`0 2000 "Burial Pubishers Ltd Al rights reserved 08876924/00 $151!)
`www.nature.com/ieu
`
`REVIEW
`
`A hypothesis for the pathogenesis of myelodysplastic syndromes: implications for
`new therapies
`C RosenfeldI and A List2
`
`'Texas Oncology, PA, Dallas, TX; and 2University of Arizona Cancer Center, Tucson, AZ, USA
`
`MDS based on cell culture, cytokine, molecular and clinical
`research is presented (Figure 1).
`
`Early events in evolution of MDS
`
`Three large (>150 index cases) epidemiologic studies suggest
`that radiation, smoking and occupational exposure to pesti—
`cides, organic chemicals and heavy metals are risk factors for
`the development of MDS."”2 Prevention of MDS will require
`
`
`
`Progenitor
`Cell
`Injury
`
`
`To guide development of new clinical strategies, a review of
`recent investigations in the pathobiology of MDS was perfor-
`med. Articles were identified through a Medline search. Stud-
`ies, including reviews, are cited in the references. A multistep
`pathogenesis is proposed. (1) Targeted injury or mutation
`within hemopoietic stem cells may be followed by an immuno-
`logic response adversely affecting progenitor survival.
`(2)
`Aocelerated proliferation and premature death of marrow cells
`is amplified by apoptogenic cytokines (INF-a, Fas ligand). (3)
`Establishment of an abnormal clone associated with telomere
`shortening. (4) Disease progression associated with loss of
`tumor suppressor activity. Opportunities for therapeutic inter-
`ventions are possible at each step. Comparisons between the
`proposed pathogenesis of MDS and severe aplastic anemia
`(SAA) are also presented. Leukemia (2000) 14, 2—8.
`Keywords: myelodysplastic syndrome; acute noniymphocytic leu—
`kemia; refractory anemia; preleukemia
`
`Introduction
`
`Three decades of investigations into the pathophysiology of
`the myelodysplastic syndromes (MDS) have confirmed the
`heterogenicity of MDS and highlighted the complexity in dis—
`ease biology.I Recent advances in technology have yielded
`provocative observations. The objective of this review is to
`integrate clinical and laboratory findings into a working
`hypothesis for the development of idiopathic MDS, differen—
`tiate idiopathic MDS from SM, and suggest new thera-
`peutic strategies.
`Interpretation of data from MDS studies remains problem-
`atic. Without a reliable disease marker, there can be questions
`regarding the accuracy of an MDS diagnosis.z Additional
`problems arise when patients with disparate biologies are
`compared. For example, patients with idiopathic MDS and
`therapy-related MDS are sometimes included in the same data
`analyses. The same is true for FAB morphologic type and cyto-
`genetics. A potential source of ambiguity in laboratory studies
`derives from the mixture of normal and malignant progenitor
`cells which are known to coexist.3 The low number of poly—
`clonal progenitor cells in most cases suggests that such studies
`are valid. However, patients
`in
`chemotherapy—induced
`remission may re-establish polyclonal hemopoiesis.”
