molecular basis of lymphomagenesis1 · molecular basis of lymphomagenesis ag receptor gene...

[CANCER RESEARCH (SUPPL.) 52, 5529s-5540s, October I. 1992]

Molecular Basis of Lymphomagenesis1

Ian Magrath2

Lymphoma Biology Section, Pediatrie Branch. National Cancer Institute, Bethesda, Maryland 20892

Abstract

Lymphoid neoplasms, like all malignant tumors, arise as a consequence of the accumulation, in a single cell, of a set of genetic lesionsthat result in altered proliferation or increased clonal life span. Themost frequently observed genetic abnormalities among the malignantnon-Hodgkin's lymphomas are translocations, which appear to be lin

eage and, to a large extent, lymphoma specific. Recombinases thatnormally mediate the process of antigen receptor gene rearrangementappear to have an important (but not exclusive) role in the mediation ofthese translocations and of other types of gene fusion (e.g., deletion ofintervening DNA). Frequently, such fusions result in the increased orinappropriate expression of crucially important proteins, many of whichare transcription factors that regulate the expression of other genes.These abnormalities, however, do not appear to be sufficient to inducelymphoma, and it is likely that the additional genetic lesions requireddiffer from one tumor to another.

The likelihood of any given clone of cells accumulating a sufficientnumber of relevant genetic lesions to give rise to a lymphoma is probablya function of its life span. Prolonged survival of a cell clone may bemediated by viral genomes (e.g., Epstein-Barr virus and human T-cell

leukemia/lymphoma virus type 1), by the abnormal expression ofcellular genes that inhibit apoptosis (e.g., bcl-2), or by the mutation

or deletion of cellular genes that are necessary for apoptosis, e.g., pS3.The background rate at which genetic lesions occur is amplified bythe interaction of inherited and environmental factors, the latter appearing to be the major determinant of incidence rates. However, inheritedfactors that influence lymphomagenesis, including variability in theability to repair DNA damage or in the fidelity of antigen receptorrecombinases for their signal sequences, may be crucial determinants ofwhich particular individuals in a given environmental setting developlymphoma.

IntroductionThe word "lymphoma," like many nosological terms in use

today, was coined in an era when gross pathology and clinicalcharacteristics were the only existing tools of disease taxonomy,such that the separation of one pathological entity from anotherwas difficult and often impossible. The subsequent preeminenceof cellular morphology as a means of lymphoma classification,which persists to the present, owes much to the seminal influence of Virchow in the mid-19th century ( 1). Morphology, however, is limited by its subjective nature, by its inability to provideinsights into pathogenesis, and by its frequent lack of precision.Lymphoid neoplasms arising from different cellular lineages,for example, are sometimes indistinguishable at a morphological level. Slowly, our reliance upon form (appearances!) is being supplemented by a new understanding of the functionalderangements that give rise to neoplastic behavior, an under-Standing that has been made possible by the rapidly developingknowledge of the molecular basis of cell proliferation and differentiation in the lymphohemopoietic system. Today, we consider that lymphoid neoplasia arises as a consequence of geneticabnormalities, almost exclusively occurring in somatic cells,that lead to prolonged cell survival, to inappropriate growth,

1 Presented at the National Cancer Institute Workshop, "The Emerging Epidemic of Non-Hodgkin's Lymphoma: Current Knowledge Regarding EtiologicalFactors," October 22-23, 1991, Bethesda, MD.

2To whom requests for reprints should be addressed.

and, frequently, to a differentiation block. These molecular genetic abnormalities and their consequences surely provide theultimate means of defining individual pathological entities, although a new classification scheme based on such abnormalitieshas yet to be proposed.

From this perspective, a lymphoid neoplasm may be definedas "the progressive accumulation of a clone of lymphoid cells

which arises as a consequence of multiple genetic changes occurring in the cell genome." This definition would exclude the

polyclonal lymphoproliferative processes that sometimes arisein patients with immunodeficiency syndromes, regardless ofwhether immunosuppression is iatrogenically induced, as inallograft recipients, inherited, or acquired. There is continuingcontroversy as to whether similar processes occur in HIV3-

infected individuals. These lymphoproliferative syndromes frequently arise as a consequence of failure of the immune systemto control the proliferation of multiple clones of EBV-infectedB-cells, rather than of intrinsic, biochemical abnormalities thatresult from genetic aberrations (2). While they may be clinicallyand morphologically indistinguishable from lymphomas, theessential difference in their pathogenesis is supported by theobservation that a fraction of those that arise shortly after allo-grafting will regress upon withdrawal of immunosuppressivetherapy. When lymphoproliferative syndromes occur late afterallografting, however, they are more likely to be unresponsiveto withdrawal of immunosuppression and to contain detectablegenetic lesions. In such cases it seems likely that a precedingphase of subclinical lymphoproliferation has led to the development of genetic abnormalities in a single cell clone, withoutgrowth of a "true" lymphoma. Lymphomas that are not

associated with EBV may also arise de novo in patients withvarious forms of inherited immunodeficiency or HIV infection(3).

