Initial transforming event in myelodysplastic syndromes may be viral: case for cytomegalovirus

Medical Oneology (1998) 15, 165-173 �9 1998 Stockton Press All rights reserved 0736~)118/98 $12.00

HYPOTHESIS

Initial transforming event in myelodysplastic syndromes may be viral: case for cytomegalovirus

AzraRaza

Rush Cancer Institute, Rush-Presbyterian-St Luke's Medical Center, Chicago, IL, USA

Myelodysplastic syndromes (MDS) are clonal hematopoietic disorders which begin in a pluripotential bone marrow (BM) stem cell. This early stem cell is believed to acquire a growth advantage over its neighbors as a result of an initial transforming event, the nature of which has remained obscure. In this paper, we propose that pathogens such as those belonging to the herpesvirus family of DNA viruses may play a role in the initial transformation of the stem cell. The case for cytomegalovirus (CMV) as a representative of this family of viruses is discussed at length and a molecular mechanism which may be involved in the oncogenic activity of CMV is proposed. No proof has been presented to implicate CMV directly in MDS, but circumstantial evidence which supports such a possibility is provided.

Keywords: myelodysplastic syndromes; viral etiology; apoptosis; proliferation; TNF-a; hematopoiesis; cytomegalovirus

Introduction The myelodysplastic syndromes (MDS) are indolent clonal disorders which predominate in the elderly and present with the clinical paradox of variable cytopenias despite generally cellular bone marrows (BM). 1'~" A novel paradigm has recently been proposed to explain the variegated nature of the pathology encountered in MDS. 3 An as yet poorly defined event(s) confers a growth advantage on a pluripotential BM stem cell whose proliferation eventually overwhelms the marrow leading to monoclonal hemopoiesis. In a certain per- centage of MDS patients, dual acting cytokines from unexplained sources further confound this picture by providing a stimulus to the early precursors to divide and induce apoptotic death of their maturing progeny. It is important to note that apoptosis is found in all

Correspondence: Azra Raza MD, Rush Cancer Institute, Rush-Presbyterian-St Luke's Medical Center, 2242 West Harrison Street, Suite 108, Chicago, IL 60612-3515, USA. Tel: 312 455 8474: Fax: 312 455 8479 Received 2 July 1998; accepted 13 July 1998

stages of the cell cycle, particularly in cells actively synthesizing DNA. 3 This situation of apparent 'antonymy' where a cell is found to be simultaneously in S-phase and apoptotic appears unique to MDS and could not be documented in a variety of other cancers such as head and neck, breast, ovarian, brain, lymphomas and de novo acute leukemias. 3 In any case, this excessive proliferation and apoptosis results in the clinical syndrome of pancytopenia (excessive intramedullary apoptosis of maturing cells) in the setting of hypercellular marrows (rapid proliferation of the transformed progenitors). The cytokines responsible for the genesis of apoptotic death of hematopoietic cells include tumor necrosis factor alpha (TNF-ct), transforming growth factor beta (TGF-[3) and interleukin-1 beta (ILI-~). 4'5 In some MDS patients, the growth advantage conferred upon the stem cell may be matched by a propensity to undergo premature apoptosis as the cells differentiate. In still other patients, the genesis of cytopenias may not be excessive intramedullary apoptosis at all, but may be excessive retention of hematopoietic cells in the BM due to

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dysregulation of adhesion molecules and/or their receptors.