`Any hypothesis for the pathogenesis of MDS must support
`some long-standing clinical observations. Why are cytopenias
`present with hypercellular marrows? Why does MDS evolve
`more slowly than AML? Why is idiopathic MDS predomi—
`nantly a disease of the elderly? Why does MDS sometimes
`respond to therapies for 5AA? Proposals for the pathogenesis
`of MDS have been suggested previously."‘9 A specific multi-
`step sequence for the development of adult-onset idiopathic
`
`Correspondence: C Rosenfeld, Texas Oncology, PA, 7777 Forest
`Lane, Building D 400, Dallas, Texas 75230, USA; Fax: 972—566—5819
`Received 28 June 1999; accepted 2 September 1999
`
`T M-CSF. mutation c-tms
`
`i G-CSF a
`l GM-CSF
`Production
`
`
`
`
`
`Cytotoxic
`Lymphocyte:
`
`T Marrow
`Macrophages
`
`
`
`
`
`
`
`T TNFq, TFas ligand
`
`1 C-Myc. ‘v Bel-2
`
`Apoplosie of
`Marrow Cello
`
`Tolomere
`Shortening
`
`Genomic Instability
`
`
`
`
`
`J. Differentiation
`
`itypermethylation
`0' p15'"'“‘
`
`Mutation p53
`
`1 Tumor
`
`Suppressor
`Activity
`
`
`
`
`Advanced
`M08
`
`A proposed multistep sequence for the development of
`Figure 1
`idiopathic myelodysplastic syndrome. M-CSF, macrophage colony-
`stimulating factor; GM-CSF, granulocyte—macrophage colony—stimul—
`ating factor; G-CSF, granulocyte colony-stimulating factor; ATG, anti-
`thymocyte globulin; TNF, tumor necrosis factor.
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`further delineation of disease-associated toxins and possible
`polymorphisms in toxin metabolism that may predispose to
`a higher risk of MDS.” Clearly, additional epidemiologic
`studies are needed.
`
`is that progenitor cells damaged by toxin
`One proposal
`exposure or spontaneous mutation evoke an immunologic
`response that further compromises progenitor cell growth and
`maturation. What is the evidence for the existence of an
`
`aberrant immunologic response when over 25 years of studies
`indicate a diminished immune state in MDS?" Incubation of
`
`marrow cells with q'closporine or removal of T cells enhance
`colony formation in some patients.'5"° Studies reported by
`Molldrem and colleagues" at the NIH indicate that sup-
`pression of CFU-GM may be mediated by CDB“ cells directed
`against MHC class I restricted antigens. The anti-CFU-GM
`response does not appear to be mediated by MDS-derived
`immune cells since, most, but not all, studies indicate that T
`cells are not clonal in MDS.‘7‘22 Clinical observations also
`
`support the notion of immune suppression of progenitor cell
`growth in MDS. Treatment with anti-thymocyte globulin or
`cyclosporine
`can
`improve
`cytopenias
`in
`select MDS
`patients.”‘26 In contrast, attempts to augment an immunologic
`response with roquinimex or IL-2 have met with very limited
`suceess indicating that the diminished immune response may
`be the result rather than the cause of MDS.”:28
`
`Non-clonal lymphopoiesis provides indirect evidence for a
`lack of stem cell involvement in MDS. Using precursors sorted
`by flow cytometry and subsequent FISH to define clonal hem—
`opoiesis, primitive progenitors (CD34‘, ThyI‘) lacked the
`cytogenetic marker whereas more committed progenitors
`(CD34‘, CD33‘) display a clonal chromosome abnormality.29
`Conceivably, these non-clonal primitive stem cells could be
`utilized as a stem cell graft for autologous transplantation.