It seems probable that a preceding phase of abnormal proliferation and/or of increased life span of one or more cell clonesis a prerequisite for the development of lymphoma, and indeed,of all neoplasms, since neoplasia is the consequence, not of asingle genetic change, but of many such changes. Preneoplasticclones must therefore survive long enough to accumulate all ofthe necessary genetic abnormalities for the emergence of thefinal pattern of growth that is manifested as a clinically apparent tumor (Fig. 1). It is because of the stochastic element in thisprocess that neoplasms are nearly always monoclonal. This rulemay be broken when the risk that the set of genetic lesions mayaccumulate in a single cell is very high, e.g., when at least one ofthe necessary genetic abnormalities is present in all cells (aninherited or germ cell mutation); when the degree of exposureor potency of one or more exogenous agents is sufficiently greatthat genetic changes occur in a high proportion of exposed cells;or when a single agent, e.g., a virus, is sufficient to cause malignancy or requires perhaps few additional genetic lesions. Inpractice, the occurrence of oligoclonality (i.e., the synchronousdevelopment of more than one malignant clone) is extremelyrare, except in the immunodeficiency-related lymphoproliferative syndromes, and a more likely event is the occasional

'The abbreviations used are: HIV. human immunodeficiency virus: EBV, Epstein-Barr virus; HTLV, human T-cell leukemia/lymphoma virus.

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MOLECULAR BASIS OF LYMPHOMAGENESIS

Ag receptor gene

rearrangement

Spontaneousmutation Environmental

factors

Immunedysfunction

EnvironmentalFactors ^\ ^ Spontaneous

mutation

NEOPLASTICCELL

Fig. I. Diagrammatic depiction of the pathogenesis of a lymphoma. In thisexample, lymphoid precursor cells develop a genetic abnormality (e.g., a translocation) and/or are infected by a virus. The risk of this occurring is probablyinfluenced by environmental factors. These initial lesions result in prolongation ofthe life span, cause DNA instability, or both, and thus increase the risk of additional genetic lesions. The life span of lymphocytes is otherwise limited in theabsence of an encounter with antigen. The genetically modified or virus-infectedcell clone subsequently accumulates additional genetic lesions, whether of exogenous or endogenous origin. An appropriate combination of genetic lesions leadsto neoplasia. Ag, antigen.

metachronous occurrence of separately generated malignantclones. The uncommon "late relapses" that occur in Burkitt's

lymphoma in Africa appear to fall into this category (4), and atleast one patient with HIV infection has developed a secondBurkitt's lymphoma (5). It is also possible that individuals who

have had a single lymphoid neoplasm are predisposed to develop a second neoplasm of the same type because of the persistence of clones of cells that contain some, but not all, of thegenetic changes required for neoplasia. Since the risk of thedevelopment of the appropriate "final" genetic lesion in the

abnormal clone may still be in excess of millions to one, theemergence of a second neoplasm of the same type during thelifetime of the individual is low. This notion is consistent withreports of the persistence of 14; 18 translocations in healthy,tumor-free patients up to 15 yr after treatment of localizedfollicular lymphoma (6).

Implications of the Need for Multiple Genetic Lesions

These considerations have a number of implications whichcan be summarized as follows, (a) The life span of a cell clonemust be sufficiently long to permit the accumulation of theseveral genetic lesions that lead to neoplasia, (b) The accumulation of genetic lesions is more likely to occur in cells that areactively proliferating, (c) The risk of the development of geneticchange increases as the degree of genetic instability increases.(d) Cure of the neoplasm does not necessarily lead to eradication of cell clones with subneoplastic genetic changes.

Life Span. A cell destined to become malignant must eitherbe of a type that undergoes essentially permanent self renewal(i.e., it possesses one of the characteristics of stem cells), or itslife span must be prolonged by one of the first (if not the first)genetic lesion to occur, including viral infection. In the lymphoid system highly efficient mechanisms exist for the elimination of cells which develop either an unwanted antigen specificity (e.g., "self") or fail to synthesize a functional antigen

receptor (immunoglobulin, in the case of B-cells). At specific

points in the differentiation process, the lack of a moleculeessential to subsequent cellular function (or the possession of aforbidden molecule) triggers a process of programmed celldeath whereby the cell enzymatically cleaves its own DNA intonucleosome-sized lengths. Known as apoptosis, this processmust be very tightly regulated and doubtless involves multiplepathways and gene products. One gene that has been shown tobe able to prevent apoptosis is bcl-2 (7), a gene that was discovered by cloning the region on chromosome 18 that is juxtaposed to immunoglobulin heavy chain sequences as a consequence of a 14; 18 translocation. This translocation is present ina high proportion (60 to 80%) of follicular lymphomas, and thederegulated expression of bcl-2 is clearly an essential component of the pathogenesis of this disease. EBV and HTLV alsocause persistence of the cell clones they infect, thus predisposing them to subsequent malignant change. In the case ofHTLV-1 and HTLV-2, the tax gene transactivates several T-cell activation genes, including the genes for interleukin 2 andits receptor, thus establishing an autocrine loop (8). In the caseof EBV, the recent demonstration that one of the EBV latentgenes, the latent membrane protein, can induce bcl-2 may bepertinent to the persistence of EBV-infected B-cells (9), particularly since EBV-transformed B-cells are probably rapidly eliminated by EBV-specific, HLA-restricted, cytotoxic T-cells. Perhaps the true EBV reservoir is a oc/-2-expressing memory B-cellwhich fails to express those EBV antigens that provoke T-cellcytotoxicity. Interestingly, p53, an antioncogene, has beenshown to be necessary for apoptosis to occur, and its loss (e.g.,by mutation or deletion) may, in some circumstances, lead toprolonged cellular life span (10).

Proliferation. The notion that prolongation of the life spanof a cell clone precedes neoplastic transformation is the equivalent of stating that a preneoplastic phase, either subclinical orclinical, must always precede the development of overt neoplasia. The preneoplastic phase would necessarily include one ormultiple clones of cells which contain at least one genetic lesion(or a viral genome) leading to prolonged life span. The probability of accumulating additional genetic lesions is increased ifthe cell clones undergo continuous or intermittent cell proliferation (e.g., as a result of antigen exposure), since changes inDNA are both more likely to occur and more likely to persist inproliferating cells. DNA repair appears to require temporarycessation of proliferation, and there is evidence that one function of the product of thep55 gene, the expression of which canbe induced by DNA damage, is to inhibit cell proliferation whileDNA repair proceeds (11). Mutations ofp53 which prevent thisfunction could, therefore, increase the likelihood that additional mutations will accumulate.