What can be said with certainty is that hematopoiesis in MDS is monoclonal where both lymphoid and myeloid cells appear to be the progeny of the same parent. 6'7 There is however, no incontrovertible proof that the 'initial transforming event' (or series of events) occurs in a pluripotential BM stem cell in MDS. Rather, the diseased nature of the parent stem cell is inferred from the abnormalities manifest in its daughters, the most prominent being dysplastic maturation and the presence of monoclonal hemopoiesis. In fact, the reason why MDS is cc,nsidered as representing a malignant state is because of its clonal nature and presence of karyotypic abnormalities which frequently undergo evolution as the disease progresses. Mono- clonality in MDS should not be confused with mono- clonality encountered in other malignant states such as acute myeloid leukemia (AML). In the case of AML, the leukemia cells are clearly descended from a single transformed parent, but residual hematopoiesis is polyclonal, s In MDS on the other hand, cells belonging to all three lineages (erythroid, myeloid, megakaryocytic) as well as probably the B-lymphocytes are monoclonal or descended from the single transformed parent cell. 6 MDS resembles the chronic phase of chronic myeloid leukemia (CML) where the normally differentiated (as opposed to dysplastic mature cells in MDS) erythroid, myeloid and megakaryocytic cells all contain the Philadelphia (]~h 1) chromosome. The transforming event in CML appears to be the bcr-abl genetic abnormality resulting from the Ph 1 translocation since the BM returns to a polyclonal state upon regression of this cytogenetic anomaly following interferon therapy. 9 In MDS, however, even the frequently encountered cytogenetic abnormalities such as those affecting chromosomes 5 and 7 may be epiphenomena or secondary events, since they often appear to be present in only a subpopulation of cells in a marrow which is otherwise monoclonal. In summary, therefore, the earliest consequence of the initial transforming event in MDS is monoclonal hemopoiesis, whilst cytogenetic abnormalities represent disease evolution. In CML, on the other hand, absence of monoclonal hemopoiesis upon eradication of the P h I chromosome appears to implicate the bcr-abl abnormality as the 'transforming event'.

What is the 'initial event' in MDS? Since there are certain karyotypic anomalies which occur with regular frequency in MDS, it is not unreasonable to look for the 'initial transforming event'

in genic changes such as loss of a tumor suppressor gene or constitutive activity of a proto-oncogene associated with the most commonly involved areas of the affected chromosomes. However, even if such a gene was identified, the question of what caused its loss or dysfunction would still remain obscure. In this paper we would like to propose that MDS can begin as a viral disease. We further propose that the virus may infect a BM stem cell, a BM stromal cell or a cell belonging to the immune system. Abnormal cytokine expression as a consequence of the infection could change the bone marrow microenvironment in such a way that only a rapidly proliferating stem cell would survive in this new setting, thus accounting for the monoclonal hemopoiesis seen in MDS. The daughters of such a rapidly proliferating abnormal stem cell might also undergo premature apoptosis either as a result of the adverse microenvironment or as a direct consequence of increased divisions in the parent, since it has been demonstrated that less than 10% of CD34 + cells dividing once in vitro were apoptotic as compared with 25% of CD34 + cells which had divided four or more times. 1~ Taken together, these abnormalities would produce the syndrome of monoclonal hemopoiesis, pancytopenia and hypercellular marrows.

If this hypothesis is correct then the first essential question pertains to the identity of the virus. We propose that the virus is probably a common human pathogen that has developed long-term residence in the host without causing clinically significant ongoing disease. The virus, however, is capable of reactivation from its latency phase and even be made 'oneogenie' in the presence of the right 'promoting conditions'. These promoting events would include immunosuppression as a consequence of aging, bone marrow transplant or other chemotoxic exposures, a second viral infection (acquired immune deficiency syndrome or AIDS) or finally any other infectious and/or non-infectious conditions which lead to upregulation of pro-inflammatory cytokines. It is also worthwhile to remember that prolonged, chronic exposure to the promoting events probably carries far more oncogenic potential than short-lived surges of cytokines or exposure to cytotoxic agents.

On the whole, it is important to emphasize that in the majority of patients with MDS, along with abnormalities of the BM stem cell, there also appears to be a dysregulation of the cytokine milieu in an otherwise exquisitely balanced and finely-tuned BM microenvironment. In fact, if we suppose that the 'initial event' in MDS is related to the BM microenvironment and