`The growth and differentiation of the progeny of clonal pro-
`genitors is further compromised by an accelerated rate of
`apoptotic cell death. In cell culture, cytokines such as TNF—a
`and IFN-y can suppress the growth of hemopoietic progenitors
`and induce Fas expression on CD34 cells."HZ What is the
`evidence for a functional role of apoptogenic cytokines in
`MDS? Elevated serum levels of TNF-a in patients with MDS
`is well documented (see Table ".3335 Increased TNF-a pro-
`duction by blood mononuclear cells in one study was restric-
`ted to patients with RA and RARS, but not RAEB or RAEBtP"
`
`Tablet
`
`Cytokine levels in MDS compared to normal controls
`
`Patlrogmdsofflc
`Clloserleldardllljst
`
`Furthermore, overexpression of TNF-a mRNA from marrow
`was detected in most cases of MDS, but not in normal controls
`or AML patients.” One probable source of TNF—a overpro-
`duction is marrow macrophages which are increased in
`MDS.”39 The increased density of marrow macrophages may
`occur in response to elevated semm levels of M-CSF.” Point
`mutations in c—fms, which encodes the M—CSF receptor, may
`also promote macrophage development in some cases.“ The
`physiological significance of TNF-a in MDS is supported by
`several lines of investigation: (1) enhanced in vitro formation
`of CFU—GM by antibody neutralization of TNF-a in MDS but
`no effect on AML CFU;‘2 (2)
`inverse correlation between
`serum TNF-a concentration and hemoglobin in one study;34
`(3) inverse correlation between clinical response to erythro-
`poietin and TNF—a levels;35 (4) inverse correlation between a
`platelet response to lL—3 therapy and TNF—a serum levels;‘3
`(5) positive correlation between TNF-a producing cells in the
`marrow and apoptosis;“ and (6) correlation between plasma
`TNF—a concentration with nucleotide oxidation in marrow
`MDS CD34+ cells.45
`
`This model suggests that secretion of TNF-a or other pro-
`apoptotic (ytokines plays a pivotal role in the ineffective hem-
`atopoiesis of MDS, but the relationship to disease progression
`remains ill defined. This implies that strategies which effec—
`tively neutralize TNF may inprove hematopoiesis. Pentoxifyl-
`line at micromolar concentrations suppresses TNF—a mRNA
`transcription. Combination therapy with pentoxifylline + cip-
`rofloxacin yielded no hematologic benefit in one study but a
`triple drug regimen of pentoxifylline + ciprofloxacin +
`dexamethasone produced hemopoietic responses in 35%
`(18/51) of patients and 28% (5/18) of responders demonstrated
`a cytogenetic response.“"7 An alternative approach is to neu—
`tralize circulating TNF-a by administration of soluble TNF
`receptors. In vitro, incubation of MDS marrow with TNFRch
`enhanced CFU-GM formation.42 Strategies which reduce the
`impact of multiple soluble mediators of progenitor cell
`apoptosis or increase the threshold for apoptosis induction
`may be more effective than single agent therapy. For instance,
`interruption of progenitor cell apoptosis could be attempted
`with soluble TNF receptor
`(to inhibit
`the initiation of
`apoptosis) plus
`amifostine
`(to raise the threshold for
`apoptosis). Another possibility for combined therapy is simul-
`taneous inactivation of more than one inducer of apoptosis.
`
`Colony—stimulating factors
`
`Pro—apoptotic factors
`
`Serum
`
`Blood cells
`
`Marrow cells
`
`Serum
`
`Blood cells
`
`Marrow oells
`
`F“
`175, U“
`[‘0
`13‘, U76
`[34
`175-93
`
`1‘”
`
`179
`
`136‘
`
`U5'
`180-82. U5‘, N39
`
`15‘, N“0
`
`led
`
`G-CSF
`GM—CSF
`M-CSF
`IL-3
`ILB
`SCF
`BPA
`FLT3 ligand
`TNF-l!
`Fas ligand
`|L_1 B
`TGF—fi
`IFN-y
`
`133—35’ 4'42-
`
`'35
`
`IS!
`
`13'
`
`ISIN' N8082- U51
`P2. 15"1
`133.51
`13"- N5‘
`13’. U‘9
`
`I, increased; 1. decreased; N, not different from nomial controls; U, undetectable; “increased predominantly in patients with refractory
`anemia; *Ievels higher in RAEBIRAEBt than in RAIRARS.
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`PWMDS
`CRosenteIdandAList
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`One potential approach includes simultaneous blockade of
`the activity of Fas ligand and TNF-a.“
`lFN—y or IL—IB are apoptogenic cytokines that could con-
`tribute to ineffective hematopoiesis in MDS. In two series,
`IFN-y gene overexpression was detected in only 5/12 and
`0/11 MDS cases?” Increased production of IL] B by blood
`and marrow cells has been reported.3"'5°'51 Increased pro—
`duction of IL—IB by cultured marrow mononuclear cells was
`detected in 13/32 MDS patients.50 Patients with RA tended to
`have the highest IL-1 B production.3°'5° lL-1 B levels have been
`correlated with the extent of apoptosis, but not proliferation.so
`Deficient production of the lL-1 B receptor antagonist by MDS
`stromal cells may give rise to unopposed apoptotic activity
`from lL-1 B.52 These studies suggest that IFN-y and IL-1B do
`not contribute to apoptosis in the majority of patients with
`MDS.