A large number of nonmalignant conditions cause persistentlymphoid hyperplasia, including chronic inflammatory diseases, chronic infectious processes, and immunodeficiency syndromes (or combinations of these) and, thereby, an increasedrisk of the development of a genetically determined neoplasm.These conditions may cause either polyclonal or oligoclonalhyperplasia due, at least initially, to continued stimulation viaphysiological pathways (antigen receptor/interleukins). Interleukin 6, for example, has been implicated in the pathogenesisof HIV-associated lymphomas. It seems probable that, in some

circumstances, e.g., chronic inflammatory conditions, predisposition to neoplasia occurs not because of the development of asingle clone with a much prolonged life span (e.g., because of agenetic lesion), but because the inflammatory process inducesmany clones with somewhat prolonged life spans. In addition,

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the inflammatory process itself may increase the risk of geneticdamage because of increased generation of free radicals (seebelow). In some immunodeficiencies, such as the WiscottAldrich syndrome, repeated occurrences of serum monoclonalgammopathy lasting for several months indicate that individualclones may occasionally become large enough for the immuno-globulin they produce to become apparent above the background of polyclonal immunoglobulin production. Such clonesare likely to be at considerably increased risk for the development of a neoplasm.

Follicular lymphomas and other "low-grade" lymphomas of

ten have long periods during which they are entirely subclinicaland, even when clinically apparent, they may have a prolongednatural history and sometimes undergo spontaneous regression(12). This, coupled to the tendency of follicular lymphomas totransform into more aggressive tumors (which have been shownto express the same immunoglobulin idiotype as the precedingfollicular lymphoma) (13) and to the existence of de novo largecell lymphomas that contain the same 14; 18 translocationspresent in follicular lymphomas, suggests that these tumorsrepresent a point on the spectrum somewhere between preneo-plastic and fully neoplastic. They do appear to be able to respond to normal B-cell growth signals, e.g., from CD4-positive

cells (13).Genetic Instability. Another element that is relevant to the

development of genetic changes in potential cancer cells is genetic instability. Today, the dynamic nature of DNA is wellrecognized and is clearly an essential component of not onlysuch processes as DNA replication (which requires the cleavageand resplicing of DNA by topisomerases as well as sequence"editing" during DNA polymerization), meiotic recombination,

and the rearrangements of antigen receptor genes, but also evolution. Indeed, one of the tools of evolutionary variation appears to be the reshuffling of functional modules, such that, forexample, a given catalytic element, when coupled to proteinmoieties with different cellular localization signals or differentbinding properties, or to different transcriptional elements,may be expressed at a different phase of differentiation or in adifferent cell lineage or alter its range of substrates. At a different level, genes must retain their integrity in spite of continuousDNA damage arising, for example, from free radicals releasedduring intermediary metabolism or inflammatory responses orfrom exposure to environmental agents. Clearly, there is anabsolute necessity for powerful and very tightly regulated enzyme systems to mediate essential genetic rearrangements andto repair DNA damage. Conversely, it is entirely feasible thatgenetic changes leading to decreased stability of DNA are anessential component of neoplasia, and variation in the regulation of these processes may well be one element that is responsible for the different propensities of individuals to developcancer. This notion is supported by the increased incidence ofmalignant lymphomas in those suffering from disorders associated with spontaneous chromosomal breakage, perhapsrelated to defective DNA repair, such as Bloom's syndrome,Fanconi's anemia, and ataxia telangiectasia (14), although im-

munological deficiency is almost certainly a contributing factorin such individuals.

Subneoplastic Genetic Changes. It seems quite probable thatgenetic lesions capable of contributing to the neoplastic state,but insufficient to cause neoplasia by themselves, occur relatively frequently in persons who never develop neoplasia. Thisis supported by several lines of evidence. Mutations in recognized preneoplastic lesions, such as colonie polyps and oral

leukoplakia, are well described (15, 16) while, in the context oflymphoid neoplasia, the recent finding, in hyperplastic but otherwise normal lymphoid tissue, of rearrangements of the bcl-2gene similar to those associated with follicular lymphomas (17)strongly supports this idea. Moreover, this finding is consistentwith the notion that an early genetic change may lead to prolongation of the life span of the affected clone and also impliesthat 14; 18 translocations are insufficient, in themselves, tocause cell transformation. In the absence of an endogenous orexogenous stimulus to proliferate, 14;18-containing cells mayrapidly enter a resting state (i.e., they are virgin lymphocytes ormemory cells). It is tempting to speculate that the clinical appearance (or recurrence) of low grade lymphomas may be aconsequence of the activation of a genetically altered memorycell of a genetically altered memory cell, doomed to persistbecause of bcl-2 expression, by exposure to antigen (or perhapsrepeated antigenic activation of the same cell done over manyyears). Additional genetic lesions may be more likely to beacquired during these periods of activation. If this were true, itwould imply that only 14;18-containing cells that repeatedlyencounter antigens able to stimulate them will lead to follicularlymphomas. Follicular lymphomas appear to express the fullB-cell repertoire of variable regions (13), so the chances that anencounter with an activating antigen will occur in the lifetime ofan individual may be low and not inconsistent with the incidence of follicular lymphomas.