Viral elioiogy for MDS A Raza

dysregulated cytokines, then it is highly probable that the altered landscape would profoundly affect the fitness of the organisms that normally reside therein. For example, mutated progenitor cells such as those bearing the bcr-abl translocation or t(14;18) abnormalities have been clearly demonstrated in healthy individuals, 11'~2 but probably remain at a low frequency because 'normal' BM microenvironmental conditions are not conducive towards their expansion. In the changed environment, however, these mutated or otherwise abnormal clones can conceivably acquire a growth advantage over their normal counterparts. This would eventually lead to monoclonal hemopoiesis, with a rapidly proliferating transformed parent whose daughters die prematurely either due to the action of excessive pro-inflammatory cytokines or as a consequence of increased divisions, or both. The syndrome of pancytopenia, hypercellular marrow, monoclonal hemopoiesis with excessive proliferation/apoptosis and abnormal cytokines expression would ensue. This situation could persist as such with increasing deterioration of the existing clinical problems such as worsening cytopenias. On the other hand, acquisition of a muta- tion in one of the daughters of the transformed parent could lead to loss of its ability to apoptose and mature, thereby giving rise to a new population of blastic cells and the transformation of a simple refractory anemia (RA) to refractory anemia with excess blasts (RAEB). Progression to acute myeloid leukemia (AML) would depend on the rapidity with which these blast cells would be able to undergo substantial clonal expansion. Monoclonal hemopoiesis would clearly predispose to such accumulation of mutations in a daughter cell. During all this disease causation and evolution, main- tenance of the abnormal cytokines milieu in the BM microenvironment would be essential for disease persistence. A second possibility is obviously related to transformation of a hematopoietic stem cell which would rapidly divide/apoptose and give rise to abnormal cytokine production eventually leading to the same scenario described above. A virus could accomplish either of these possibilities by infecting any early BM progenitor cell or a BM stromal cell and we propose that a DNA virus belonging to the herpes virus family such as cytomegalovirus could be one such pathogen.

The case for cytomegalovirus as the 'initial transforming event' in MDS Human cytomegalovirus (CMV) is a ubiquitous pathogen belonging to the herpes virus family that infects 40-100% of adults without producing clinically appar-

ent disease in the majority of immunocompetent individuals] 3'14 In immunocompromised individuals such as neonates, transplant recipients or patients with acquired immune deficiency syndrome (AIDS), the virus is reactivated from the dormant state to cause a variety of pathologies. For example, in patients under- going bone marrow transplant, CMV can be associated with delayed platelet engraftment, marrow suppression and graft failure. ~5 The pathogenesis of marrow suppression in these cases has not been fully under- s t o o d . 16"17 Several issues have become clearer recently regarding the latent repository sites of the virus as well as mechanisms of reactivation. CMV has been demonstrated to infect and persist in a variety of hematopoietic cells including peripheral blood leukocytes, TM

monocytes/macrophages, 1~ in interleukin-2 (IL-2) treated cultures of purified T-lymphocytes 2~ CD34 + multi potent progenitor cells 2~-24 and BM stromal cellsY The virus can be reactivated in a number of ways: For example, it has been shown that CD33 § cells supporting latent virus undergo reactivation when differentiated in the presence of human fibroblasts or by addition of certain pro-inflammatory cytokines such as TNF-ct, interleukin-4 (IL-4) or granulocyte-macrophage colony-stimulating f a c t o r (GM-CSF). 26 This appears to suggest that cell differentiation pathways act as determinants of reactivation. Another significant observation to emerge from this study is that there is a clearly defined BM derived myelomonocytic progenitor cell which is the precursor for both stroma (dendritic cells) and parenchyma (granulocyte and macrophage) and it is this cell which serves as a repository for the latent v i r u s . 26 These precursor cells coexpress CD33 and CD15 or CD33 and CD14 along with dendritic cell markers CDla and CD10.

CMV infections can be reactivated in a variety of immunocompromised states with frequently cata- strophic consequences. The obvious example is a BM transplant recipient in whom failure to engraft, graft- versus-host disease and many opportunistic infections result from reactivated CMV. Because the virus affects both stromal and parenchymal cells the resulting hematopoietic suppression may be due to inhibition of progenitor cell growth or due to the abnormal cytokines production and dysfunction of the stromal cells. Finally, since both lymphoid and myeloid cells are clearly affected by CMV and progenitors of monocytes-macrophages, and dendritic cells serve as a reservoir for the latent virus, the result is that active CMV infection can have far-reaching consequences for most lymphohematopoiesis.