`
`Several investigators have reported increased marrow cell
`apoptosis in MDS and have implicated the potential role of
`the Fas/Fas ligand system.53‘5° Increased Fas expression was
`detected on marrow CD34 cells from MDS patients.‘2'55'5°
`Lack of correlation between Fas expression and apoptosis sug-
`gests that multiple mediators of cell death are operational.55
`Gupta and coinvestigatorss9 have examined Fas
`ligand
`expression in marrows of MDS patients. The mean percentage
`of Fas ligand expressing cell was higher in MDS (17%) than
`from normal controls (6%). In contrast to normals where Fas
`ligand was detected mostly in lymphocytes, Fas ligand was
`expressed in erythroblasts, myeloblasts, megakaryocytes, mat—
`uring myeloid cells and dysplastic cells. In another study, Fas
`ligand expression in MDS patients was localized to marrow
`macrophages."0 Further
`investigations revealed more Fas
`ligand positive marrow cells in RA/RARS (9%)
`than in
`RAEB/RAEBt (20%).59 However, this appears to be inconsist-
`ent with the finding that the extent of marrow cell apoptosis
`inversely correlates with clinical stage of MDS. A higher
`degree of apoptosis was seen in early FAB classes for both
`CD34“ cells or marrow aspirates in most studies.‘*56 Further-
`more, as MDS clinically progresses, apoptotic signals decrease
`(Fas antigen, c—Myc oncoprotein) whereas anti-apoptotic sig—
`nals increase (bcI—2 oncoprotein).“'55-°' Fas—associated phos-
`phatase-1 (fap-I) is a negative regulator of fas. Recent studies
`have shown that fap-I expression is reduced in MDS marrow
`cells compared to marrow cells from either normals or AML
`than has progressed from MDS.62 Several investigators have
`suggested that the clinical sequelae of apoptosis is cytopenia.
`Since MDS is usually detected by cytopenia and apoptotic
`activity is most pronounced in the early phases of MDS, it is
`possible that apoptosis precedes the clinical recognition of
`MDS.
`
`Amifostine is a phosphorylated aminothiol with dual bio-
`logic activities including free radical scavenging, by addition
`of reducing equivalents, and inhibition of TNF—a and other
`inflammatory cytokine elaboration.63 In this way, amifostine
`may protect against TNF-induced apoptosis in MDS.43 In one
`trial, amifostine responses were noted in 83% (15/18)
`patients.“ In another study with 12 patients, none satisfied
`the criteria for a partial or complete response."5 Theoretically,
`amifostine activity should be more pronounced in early, rather
`than,
`late MDS. However, the potential application of this
`agent
`in MDS awaits further
`investigation.
`Inhibition of
`apoptosis by agents that decrease the c-Myc/Bcl-Z ratio could
`also be attempted. There are candidate agents to decrease
`c—Myc
`(Vitamin D3, agents that potentiate intracellular
`cAMPcontent and antisence oligonucleotide)“
`Extensive cell culture studies have been performed to inves-
`
`.b
`
`Leukemia
`
`tigate the cause(s) of cytopenia in established MDS. Numerous
`studies indicate deficient growth of myeloid, erythroid and
`megakaryocytic colonies."9 Similar to AML, blast cell colonies
`(CFU-L) can be detected in MDS.7o One cause for cytopenias
`may be related to diminished capacity for differentiation."