Follicular lymphomas also provide support for the possibilitythat successful treatment of lymphomas could result in theelimination of the fully transformed clone, but permit the persistence of cell clones containing less than the full complementof genetic abnormalities required for neoplasia. Recently, it wasreported that cells bearing 14; 18 translocations (detected bypolymerase chain reaction) can be detected in long-term survivors (10 to 15 yr) of localized follicular lymphoma (18). Suchcells either represent "dormant" lymphoma cells, possibly

awaiting activation by the relevant antigen, or, more likely,belong to a premalignant clone which will only become malignant if additional genetic lesions are accrued.

Nature of the Genetic Lesions Associated with LymphoidNeoplasia

The genetic lesions that occur in the lymphomas are, to ahigh degree, lineage and probably also differentiation stage specific. There are two explanations for this, (a) It appears highlyprobable that only genes that are transcribed (i.e., in regions ofopen chromatin structure, providing enzymes with access to theDNA) are susceptible to genetic damage, and (b), in any givenlineage, only a finite set of genetic abnormalities will be relevantto the development of neoplasia.

The factors which govern whether a genetic lesion will arisein an individual are multiple and include the total number ofsusceptible cells in the individual, the life span of the cells, theirproliferative state, the relative efficiency (inherited) of such processes as DNA repair and antigen receptor gene rearrangement(see below), the ability to scavenge endogenously or exoge-nously generated free radicals, and the presence of environmental agents that may alter any of these attributes or causeDNA damage directly. Ultimately, however, there are two major considerations: the nature of the genetic changes and thegenes that are involved. The types of genetic abnormality thatcan occur include point mutation, deletion or insertion, geneamplification, and gene fusion. Gene amplification has not, to

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date, been shown to be a common genetic abnormality in lym-

phoid neoplasia, and it will not be discussed further.

Mutation. Point mutations, and some kinds of deletion andinsertion, are usually generated by interaction of DNA withhighly active (i.e., charged) radicals, either of intracellular orextracellular origin. To a degree, the origin of the damage canbe deduced, since endogenously generated mutations are usually transitions (i.e., substitution of one pyrimidine base foranother or one purine for another), while exogenously generated mutations tend to be transversions (i.e., substitution of apurine for a pyrimidine or vice versa). Exogenously inducedmutations can also be inferred from the high frequency of aspecific mutation in tumors from patients from the same geographical region or subpopulation, but a low frequency of thesame mutation in the same tumor type from a different population. An excellent example of a point mutation that is verylikely to have been exogenously induced is the G to T transversion that occurs in codon 249 of the/>53 gene in hepatocellularcancer from South Africa and China, but not in hepatocellularcancer from Taiwan and Japan (19, 20, 21).

It is perhaps pertinent, in this context, to point out thatcancer chemotherapy entails the use of agents that are highlymutagenic. As such, it is not surprising that these agents havebeen incriminated in the production of second malignancies,notably in Hodgkin's disease, but also in a number of other

tumors (22, 23). However, the possibility that the emergence ofa second tumor results from the accumulation of additionalgenetic changes in a premalignant clone of cells that has notbeen eliminated by treatment, and which is environmentallyinduced or constitutional, should always be considered. Thatthis possibility is real is demonstrated in familial cancers, wherethe same genetic lesion may predispose to more than one typeof tumor, for example, the occurrence of osteosarcoma in patients who survive familial retinoblastoma (24), or the occurrence of multiple tumor types (sarcomas and carcinomas) infamilies with inherited p53 abnormalities, one of the lesionsresponsible for the Li-Fraumeni syndrome (25, 26).

Gene Fusion. Gene fusion (i.e., the fusion of one gene or partof a gene with another or part of another) occurs as a result ofdeletion of intervening DNA or inversion of a segment of achromosome (when both involved genes are on the same chromosome) or chromosomal translocation (when the relevantgenes are on different chromosomes). In the lymphoid system,such processes quite frequently bear the molecular hallmarks ofbeing mediated by the recombinases that are normally involvedin the process of antigen receptor gene rearrangement. Consistent with this is the tendency for the genes that define lymphoidcells, namely, these same antigen receptor genes (immunoglo-bulin and T-cell receptor genes), to be involved in the geneticfusions that are so characteristic of different types of lymphoma(27). In part, this may relate to the fact that the antigen receptorgenes must undergo recombination of variable and constantregion coding segments in order to generate functional antigenreceptor molecules and to give rise to the required diversity ofantigen binding sites. Regions of DNA that are to be recom-

bined must be accessible to the enzyme systems that mediate therecombinations and are therefore more likely to be the sites ofDNA breakage and religation. The process of antigen receptorgene rearrangement is essentially continuous and involves verylarge numbers of cells. In the B-cell lineage alone it has beencalculated that, in humans, some 5 x 10'Â°cells are generated

each day from 5 X IO6 precursor cells (28). Only 5 x IO6 cellsenter the B-cell pool, the rest presumably being eliminated byapoptosis.