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CMV particularly targets monocytes as repository sites for its latency phase. Once the virus enters monocytes, replication appears to stop after switching from immediate early to early phase of the v i r u s cycle. 27 Viral entry into monocytes appears to be particularly facilitated via the CD13 surface antigen, a human metalloprotease amino peptidase. 28 It has also been shown that among undifferentiated peripheral blood mononuclear cells, those expressing the CD14 marker are most susceptible te CMV infectionY thereby suggesting that CD14 may be a cofactor in viral entry. Interestingly, all infected CD14 cells were shown to express CD13 as well. 2s

CMV is also present in macrophages residing mostly in lung and spleen in normal subjects, 3~ but include skin 31 colon 32 and brain 33 in AIDS patients. It has been hypothesized that the infected macrophages escape immune surveillance because the virus disrupts the microtubular system and modifies the cytoskeleton of these cells. 34 Interestingl3; when virus-bearing monocytes are differentiated into macrophages in vitro, full viral replication o c c u r s . 35 A similar phenomenon may also occur in vivo as mon ocytes become macrophages during inflammatory episodes, since the process of differentiation appears to nudge the virus from a latent to an active phase.

CMV can dysregulate cytokine production by infection of stem cells, stromal cells such as fibroblasts, monocytes and macrophages as well as differentiated hematopoietic cells such as polymorphonuclear leukocytes (PMN). The most dramatic effect is on TNF-a whose expression is enormously upregulated following infection of myelomonocytic cells. This probably results from transactivation of the TNF-a promoter via IE proteins. 36 Abundant transcripts of TNF-ct have been documented in colonic macrophages of AIDS patients suffering from CMV colitis. 32 A vicious cycle ensues with TNF-ct production inducing CMV infection and resulting enhanced viral replication. This effect appar- ently results from TNF-a p75 receptor induced translocation of NFkB which then interacts with the CMV immediate early or rE promotor 37 to increase further viral replication that in turn leads to increased transactivation of the TNF-ct promoter. 36 It is perfectly conceivable that presence of excessive proinflammatory cytokine such as TNF-ct during a variety of illnesses may promote CIvIV replication in v ivo . 38'39

In summary, therefore, latent virus can be reactivated either when progenitor cells carrying the virus are co-cultured in the presence of permissive human fibroblasts, or pro-inflammatory cytokine, or when the

infected cell undergoes a process of differentiation, for example a monocyte maturing into a macrophage. The reactivated virus may cause myelosuppression, the precise mechanism of which remains obscure and probably is multifactorial. Either the virus is directly cytopathic to the cells infected or renders them unresponsive to growth factors. On the other hand, an increased production of inhibitory growth factors such as TNF-~t, TGF-[3, macrophage inhibitory protein (MIP)-lc~ accompanied by decreased production of viability factors such as G-CSF, GM-CSF, and stem cell factor (SCF) by infected stromal cells would indirectly suppress myeloid activity as well. n~ In fact not only has viral infection of human bone marrow stromal cells been demonstrated to occur, but a decrease in their capacity to support normal hematopoiesis has also been f o u n d . 4~ This has been hypothesized to result from both dysregulated cytokine production by stromal cells as well as disruption of stromal cell-hematopoietic cell interactions. 4~ One interesting aspect of this study relates to TNF-c~ production by stromal ceils which was in fact found to be quite low for both CMV-infected as well as non-infected cells. However, upon lipopoly- saccharide (LPS)-induced stimulation, the secretion of TNF-a by CMV-infected stromal cells was five-fold higher than that of control stromal cells. This leads one to wonder whether an LPS challenge to infected stromal cells harboring the latent virus results in reactivation of the virus and a highly exaggerated TNF- ct response as noted above. This excessive production of TNF-a (as well as other pro-inflammatory cytokine) would then perpetuate the vicious cycle of increased viral replication followed by increased transcription of the cytokine. Prolonged and sustained, this cycle may lead to the abnormal cytokine milieu which only supports a highly proliferative stem cell and eventually monoclonal hemopoiesis, thus evolving into the syndromes of myelodysplasia.