`Therapeutic trials of differentiating agents have not demon-
`strated consistent efficacy in ameliorating cytopenian"4
`Interpretation of the results from investigations of colony—
`stimulating factor levels in MDS patients is difficult to discern.
`Results appear inconsistent (Table 1) and CSF levels may be
`altered by clinical events (ie infections). Furthemrore, in most
`studies, serum CSF levels did not correlate with clinical para—
`meters.3"'75v'"6 Given these limitations, the decreased cellular
`production of G-CSF, GM-CSF and burst-promoting activity
`(BPA) plus low serum levels of SCF suggest that low CSF levels
`may be related to cytopenias and provide a basis for CSF ther—
`apy of MDS. Diminished elaboration of CSF may arise from
`apoptosis and functional abnormalities of stromal cells.“7775
`Studies indicate decreased production of GM—CSF and G-CSF
`by MDS monocytes.79'°° The use of colony—stimulating factors
`for the treatment of MDS is beyond the scope of this paper.m
`
`MDS disease progression
`
`The next proposed step is reduction of telomere length. Tel-
`omeres are located at the ends of eukaryotic chromosomes,
`and function to stabilize chromosomes. Telomere length is
`progressively reduced by cell divisions. Accelerated apoptosis
`and proliferation may lead to the reduction in telomere length
`observed in MDS.53'“'°7 Reduction in telomere length is prob-
`ably not directly responsible for disease progression since only
`42% of patients with RA have telomere length reduction.”
`Instead, telomere shortening may give rise to genomic insta-
`bility that leads to cytogenetic evolution and disease pro-
`gression. This assertion is supported by the finding that telo-
`mere
`shortening
`is
`associated with
`advanced MDS,
`cytogenetic abnormalities, percentage of marrow blasts, leu-
`kemic transformation and poor prognosis?”7 Since telomer-
`ase activity (a DNA polymerase that can synthesize the telo-
`meric sequence) is normal to low in most patients, telomere
`length may be a better reflection of the pathophysiology in
`MDS.“ Progenitor cells with shortened telomeres may be
`more susceptible to elimination by telomerase inhibitors.
`Telomerase inhibitors are currently in development.”
`Progression to advanced MDS and AML has been linked to
`inactivation of the tumor suppressor genes, p1 5'"'“" and to a
`lesser extent, p53, and thereby contribute to clonal expansion
`(see Figure 1). p53 is a tumor suppressor gene which serves
`as a major control of the GI checkpoint.” Studies indicate that
`mutations of p53 occur in less than 20% of MDS case's."‘”’3 At
`the molecular level, p53 mutations in MDS are usually point
`or missense mutations of one allele, associated in some cases
`with 17 p deletion of the alternante allele.94 Evidence that sug-
`gests mutation of p53 is related to progression of MDS
`includes the limitation to advanced MDS (RAEB, RAEBt),
`association with complex cytogenetic abnormalities, and risk
`of secondary leukemia."‘*9' In view of the low frequency of
`mutations, introduction of wild~type p53 into MDS cells by
`gene therapy offers limited therapeutic potential.” Bishop and
`coinvestigators‘” investigated whether a rebound in p53 after
`brief inactivation of p53 RNA by antisense oligonucleotide
`could inhibit clonal proliferation.
`In a phase I
`trial
`that
`included 10 high risk MDS patients (three RAEB, seven RAEB-
`t), there was one transient response detected after intravenous
`infusion OL(1)p 53.
