reported Deleted Regions on Chromosome 5q31-3420-26 Disease Group Studied
Size of Deleted Region
Flanking Genes/ DNA Markers
Genes of Interest Mapped Within the Region
Le Beau et al20
MDS/AML
2.8 Mb
IL-9 D5S166*
CSF2, IL-3, IL-4, IL-5, IL-9
Zhao et al21
MDS/AML
1-1.5 Mb
D5S479* D5S500*
EGR1, CDC25C
AML
700 kb
D5S500* D5S594*
CDC25C, HSPA9, EGR1, CTNN1
Boultwood et al24
del(5q) Syndrome
5.6 Mb
FGFA NKSF1
GLR, ADR2, CSF1R, SPARC
Jaju et al26
del(5q) Syndrome
3 Mb
ADR2 IL-12b
CSF1R, SPARC, GR11
Boultwood et al25
del(5q) Syndrome
1.5 Mb
D5S413* GLR1
MEGF1, G3BP, SPARC, CSF1R
Study
Horrigan et al22
*Represent DNA markers. Abbreviations: ADR2 = adrenergic -2-, receptor, surface; CDC25C = cell division cycle 25C; CSF2 = colony-stimulating factor 2; CSF1R = colony-stimulating factor 1 receptor; CTNN1 = catenin (cadherin-associated protein), 1, 102 kDa; EGR1 = early growth response 1; FGFA = fibroblast growth factor acidic; G3BP = Ras-GAP SH3 domain binding protein; GLR = glycine receptor; GLR1 = glycine receptor 1; GR11 = glutamate receptor; HSPA9 = human mortalin; kb = kilobase; MEGF1 = protein 1 with multiple EGF-like domains; NKSF1 = natural killer cell stimulatory factor–1; SPARC = secreted protein acidic and richin cystein
Clinical Leukemia • September 2006
29
MDS and Deletion 5q Abnormality
Figure 2
Single-Nucleotide Polymorphisms in a Patient with Myelodysplastic Syndrome Copy Number
Loss of Heterozygosity
A B C D E
A B C D E
5p15.33 5p15.32 5p15.31 5p15.2 5p15.1 5p14.3 5p14.2 5p14.1
Deleted Segment on 5q
5p13.3 5p13.2 5p13.1 5p12 5p11 5q11.2 5q12.1 5q12.3 5q13.1 5q13.2 5q13.3 5q14.1 5q14.2 5q14.3 5q15
5q22.3 5q23.1 5q23.2 5q23.3 5q31.1
Deleted Region
5q21.1 5q21.2 5q21.3 5q22.1
5q31.2 5q31.3 5q32 5q33.1 5q33.2 5q33.3 5q34 5q35.1 5q35.2 5q35.3
Single-nucleotide polymorphism analysis identifying a large region of LOH on chromosome 5q in a patient with MDS with no history of 5q deletion. The copy number is shown in pink, highlighting a fall in copy number. The yellow figure relates to retention of heterozygosity and outlines a region of LOH on the long arm of chromosome 5.
identified in 6% of cases of patients with MDS associated with del(5q) and in 5% of other subtypes of MDS, except in refractory anemia with ring sideroblasts with thrombocytosis, in which this mutation occurs in > 60% of patients. JAK2 mutation in del(5q) syndrome is associated with proliferative bone marrow.14-17 The presence of additional gene mutations can, therefore, result in a growth advantage to the cell, culminating in disease progression. The hypothesis is supported by 2 studies comparing the incidence of additional gene mutations in isolated del(5q) or del(5q) in the presence of a complex karyotype. Crescenzi et al performed interphase fluorescent in situ hybridization (FISH) on 30 cases of MDS and acute myeloid leukemia (AML) and found no additional deletions in all 19 cases of isolated del(5q), whereas abnormalities, including p53, AML1, and NF1 mutations, were identified in a high proportion of those with a complex karyotype.18 In a study by Fidler et al, no mutations of NRAS, TP53, or FLT3 were identified in cases of del(5q) syndrome.19 The absence of additional gene mutations in del(5q) syndrome results in stable disease with a favorable prognosis and low risk of leukemic transformation. Identifying novel candidate genes central to the pathogenesis of del(5q) syndrome remains a matter of much research. The uniform
30
nature of the disease and the consistently deleted segment on chromosome 5q pointed toward the loss of a key tumor suppressor gene from this region. However, as outlined later, studies to date have failed to identify a gene on 5q31-q33 that is responsible for the 5q phenotype. Haploinsufficiency is a state arising from the deletion of a gene on 1 allele, leading to a decrease of the gene product by 50%, which is subsequently incapable of providing normal function. Haploinsufficiency is thought to play a key role in the pathogenesis of MDS and might also provide a mechanism for good responses to lenalidomide.