Clinical association of myelodysplasia and CMV infection CMV infection in immunocompetent adults is associated with a self-limited infectious mononucleosis-like syndrome with only subclinical hematologic abnormalities. Cases of thrombocytopenia have been reported in otherwise immunocompetent individuals with active CMV infection, and often the thrombocytopenia is quite profound. 41-47 Interestingly, there is one case report 41 of a 41-year-old previously healthy male who developed acute CMV-induced thrombocytopenia as well as marked dysplastic changes in the BM. In this


case, there was evidence to suspect both direct cytopa- thologic injury to bone marrow cells and autoimmune mechanisms (presence of platelet associated IgG and anti-DNA antibodies were positive) responsible for the profound thrombocytopenia and myelodysplasia. Autoimmunity is known to accompany viral infections and CMV has been implicated in such autoimmune disorders as acute myocarditis, acute inflammatory polyneuropathy, and Sjogren's syndrome. 47 It is also worthwhile to remember that myelodysplastic syndromes are also associated frequently with confounding immunologic defects represented by the detection of circulating immune complexes, lowered T-cell counts and accompanying immune disorders. 48 The patient mentioned above 41 improved following treatment with prednisolone with complete resolution of thrombocytopenia, myelodysplastic changes in the BM and evidence of autoimmune disturbances. In summary, therefore, acute infections with CMV can also lead to severe hematological disorders such as mononucleosis, hep- atosplenomegaly, lymphadenopathy, thrombocytopenia, aplastic anemia and hemolytic anemia, t3

Pathogenic infections with CMV occur predom- inantly in neonates, and immunocompromised hosts such as transplant recipients or patients with AIDS. Leukopenia, hemolytic anemia and mild thrombocytopenia are well recognized hematologic complications in new-born infants, reflecting an immature immune system as a predisposing factor in this group. 49 A rather severe illness is encountered with CMV infection in the immunocompromised setting. The incidence of CMV infection is high (68%) in bone marrow transplant (BMT) recipients, s~ and in approximately half the patients CMV can be isolated from some sites by a median of 53 days post-transplant, s~ In BMT and other organ transplant settings, CMV is found to be frequently the cause of interstitial pneumonitis, neutropenia, delayed engraftment, thrombocytopenia, failure of engraftment, 13's~- and less frequently of hepatitis and enteritis9

Myelodysplastic changes in the bone marrows were found to be associated with active CMV infection in three patients following allogeneic BMT. s4 The hematopoietic cells were shown to be of donor origin in all three cases and moderate anemia developed despite normo-hyperplastic marrows showing trilineage dysplasia. CMV was detected in each case using PCR. The authors of this study hypothesized that CMV could have impaired hemopoiesis either by directly infecting the stromal cells with subsequent perturbation of cytokines or by directly infecting hematopoietic pro-

genitors with resulting myelosuppression and morpho- logic changes of dysplasia. Obviously, use of a variety of drugs (antiviral, immunosuppressant, chemotherapy) as well as graft-versus-host disease could not be ruled out as possible causes for the dysplastic changes and myelosuppression. Ganciclovir administration has been associated with decreased blood counts in virtually all treated patients 5s and recombinant human G-CSF has been suggested as causing MDS and/or AML in patients with aplastic anemia or congenital neutropenia. 56

Mechanism of CMV-induced myelodysplasia Viral infections in general are associated with hematopoietic suppression manifested by leukopenia and thrombocytopenia. ~7 The mechanism of this pancytopenia is poorly understood. Recognition of CMV infections in transplant recipients with accompanying morbidity and mortality prompted an urgent investigation of the immunology and pathology of this virally- induced myelosuppression. Mechanisms proposed to explain these hematologic abnormalities have ranged from alteration of accessory cell function via the production of inhibitory cytokines by the virus, to perturbation of stromal cell function resulting in a fall in viability factors, or alteration of adhesion molecule expression, to direct infection of hematopoietic progenitor cells. 5s Profoundity of cytopenias and other disease manifestations caused by active CMV infection depend on a variety of factors including the type of cells infected and the immunocompetence of the host. Whilst CMV is clearly associated with hematologic suppression as well as production of pro-inflammatory cytokines such as TNF-c~ and MIP-lcc, this is clearly a far cry from being the cause of myelodysplastic syndromes. We propose the following hypothetical possibility.