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`As noted earlier, Fas expression decreases as the blast per-
`centage increases.55 The inhibitory effect of TGFB on leu-
`kemic colony formation in early MDS is diminished after
`transformation to AML.97 Transcription of p15'"'“", a cyclin—
`dependent kinase inhibitor represents one mechanism by
`which TGFB exerts its inhibitory effect. Hypermethylation of
`the p 15""K‘b gene occurs in 38—50% of MDS patients and
`may contribute to loss of proliferative regulation.”'99 Evidence
`implicating a causal role for inactivation of p 15'”'“" with dis-
`ease progression is provided by the increased frequency of
`hyperrnethylation observed in advanced MDS and secondary
`AML compared to early MDS. Agents which promote hypo-
`methylation of DNA may impact the evolution of the leukemic
`clone by derepression of p 15 transcription.'°° 5-Aza-2’-
`deoxycytidine and 5-azacytidine promote DNA hypomethyl-
`ation by inhibition of DNA methyltransferase.‘°' However,
`most of the activity of 5-azacytidine, a drug demonstrated to
`have clinical efficacy, is through mechanisms other than DNA
`demethylation.'°"I02 Incubation of cell
`lines harboring
`methylation silenced p 15 with 5-Aza-2’-deoxycytidine leads
`to re-expression of p15.103 In one study, a 72 h infusion of
`5-Aza-2’-deoxycytidine to 29 high risk MDS patients yielded
`a 29% complete response rate and 24% partial responses.'°‘
`Prolonged myelosuppression was the sole cause for a 17%
`drug-related mortality. Using a lower dose of 5-Aza-2'-deoxy-
`cytidine in 61 patients produced similar clinical effects with-
`out marked myelosuppression.”5 Additional studies with 5-
`Aza—2'—deoxycytidine should be performed.
`
`Clinical relevance of model
`
`How does this model account for MDS being a disease of
`the elderly? Accumulated environmental exposures provide a
`cumulative probability of mutational events that
`increases
`with time. Age-related telomere shortening fosters heightened
`susceptibility to genomic instability that can lead to emerg-
`ence of clonal disease. A similar explanation can account for
`the differences between MDS and SAA. The younger age of
`SAA patients may reflect exposure to a progenitor cell toxin
`or a genetic event in a patient not susceptible to genomic
`instability from age-related telomere loss. This proposal is sup—
`ported by the finding of shorter telomere lengths in MDS than
`SAA.'°" Another difference between MDS and SAA may be
`the milieu of hemopoietic inhibitory cytokines that drive
`apoptosis. In SAA, lFN—y gene overexpression was detectable
`in most patients.‘9"°7 As described earlier, lFN—y mRNA was
`usually not detected in MDS patients, but TNF-a was over-
`expressed in 11/14 case5.37"9 These data suggest a tendency
`for
`lFN—y—mediated apoptosis in SAA and TNF-a—driven
`apoptosis in MDS. Some features of MDS and SAA are shown
`in Table 2. We propose that MDS and SAA may have a similar
`
`Table 2
`
`Pathophysiologic factors in MDS and SAA
`
`I Hemopoietic inhibitors
`lProgenitor cells
`Apoptosis of marrow cells
`Telomere shortening
`Clonal hemopoiesis
`Production of G—CSF and GM—
`CSF
`
`MDS“
`
`SAA
`
`Yes
`Yes
`Yes
`Yesb
`Yes
`Often 1
`
`Yesa‘i‘a‘“
`Yes“
`Yes"°-“'
`Yes106
`Rare‘ ‘2
`Normal or I"3
`
`“See text for details.
`I’More pronounced telomere shortening in MDS than SAA (see text).
`
`Patlngmsisotflc
`Cltoserleldaidllljst
`
`pathophysiology but the disease expression is dependent on
`patient age.
`There are a few other points suggested by the model. Mar—
`row failure has a multifactorial etiology including decreased
`marrow production of colony-stimulating factors,
`increased
`elaboration of hemopoietic
`inhibitors
`and accelerated
`apoptosis of progenitor cells. As proposed previously, qto—
`penia with a hypercellular marrow can be attributed to simul—
`taneous progenitor cell apoptosis, increased proliferation with
`a differentiation block.“'53'7‘
`
`The sequential development of MDS does not explain all
`observations. This proposal provides a basis for designing new
`studies for what remains an enigmatic disease.
`
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