The existence of a mechanism for cutting and splicing DNAcarries with it a risk that the wrong regions will be cut andspliced. Since the consensus signal sequences that are adjacentto the DNA segments that must be spliced together as part ofthe normal process of antigen receptor gene rearrangement arealso found quite widely distributed throughout the genome, it isnot difficult to envisage that genes with no known relationshipto antigen receptor genes may sometimes by spliced together atthat time in cellular differentiation when antigen receptor recombinases are expressed. Indeed, this has been shown to be soin the case of the gene known as tal-I or sci, situated on chromosome 1, which is fused with a gene that has been named sil(sci interrupting locus), also on chromosome 1, by the deletionof intervening DNA in some 20 to 30% of T-cell acute lympho-blastic leukemias (29, 30). While this particular genetic abnormality, like physiological antigen receptor gene rearrangement,is intrachromosomal, it has been shown that antigen receptorrecombinases are able to mediate interchromosomal recombinations (31 ). Thus, the antigen receptor gene recombinases areat least capable of mediating the most obvious genetic lesions inlymphoid malignancies, chromosomal translocations (27, 32).However, recombinase signal sequences have not been detectedat translocation breakpoints in all translocations occurring inlymphoid neoplasia. Whether these enzymes mediate translocations believed to occur close to the timing of antigen receptorgene rearrangement during cell differentiation, even if recombinase signal sequences are not evident, is a debated issue.

One of the implications of the possible mediation of genefusion by antigen receptor gene recombinases is the necessitythat such genetic aberrations must occur early in lymphoiddifferentiation, i.e., during that phase of differentiation whenthe recombinases are present in the cell. The stage at which therecombinases first appear has not been precisely identified.Since regulation of their function is probably through access tospecific genes via alterations in eliminatili structure, they arelikely to be expressed before a cell actually undergoes physiological recombination of antigen receptor genes. Pathologicalrecombination at this early stage of differentiation and resultantderegulation of the expression of a gene which may be appropriately expressed at this early stage may lead to its subsequentinappropriate expression as the cell undergoes further differentiation. This scenario is much more likely than fusion occurringat a differentiation stage when one of the genes is not normallyexpressed; fusion is intuitively unlikely when one of the partners is protected by a closed chromatin structure. If a geneticfusion has occurred that results in continued expression ofgenes that induce proliferation, the cell may only be recognizedas neoplastic at the point in the differentiation pathway whenits proliferation should normally cease, perhaps several stepsbeyond the stage in which the genetic change occurred. A mutation in a gene that prolongs the life span of a cell, but does notinfluence proliferation, may result in the accumulation of cellsat a terminal stage of differentiation, or at a point where furtherdifferentiation requires an external signal (e.g., antigen).

It is not necessary to surmise that all instances of gene fusionare mediated by recombinases that normally mediate antigenreceptor gene rearrangements. Other enzyme systems existwhereby genetic recombination can occur, for example, thosethat mediate the excision and reinterpolation of mobile geneticelements (e.g., transposons) and viral sequences. The possible

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MOLECULAR BASIS OF LVMPHOMAGENESIS

role of such recombinases in cellular gene fusion pertinent toneoplasia is largely unexplored.

Consequences of Genetic Abnormalities

The repertoire of genetic abnormalities that occur in lym-phoid neoplasia is constrained by functional considerations. Inany given cell type, only certain functional changes have relevance, and it is genes whose altered function can contribute toneoplasia that are referred to as oncogenes (genes whose expression normally promotes growth and which are associatedwith cancer when there is an enhancement of function) and asantioncogenes (genes whose expression normally inhibitsgrowth and loss of function is associated with cancer). Theterms "oncogene" and "antioncogene" are correctly applied

only in the context of pathologically altered function. In practice there are three general types of functional change to consider: enhancement; inhibition; and alteration of a function. Avariety of genetic lesions may result in inactivation of a gene,including a wide range of intragenic lesions, deletion of thegene, or altered expression of a second gene that regulates itstranscription. Enhancement of activity is the result of a muchsmaller subset of genetic lesions, since they must be quite specific. Such lesions may cause altered expression of a gene thatnormally regulates the first, juxtaposition to heterologous enhancer sequences or to the promoter of another gene (sometimes resulting in a fusion protein), deletion or mutation ofthe regulatory region of the gene, or mutation of those regionsof the protein product that are related to its enzymatic or protein binding properties. The inappropriate expression of a gene(e.g., at a stage of differentiation or a phase of the cell cyclewhen it should not normally be expressed), or lack of its expression, may lead to altered growth, prolonged life span, orinhibition of differentiation.

Deregulation of Transcription Factors and Genes Affecting theCell Cycle and Life Span

Genetic abnormalities associated with lymphomagenesis frequently, but not invariably, result in the deregulation of putativeor proven transcription factors such as c-myc, pbx, the rhom-botin genes, and sci (tal-\) (33-36). Clearly, such genes mayaffect a broad variety of molecules involved with any and everyfacet of the life of the cell, including proliferation and differentiation. Expression of the c-myc gene, for example, appears tobe associated with the ability of the cell to enter the cell cycle,i.e., to be "competent" to undergo DNA replication. There are

two major mechanisms whereby such genes are deregulated:juxtaposition to regulatory elements derived from an antigenreceptor gene and the formation of a fusion gene.

Role of Antigen Receptor Genes in Deregulation. A repetitivetheme in lymphoid neoplasia is the presence of a chromosomaltranslocation involving an immunoglobulin or T-cell antigenreceptor locus on one chromosome and a transcription factoron the partner chromosome. It seems highly probable that thetranscription factor is then regulated as if it were the antigenreceptor gene (Fig. 2A). In some cases, structural damage ispresent in the regulatory region of the transcription factor inaddition to it being juxtaposed to an antigen receptor gene.Such abnormalities may be relevant to the usurpation of itsregulation by enhancer regions within the antigen receptor locus (37).