More than 1 region on chromosome 5q is reported to be deleted in myeloid disorders with del(5q). The critically deleted segment in del(5q) syndrome is shown to flank band 5q32, whereas in other subtypes of MDS and AML, the deleted region localizes to band 5q31.20-26 Table 2 summarizes the reports of the critically deleted segments on chromosome 5q, and Figure 1 shows a diagram representing the regions deleted in AML and MDS compared with del(5q) syndrome. Early studies of the critically deleted segment in del(5q) syndrome identified a region of 5 megabase (Mb); however, a more recent report describes a region of 1.5 Mb flanked by the genes D5S413 and glycine receptor 1, both distinct and telomeric to the deleted regions identified in MDS and AML.24-26 Chromosome 5q harbors several genes encoding for growth factors, cytokines, transcriptional regulators, and tumor suppressor genes.27-31 Examples include c-FMS, colony-stimulating factor 2, the early growth response gene, and the interleukins (ILs) IL-3, IL-4, IL-5, and IL-9. Initial reports of the tumor suppressor gene interferon regulatory factor (IRF-1) stimulated interest as this gene mapped to 5q31.1 and was shown, in a study of MDS and AML, to be the gene consistently deleted.32 However, a study of IRF-1 in del(5q) syndrome showed no homozygous loss of the IRF-1 gene, and further studies demonstrated the genes for IRF-1, colony-stimulating factor 2, IL-3, IL-4, and IL-5, all in fact mapped outside the critically deleted segment for del(5q) syndrome (Figure 1).25,26 Other tumor suppressor genes mapped to the long arm of chromosome 5 include the mutation in colorectal cancer and familial adenomatous polyposis genes; however, neither have been shown to be deleted in del(5q) syndrome.33-35 The most recent report by Boultwood et al, in which the 1.5 Mb region was described, reports 40 genes that map within the commonly deleted region in del(5q) syndrome, of which 33 are shown to be expressed in CD34+ cells and, therefore, represent potential candidate genes.25 Genes of interest include the putative tumor suppressor genes protein 1 with multiple EGF-like domains and secreted protein acidic and richin cystein. Protein 1 with multiple EGF-like domains represents a member of the cadherin gene family responsible for cell adhesion, and secreted protein acidic and richin cystein is a protein involved in cell-matrix interactions; however, to date, no mutations have been identified in these genes.
Gene Expression Potential benefits of gene expression profiling (GEP) include the identification of novel candidate genes involved in the pathogenesis of MDS and providing unique profiles of different subtypes of MDS, thereby aiding the classification and prognosis of disease.
Clinical Leukemia • September 2006
Wendy Ingram, Ghulam J. Mufti The ability to distinguish between normal controls, low-risk and high-risk MDS, and AML on the basis of differences in patterns of gene expression of CD34+ cells is reported by 2 separate groups.36,37 Hofmann et al identified 11 genes that predicted the risk of the disease and propose that GEP could provide a useful tool in risk evaluation at the time of diagnosis.36 The use of GEP in del(5q) syndrome has been reported in 2 studies. Pellagatti et al analyzed 21 cases of MDS, 7 of which had a diagnosis of del(5q) syndrome.38 Gene expression profiling of the neutrophils using complementary DNA technology was performed, and the results of the 7 cases were compared, with 6 patients having refractory anemia and normal karyotype. A significant difference in the expression of 71 genes was identified, with a difference in expression profile of 5 genes. However, only 1 of the 5 genes, the ATX1 antioxidant protein 1 homologue gene, was mapped to the critically deleted segment of chromosome 5q. A further study by the same group recently described the results of GEP of CD34+ cells in 55 cases of MDS, 20 of which had del(5q) syndrome.39 Expression profiles obtained from the del(5q) syndrome cohort were quite distinct from other subtypes of MDS. Approximately 40% of genes found to be downregulated were mapped to chromosome 5q, most likely reflecting the loss of 1 allele and supporting the hypothesis of haploinsufficiency as the pivotal mechanism in the pathogenesis of the disease. Of particular interest was the overexpression of histone genes, genes encoding for proteins in the actin cytoskeleton, ARPC2, CORO1C, CAPZA2, as well as megakaryocyte and platelet genes PF4V1, PPBP, and CD61.39 In addition, interferon-stimulated genes, IFIT1 and IFITM1 were significantly upregulated in all cases of MDS, which might provide a potential diagnostic marker of MDS and might contribute to the clinical features of MDS, such as peripheral blood cytopenias. The abnormal expression of genes identified by GEP provides further insight into the molecular pathogenesis of MDS, in particular to disease evolution; however, to date, it has failed to identify a critical novel candidate gene or the mechanism behind the selective deletion in del(5q) syndrome. The introduction of a high resolution, genome-wide, array-based technology utilizing single-nucleotide polymorphisms (SNPs) has been applied to solid tumors and leukemias.40-47 The data generated, expressed as a change in copy number, is interpreted as a deletion of a region of the chromosome, loss of heterozygosity (LOH), or uniparental disomy. A report of 10K SNP analysis in AML demonstrated LOH as a result of uniparental disomy in 20% of cases.47 A further report by the same group identified 4 homozygous mutations in AML at distinct loci and hypotheses that the mutation precedes mitotic recombination, which subsequently acts as a second “hit” responsible for the removal of the wild-type allele.48 Single-nucleotide polymorphism analysis in MDS is currently being explored. The use of 500K SNP analysis in cases of MDS with normal cytogenetics is being analyzed by our group. Of interest is the ability to identify significant deletions, including del(5q), or regions of LOH that are not detected on routine cytogenetic analysis using G-banding technique (Figure 2).