An important advancement in our understanding of the patho-physiology of MDS is provided by the recent recognition of excessive intramedullary apoptosis of hematopoietic cells in this disease. An interesting observation in this regard is the unusual suicidal death of cells actively engaged in DNA synthesis, a process which has now been termed 'antonymy'. s9 We have shown previously that large numbers of S-phase cells in MDS marrows are apoptotic and that this situation is unique to MDS and absent in a variety of other malignant states such as brain tumors, breast cancers, head and neck tumors, lymphomas and acute myeloid leukemia. 6~ Thus it appears that in MDS patients, the diseased parent cell and its progeny are engaged in

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abnormally high proliferation which is successfully aborted by an equally rapid rate of apoptosis. The rapid proliferation affects cells at all stages of maturation as does the increased apoptosis. This 'antonymous' situation may be explained on the basis of a virally-induced mechanism.

Viruses cannot assemble the proteins essential for their replication in a resting or quiescent cell because of the absence of key required components such as DNA polymerase and deoxyribonucleotides. As a result, if viruses infect or otherwise become activated in a resting cell, they must induce at least the appearance of S-phase activity in order to gain access to the cellular proteins required for viral replication. Because viral proteins are not effective enough to actually propel a resting cell through the entire cycle including mitosis, the cell ends up simulating an S-phase environment sufficient to permit viral replication, but usually without being followed by actual mitosis. DNA viruses accomplish this series of events by transcribing proteins early in the course of infection called IA (EIA) proteins which can interact with cellular transcription factors to initiate DNA synthesis. Transition of a resting cell from G1 to S is under the well orchestrated control of P16/RB/E2F regulatory pathway in normal cells and is one of the most frequent targets of genetic alterations in human cancers. 6a The transcription factor in this pathway is the E2F family first identified as a group of proteins with EIA-inducible cellular activity. 62 E2F 1 is the most well characterized member of this family and clearly influences the transcription of S-phase genes that drive the cell through the G 1 check po in t . 63~6

DNA viruses exploit this ability of transcription factors to initiate DNA synthesis and induce overexpression of EzF 1. However, one consequence of such an abnormal proliferative signal may be: activation of p53 dependent apoptosis and in fact Kowalik et al showed that endogenous p53 is sufficient to induce apoptosis upon E2F 1 overexpression. 66 Moreover, reversal of this apoptosis can be partially achieved upon introduction of a mutant p53 overexpression. 67 Since the apoptosis is not completely reversed in the absence of a wild type p53, it is clear that events independent of this pathway may also be involved in the indaction of apoptosis. In fact, it was shown that E2F1 itself possesses specific pro- apoptotic activity, a property which is not shared by other E2F family members even though they are equally effective in inducing S-phase activity in resting cells. 67 In summary, therefore, overexpression of E2F 1

may lead a cell to proliferate more rapidly but also to be susceptible to apoptosis either because of an intact

p53 pathway or via a direct pro-apoptotic activity of E2F1.

We propose that CMV may initiate cellular events that could ultimately lead to MDS by a somewhat similar mechanism as follows. Reactivation of latent CMV could occur either while the resting, quiescent bone marrow stem cell serving as its repository is recruited into cycle and undergoes some level of differentiation, or there are pro-infammatory cytokines in the vicinity of the quiescent BM stem cell containing the latent virus. Upon activation in an otherwise resting stem cell, the virus simulates an S-phase environment in the cell via overexpression of E2F1. In fact, CMVIE1 gene product has been shown to interact with E 2 F 1 to induce transcription of S-phase related proteins already 68 thereby simulating DNA synthesis phase activities. This may allow the stem cell to begin DNA synthesis and lead to rapid proliferation. In the presence of intact p53, however, the daughters of such a cell would also be sensitive to premature apoptosis. In fact, a number of the daughters may demonstrate the dual activity of DNA synthesis and apoptosis simultaneously, an antonymous situation which has been uniquely associated with MDS as described before. Viral activity would lead to production of pro-inflammatory cytokines which in turn would promote viral activity.

In other words, a reactivated DNA virus in a quiescent BM stem cell could result in clonal expansion of such a cell via transactivation of cellular E2F1 resulting in monoclonal hemopoiesis. Presence of intact apoptotic pathways (p53) or proinflammatory cytokines would terminate a number of the daughters from completing all stages of maturation, leading to excessive proliferation and equally excessive apoptosis of hematopoietic cells. The source of the cytokines could either be the surrounding stromal cells or the transformed cell and its daughters.