Hybrid Transcription Factors. A second theme that is frequently observed in lymphoid neoplasia is the fusion of two

Fig. 2. Diagrammatic depiction of two types of genetic fusion that result inaberrant expression of genes, in this case, transcription factors. In A. a translocation has resulted in the juxtaposition of enhancer sequences (/ ) present in onegene (in lymphoid neoplasia, frequently an antigen receptor gene) to a transcription factor, causing its inappropriate expression (deregulation) and consequentinappropriate expression of the genes whose transcription it normally regulates.The deregulated transcription factor may either increase or supress transcriptionof other genes, although in this representation it is depicted as a transactivator. InB. a fusion gene has been produced, either by translocation or intrachromosomaldeletion. This results in the downstream component of the fusion gene beingexpressed as if it were the upstream component, since hybrid transcripts initiateat the promoter of the upstream partner. The downstream partner must containfunctionally competent regions (e.g., DNA binding regions). Once again, theeffect of the fusion gene could be to inappropriately activate or suppress the genesthe downstream partner normally regulates. />,promoter.

genes coding for transcription factors. In this circumstance, thefusion protein is expressed as if it were the upstream partner,which bears the regulatory regions, including promoters, thatnow govern the expression of the fusion gene. The pattern ofDNA binding, however, is probably determined by the downstream partner (Fig. IB). In effect, the genes regulated (up ordown) by the downstream transcriptional factor will be so regulated at a time in the differentiation process, or possibly cellcycle, when the upstream gene is normally expressed, ratherthan when the downstream component is normally expressed.It requires little imagination to see what damage could bewrought by the inappropriate expression of a gene that controlsthe expression of many other genes. In some cases, inappropriate expression of genes, even though they are irrelevant to theneoplastic state, may account for the aberrant immunopheno-types that are sometimes seen in lymphoid neoplasms.

Deregulation of End-Function Genes. While inappropriateexpression of transcription factors could result in a wide rangeof aberrant functional consequences, only a limited number ofsuch aberrations would be relevant to neoplasia, includingalterations in the regulation of proliferation, differentiation,and life span. Abnormal expression of genes that participate in

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these functions may also be occasioned by genetic aberrationsthat affect them directly. Not surprisingly, similar mechanismsof deregulation to those described already apply. The abnormalexpression of bcl-2, consequent upon its juxtaposition to im-munoglobulin sequences, and the role of this gene in the inhibition of apoptosis have been mentioned above. A more recentlydescribed example is the 11;14 chromosomal translocation inwhich a gene, bcl-l, which has strong homology to the family ofgenes known as "cyclins," involved in the regulation of cell

cycle progression, is juxtaposed to regions of the immunoglo-bulin heavy chain region (38). The 11;14 translocation ispresent in a type of small cell lymphoma referred to as a lym-phocytic lymphoma of "intermediate differentiation," a subtypeof which is referred to as a "mantle zone" lymphoma (centro-

cytic lymphoma in the Kiel classification) (38, 39).

Altered Function of Genes Involved in Cellular Proliferationand Differentiation

In addition to the deregulation of the expression of genes thatregulate vital cellular processes, the function of the genes thatdirectly mediate these processes may be modified by geneticchanges. In this case, the protein product of the gene must beaffected such that there is either a loss of function (e.g., in thecase of genes that are necessary for apoptosis to occur or thatinhibit cellular proliferation or promote differentiation) or anincrease in function (e.g., in the case of genes that inhibit apoptosis or differentiation or promote cellular proliferation). Alarge number of genes are involved in these processes, many ofwhich have already been recognized as oncogenes or antionco-genes. Detailed reviews have been provided elsewhere (40, 41).In brief, oncogenes include molecules capable of functioning asligands for growth factor receptors (e.g., sis), as activatedgrowth factor receptors present on the cell surface (e.g., erbA.and erbB), as activated signal transducing molecules, normallyassociated with the cytoplasmic aspect of the cell membrane(e.g., ras), as cytoplasmic signalling molecules (e.g., src) or astranscription factors (e.g., myc, myb, fas, jun, pbx, rhom-\, andrhom-2) normally present in the cell nucleus. Not surprisingly,abnormalities of genes involved in the same functional pathwaymay give added or synergistic pathological effects. Antionco-genes include the retinoblastoma gene, rb, the Wilms' tumor

gene, and the gene involved in carcinoma of the colon. Onlymutations in the rb and p53 genes have so far been described inlymphoid neoplasia (42).

The Special Case of pS3. The p53 gene is a special casebecause it appears to be able to contribute to neoplasia in twomodes, either as an oncogene or an antioncogene (43, 44). Thisis probably a consequence of the type of mutation that occurs inthis gene, for some mutations result in loss of function (namely,its ability to inhibit cellular proliferation) and some in the gainof a new function (e.g., the binding of a heat shock protein,HSC70, although the significance of this to oncogenesis is unclear). There is no doubt that/>53 plays an important role in theinhibition of cellular proliferation and has different conforma-

tional forms that it assumes in different phases of the cell cycle(45). Some mutations may result in its assuming permanently aconformation that should normally be present only transiently,during a specific phase of the cell cycle, and it may then beunable to inhibit cellular proliferation. It has also been shown inexperimental systems that some mutated forms of p53 can cooperate with ras genes in the induction of malignant transformation. While it has been postulated that this is a dominant

negative effect, i.e., the mutated protein simply binds to andconsequently inactivates the normal protein, thus preventinginhibition of cellular growth, this may be an oversimplification.Apart from its relevance to cellular proliferation, mutations inp53 could affect several pathways relevant to lymphomagenesis.p53 appears to be required for normal B-cell differentiation(46) as well as for apoptosis (10), and its lack (e.g., by "loss-of-function" mutations) could thus impair differentiation and lead

to prolonged life span. Mutation may also be relevant to theemergence of chemotherapy-resistant clones, since induction of

apoptosis appears to be one mechanism whereby cells are killedby chemotherapeutic agents (47). Thus, lack of p53 could leadto lack of chemotherapeutic efficacy by preventing apoptosis. Inaddition, mutated p53 has been shown to transactivate themdr-l gene involved in pleiotropic drug resistance, whereaswild type/753 may suppress mdr-\ (48).

p53 mutations appear to be relatively uncommon in lymphoid malignancy. Only the small noncleaved cell lymphomasfrequently possess p53 mutations (30% in primary tumors and70% or more in derived cell lines). Diffuse small cell lymphomas and chronic lymphocytic leukemia hnvep53 mutations in asmall percentage of cases (49, 50).