Mechanism of Action of Lenalidomide Lenalidomide is an orally active thalidomide analogue belonging to the class of immunomodulatory drugs. The development
of immunomodulatory drugs has stemmed from previous reports describing clinical responses after the use of thalidomide in MDS. In particular, erythroid responses are observed with thalidomide, with up to a third of patients responding and transfusion independence seen in a minority of cases.49-52 The mechanism by which thalidomide exerts its activity is not well understood. Groups have analyzed a variety of factors, including levels of tumor necrosis factor–, basic fibroblast growth factor, vascular endothelial growth factor, and bone marrow apoptosis; however, studies have failed to clarify the precise mechanism of action.49,53 In addition, thalidomide is generally poorly tolerated in patients with MDS, hence the need for novel agents. Immunomodulatory drugs are a group of drugs with potent immunomodulatory and antiangiogenic properties that lack the teratogenic and neurotoxic side effects of thalidomide. Most of the preclinical studies of lenalidomide are reported in myeloma cell lines. In vitro evidence of decreased IL-6, IL-12, and tumor necrosis factor– production; enhanced T-cell activation through increased production of IL-2 and IFN- augmentation of natural killer cell cytotoxicity; activation of caspase 8–mediated apoptosis; and an inhibitory effect on growth factor–induced Akt phosphorylation highlight the wideranging mechanisms of action of these compounds.54-56 Of particular relevance to del(5q) syndrome is the recent literature on the in vitro activity of lenalidomide on tumor cell lines. Cell lines with chromosome 5 deletion, including T-cell, B-cell, and myeloid lineage, all showed inhibition with lenalidomide in vitro. A G0/G1–induced cell cycle arrest with inhibition of Akt and Grb2-associated binder 1 (Gab1) phosphorylation and prevention of the binding of Gab1 to its erythropoietin (Epo) receptor was observed with Namalwa CSN.70.57 Namalwa CSN.70, a Burkitt’s lymphoma cell line, was found to be the most sensitive cell line to lenalidomide. The use of a sensitive cell line potentially enables the mechanism of action of lenalidomide to be explored in more detail. No effect of lenalidomide on Gab1 in UT-7 cells was observed, whereas data by List et al report enhanced STAT phosphorylation and increased Epo sensitivity in the presence of lenalidomide in UT-7 cells.58 The differences in observations between the 2 studies do not show conflicting data, because Gab1 is not involved in Epoinduced STAT5 activation. Preliminary data presented by Boultwood et al demonstrate upregulation of the SPARC gene in erythroblasts after treatment with lenalidomide.59 The SPARC gene is known to be mapped to the commonly deleted region of chromosome 5q identified in del(5q) syndrome and might explain the enhanced STAT5 phosphorylation and Epo sensitivity seen with lenalidomide. An additional gene of interest is CDC25C, which localizes to chromosome 5q. Its role in relation to response to treatment with lenalidomide is currently being explored by List et al. In a further study by Jädersten et al published only in abstract form, the effect of lenalidomide on CD34+ cells from healthy donors was compared with patients with MDS with deletion 5q31.60 A selective inhibition of the del(5q) clone was observed, compared with no effect on normal CD34+ cells. The potential for selectivity of the abnormal clone by lenalidomide might provide valuable insight into the mechanism of action of the drug and further our knowledge of critical genes central to the pathogenesis of del(5q) syndrome.