The above situation would be quite reminiscent of the modus operandi attributed to another member of the herpes virus family, the Epstein-Barr virus (EBV). Recent elegant studies have demonstrated that latent membrane proteins of EBV such as LMP-1 residing in the cell membrane can activate the TNF-a receptor associated factors (TRAF) to mobilize NFKB and initiate DNA synthesis with consequent immortalization of B-cells in lymphomas associated with immunosuppressed states. 69 In the case of CMV and MDS, we propose an activation and overexpression of the transcription factor E2F 1 leading to induction of proliferation followed by frequent premature apoptosis in


the daughter cells. Because the affected cell is a stem cell to begin with, immortalization may not even be necessary. Excessive proliferation, excessive apoptosis, monoclonal hemopoiesis and excessive amount of pro- inflammatory cytokines which have been shown to be the hallmarks of MDS could ensue as a result of this viral activity. As in the case of EBV and lymphomas, the virus may not be actively replicating, but the disease follows events initiated by latent proteins of a dormant virus. This is all the more reason to emphasize that we hypothesize the role of a virus only as an initiating event in MDS and do not wish to propose that MDS is a viral infection which could be curable with anti-viral therapy. That would be as distal to the problem as saying that because smoking causes lung cancer, stop- ping the habit would cure an established malignancy.

Summary and future directions A hypothesis that MDS can begin as a viral disease has been proposed in this paper. Further, a DNA virus belonging to the herpes virus family such as CMV has been considered as fulfilling the role of such a pathogen. No proof has been presented for either proposal; however, circumstantial evidence and a possible molecular mechanism have been offered in support of the hypothesis. The herpes virus family members appears as possible etiologic agents since they fit the role of being common human pathogens that can remain dormant in repository sites frequently involving an early BM stem cell, and are capable of reactivation under select circumstances. In their latent states, these viruses encode proteins which have been shown to affect cellular functions, thereby making them danger- ous pathogens over a lifetime of the host. These latency associated activities could end up with possible cata- strophic consequences for lymphohemopoiesis when combined with secondary events that compromise the immune system such as a second viral infection or a degenerating immune system as a consequence of aging. EBV, CMV, herpes virus 6 and 8 have all been shown to cause bone marrow disturbances. 7~ It has further been suggested that the syndromes of myelodysplasia may result either as a consequence of abnormal function of a hematopoietic stem cell or a BM stromal cell. An early pluripotential BM stem cell which is a common precursor for both dendritic and myeloid cells has been shown to serve as a repository site for latent CMV infection, thereby making this virus an attractive possible candidate.

Clearly, the myelodysplastic syndromes represent enormous clinical heterogeneity in their presentation, natural histories and response to therapy and this is matched by an almost parallel biologic heterogeneity. Given the highly variegated nature of this disease, it is not unreasonable to expect variable etiologies for these syndromes as well. There is no doubt that exposure to benzene 73 and other toxins is associated with develop- ment of MDS, and in such cases, the viral etiology is clearly not applicable. However, in other cases of primary de n o v o MDS, the virus may be a distinct possibility as the agent for the initial transformation of a BM stem cell. The purpose of summarizing a possible viral role in MDS is to reopen an otherwise much neglected area of research. Since not all MDS patients present with identical pathologies, it is worthwhile to investigate more than one possible initiating event and consider the clinical syndromes as a final common pathway rather than a uniform illness. Chemical and toxic exposures have been vigorously investigated in MDS. It is time to include pathogens such as viruses in the list of possible causes for these otherwise frustrating disorders. In the present paper, an emphasis was placed on CMV amongst the possible viruses. However, a lentivirus, or retrovirus or other DNA viruses need to be considered with equal vigor.

Acknowledgements This work was supported by a grant from the National Cancer Institute (POICA 75606), The Markey Charitable Trust (94-8) and the Dr Roy Ringo Grant for basic research in MDS. The author wishes to thank Ms Lakshmi Venugopal and Ms Sandra Howery for excellent administrative and secretar- ial assistance.

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Initial transforming event in myelodysplastic syndromes may be viral: case for cytomegalovirus