Association of Specific Genetic Lesions with ParticularLymphoma Subtypes

The phenotype of a cell results from the expression of alimited set of genes, and somatic genetic changes of consequence are not only more likely to occur in these genes, becauseof their open chromatin structure, but are unlikely to affect thecell if they occur in genes that are not expressed. Moreover,different genetic changes will have different cellular consequences, e.g., deregulation of myc causes continued proliferation, whereas deregulation of bcl-2 prolongs life span. It is not,therefore, surprising that a number of distinct pathological entities can be defined, each of which has a characteristic histolÃ³gica! appearance, immunophenotype, nonrandom chromosomal translocation (Fig. 3), and clinical behavior pattern. Inaddition, each lymphoma can be reasonably well equated with aparticular normal counterpart cell (Figs. 4 and 5). This statement needs to be qualified by the recognition that what appearto be identical chromosomal translocations can occur in morphologically different lymphomas. It is important to note thatthis is not the case for lymphomas that are of different celllineage. Within a lineage, however, the same translocation mayappear in tumors with different histology, e.g., 8; 14 translocations in small noncleaved cell lymphomas and a subset of diffuse large cell lymphomas, and 14; 18 translocations in all histological categories of follicular lymphomas (including small,mixed, and large cell) as well as a subset of diffuse large celllymphomas and small noncleaved cell lymphomas (51, 52).

There are several possible explanations for these findings.For one thing, there is a morphological continuum betweensmall noncleaved cell lymphomas and large cell lymphomas(53), on the one hand, and small, mixed, and large cell follicularlymphomas on the other (54). This morphological, as well asgenetic, similarity implies that the lymphomas in each seriesmay be closely related to each other with respect to their patho-genesis. Interestingly, there is evidence that there is a geneticspectrum too, the translocations of different histolÃ³gica! subtypes varying with respect, for example, to the locations ofchromosomal breakpoints (55, 56). It is entirely possible thatthe associated genetic lesions also differ. Indeed, in lymphomas

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Precursor cell

MC PC

Fig. 3. Diagrammatic depiction of differentiation pathways of cells of thelymphoid lineage and the association of specific chromosomal translocations withparticular phenotypes. Each translocation is more likely to arise in a specific celltype, because (a) the level and pathway (lineage) of differentiation determine thechromatin pattern in DNA regions that may be involved in a translocation, (b)enzymes that mediate the translocation may be differentiation stage specific, and(c) the functional consequences of the translocation have relevance limited to onelineage and possibly to only a particular range of differentiation stages. Thehypothetical multipotential cell shown here is able to differentiate into T-cellprecursors, in which chromosome translocations involving the T-cell antigenreceptor genes (14ql l, n and i: 7q35, lÃ¬;and 7pl3. y) can provide a critical lesionin the pathogenesis of T-cell acute lymphoblastic leukemia (ALL) or lymphoblas-tic lymphoma (II}. or into B-cell precursors in which chromosome translocationsinvolving the immunoglobulin heavy chain genes on chromosome 14q32 providea critical lesion for the development of various B-cell neoplasms, including smallnoncleaved cell lymphoma (SA/O, large cell lymphoma (Â¿O. mantle zone lymphoma (MZL), and small cleaved cell lymphoma (SCC). PB, pre-B-cell; VL, virginlymphocyte; AL, activated lymphocyte; MC, memory cell; and PC, plasma cell.

BONEMARROW

Â«CUTELYMPHOBLASTIC

LEUKEMIA

^ N N

ITHYMUS

.\N \XVXX NX V

'

tiÃ®kxxxx

\X X X XJ\XX

v X X XXXXXXXX'^ X X X N At X X X \ 'v X X X V^. X X X X '\X X X X X X X X X V

ACUTELYMPHOBLASTIC

LEUKEMIA

LYMPHOBLASTIC

CELLLYMPHOMASKIN

PERIPHERAL T CELLLYMPHOMA5

LYMPHOID TISSUE

Fig. 4. Diagrammatic depiction of the cellular origins of lymphoid neoplasmsarising from various stages of differentiation of T-cells. The most immatureprecursors arise in the bone marrow; migrate to the thymus where further antigen-independent differentiation occurs, and then exit to peripheral lymphoid organsincluding the skin, lymph nodes, liver, and spleen. Reproduced with permissionfrom Ref. 73.