Clinical Leukemia • September 2006
31
MDS and Deletion 5q Abnormality
Table 3
Summary of Trials of Lenalidomide in MDS del(5q)62,64 Study
List et al62 (MDS-001) List et al64 (MDS-003)
Number Erythroid Response (%) of Patients
Cytogenetic Response (%)
Complete Cytogenetic Response (%)
All cases
43
56
55 (11 of 20)
50 (10 of 20)
del(5q) Cases
12
83
83
75
All cases
148
64*
76
55
Isolated del(5q)
111
69*
Not applicable
Not applicable
Complex karyotype
37
49*
Not applicable
Not applicable
*Denotes percentage of cases with transfusion independence.
Clinical Trials of Lenalidomide in MDS Associated with del(5q) Hematologic improvement is reported in up to a third of patients who receive thalidomide.49-52 Dose increase is, however, limited by its toxicity. Lenalidomide is an amino-substituted thalidomide analogue that lacks teratogenicity in animal models and is associated with fewer side effects.61 In a pivotal study of lenalidomide in MDS reported by List et al, 3 dose regimens (10 mg per day, 25 mg per day, or 10 mg per day for 21 days repeated every 28 days) were analyzed in 43 patients, including 12 cases with deletion 5q31.1 (del[5q31.1]).62 Enrollment criteria included symptomatic anemia (hemogobin < 10 g/dL) or transfusion-dependent anemia ( 4 units of red blood cells within 8 weeks). The overall response rate was 56%, with 20 of 32 transfusion-dependent cases achieving transfusion independence. The erythroid response, assessed using the International Working Group criteria,63 in the del(5q) cohort was significantly better than other groups at 83% versus 57% in those with normal cytogenetics. In addition, a cytogenetic response was observed in 10 of 12 patients with del(5q31.1), with a complete cytogenetic response confirmed by interphase FISH in 5 of 12 patients. The drug was well tolerated; however, significant myelosuppression occurred at increased doses, requiring interruption in 77% of the cohort receiving 25 mg daily.62 A multicenter phase II study of lenalidomide was subsequently performed in 148 patients with MDS with del(5q31.1).64 Patients with low or intermediate-1 risk by IPSS, transfusion dependence (> 2 units in the past 8 weeks), and del(5q) with or without additional cytogenetic abnormalities were eligible. The drug was administered at 10 mg per day or 10 mg per day every 21 days. Preliminary data are available in abstract form. Of the 122 evaluable cases, 64% achieved transfusion independence, of which a cytogenetic response was noted in 76% and a complete cytogenetic response in 55%. Cytopenias were the most common adverse event recorded. The median response duration was not reached at a median follow up of 9.3 months. A summary of the clinical trials of lenalidomide is shown in Table 3.62,64 The studies of lenalidomide to date have shown it to be a highly effective compound in the treatment of MDS, in particular, when associated with the 5q abnormality. Reports of its use in the presence of a complex karyotype involving del(5q) also show promising results. The phase II study (MDS-003) outlined earlier, included 37 cases of complex karyotype, with transfusion independence seen in 49% of cases compared with 69% in isolated del(5q).64 A report of the use of
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lenalidomide in a patient with a complex karyotype and < 5% bone marrow blasts is described by Giagounidis et al.65 The patient achieved complete cytogenetic remission at 6 months of treatment confirmed by interphase FISH, normalization of bone marrow megakaryocyte morphology, and transfusion independence. A 3-arm multicenter study of lenalidomide is currently under way in Europe, with randomization among 5 mg, 10 mg, or best supportive care. A survival advantage will, however, require long-term follow-up because of the prolonged survival in this cohort. In addition, quality of life studies will provide crucial information in evaluating the benefits of lenalidomide.
Conclusion To date, no consensus has been reached regarding potential candidate genes involved in the pathogenesis of del(5q) syndrome. It is clear that secondary hits are important in the progression of the disease; however, in the setting of del(5q) syndrome, haploinsufficiency might account for the characteristic clinical features recognized in this disease. In addition, in vitro studies of lenalidomide on tumor cell lines or bone marrow from patients with del(5q) syndrome could provide crucial information to key genes involved in the pathogenesis of the disease, while also furthering our knowledge of the mechanism of action of the drug. Of particular interest is the recent finding of the upregulation of the SPARC gene after treatment with lenalidomide. The use of gene expression profiling and SNP analysis has enabled the investigation of vast numbers of genes and might provide a useful tool in aiding the classification of MDS, predicting prognosis, and in particular through the analysis of changes in gene expression prelenalidomide and postlenalidomide therapy, could identify genes worthy of further study.
Acknowledgements We thank Alan List for his critical review of the manuscript and Natalie Twine for help in preparing the figures for illustration. Wendy Ingram is sponsored by the Leukaemia Research Fund.
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