where more than one genetic lesion has been described, one ofthe lesions may be present in only a subset of tumors [e.g., p53mutations in small noncleaved cell lymphomas (49, 50)]. In thecase of follicular lymphomas, the development of additionalgenetic lesions (e.g., an 8; 14 translocation, or simply more ane-uploidy) is associated with histolÃ³gica! evolution from follicularto diffuse morphologyâ€”a form of evolution rather like a blast

crisis in chronic myeloid leukemia. These transformations areassociated with a rapid downhill clinical course (57-59). It isprobable that similar considerations apply to more aggressive(intermediate or high grade lymphomas), although this is lessobvious because, as a group, their behavior is readily distinguished from that of the low grade lymphomas. However, theclinical course of individual patients with diffuse aggressivelymphomas varies quite markedly, and recurrent tumors mayproliferate much more rapidly than primary tumors. We mayconclude that the borderline between one type of lymphomaand another, arising from the same lymphoid lineage, is oftenblurred, both with respect to histology and genetic abnormalities, although broad distinctions can quite readily be made. Weshould not, perhaps, be surprised at this, since the distinctionsmade between the differentiation stages of normal lymphoidcells are similarly somewhat artificial. The use of genetic lesionsfor taxonomic purposes does, however, have the advantage ofgreater objectivity. In addition, subsets of histologically identical tumors (e.g., follicular lymphomas with and without a 14; 18translocation or molecular variants of the small noncleavedlymphomas) can be readily distinguished (60, 61). Whetherthese different subsets of tumors behave differently at a clinicallevel, or have a different response to treatment, remains to bedetermined.

Correlation of Pathological Anatomy, Clinical Features, andGenetic Abnormalities

While there is much to be learned of the genetic lesionsassociated with lymphoid neoplasia, a clear pattern is beginningto emerge. The broad distinctions of low, intermediate, andhigh grade lymphomas made by the National Cancer InstituteWorking Formulation are somewhat artifactual, since there isconsiderable overlap between the intermediate and high gradecategories with respect to clinical behavior. There do seem tobe, however, more and less aggressive groups, based on theirclinical behavior, even though within these groups there is arather broad spectrum of aggressiveness. Follicular lymphomasand small cell lymphomas, for example, which fall into the lowgrade category, are compatible with long survival, even withminimal or no therapy. Patients with these forms of lymphomausually die as a result of conversion of the lymphoma to a moreaggressive form, or from the complications of treatment rather

IMALL CELL LVMFHOUA

ACirrtLVyPHOflLAlTI

1! UK MlÂ» PrCVL

MANTLE ZONE ITMPHOMA

PC

MC â€¢¿�

IB

tvMUNOILAITIC

Fig. 5. Diagrammatic depiction of the cellular origins of lymphoid neoplasmsarising from various differentiation stages of B-cells. The most immature precursors arise in the bone marrow (precursor cell. PrC) and differentiate withoutrequirement of antigen. On becoming virgin lymphocytes, the cells recirculatethrough primary follicles. If exposed to antigen, a germinal center develops, theremnants of the primary follicle forming the mantle zone. Activated cells give riseto immunoblasts (IB) which either become memory cells (MC), which also recirculate, or plasma cells, which migrate to the bone marrow. PC, plasma cell; VL,virgin lymphocyte. Reproduced with permission from Ref. 73.

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than from the underlying process itself. Interestingly, to datethere is no convincing evidence that any patient with more thanlocalized low grade lymphoma has been cured. On the otherhand, patients with intermediate or high grade lymphomas usually have a very rapid clinical course if untreated, but respondwell to chemotherapy and can be cured in a high proportion ofcases (60% or more).

There are clearly basic differences in the types of geneticlesions that are associated with low grade lymphomas and thoseassociated with intermediate and high grade lymphomas. Theformer result primarily in the slow accumulation of neoplasticcells in anatomically appropriate areas. Thus, follicular lymphomas and high grade lymphomas are, for the most part,confined to the reticuloendothelial system, including the lymphnodes, liver, spleen, and bone marrow. Moreover, the architectonics of the cellular accumulation bears a considerable resemblance to that of the normal counterpart cells. Follicular lymphomas are so called because of their histological andphenotypic similarity to normal secondary follicles in lymphoidtissue, while mantle zone lymphomas often assume a quasifol-

licular pattern quite consistent with the immunophenotypic resemblance of these lymphomas to the cells which normallycompose the primary follicles and the mantle zone of secondaryfollicles (39).

Low grade lymphomas tend to be widely disseminated (lessthan 10% are localized at presentation), consistent with therecirculation of their normal counterpart cells through the lymphoid tissue. Moreover, they retain, in large measure, the migratory and homing characteristics of their normal counterpartcells. Indeed, there is evidence that the follicular pattern offollicular lymphomas is dependent upon the presence of dendritic reticular cells, which are always present in both normaland neoplastic germinal follicles (62). Interestingly, there isgood evidence that the chromosomal translocation associatedwith the majority of follicular lymphomas, t(14;18), occursclose to the time of immunoglobulin gene rearrangement (thebreakpoint occurs at a J region adjacent to a heptamer-nono-mer signal sequence), suggesting that the neoplastic clone undergoes differentiation prior to its manifestation as a tumor. Itwould, therefore, appear that the central abnormality of theneoplastic clone in low grade lymphomas is a tendency to abnormal accumulation at a specific stage of differentiation (inthe case of mantle zone and follicular lymphomas, at the stageof recirculation through lymphoid follicles). This is entirelyconsistent with the deregulation of the bcl-2 gene that is theconsequence of the 14; 18 translocation (although this translocation is probably insufficient, by itself, to induce a lymphoma).Perhaps all low grade lymphomas result from genetic lesionsthat have a similar functional consequence.

In contrast to the low grade lymphomas, other lymphomasare associated with more aggressive growth and less adherenceto the anatomical distribution of the presumptive normal counterpart cell, although high grade lymphomas manifest evidenceof some retention of the migration characteristics of their lineage, e.g.: the frequent involvement of the thymus in lympho-blastic lymphoma, the neoplastic counterpart of precursor T-cells and of the skin in several T-cell lymphomas of peripheralT-cells, e.g. mycosis fungoides, SÃ©zarysyndrome, and the T-celllymphoma associated with HTLV-1 (Fig. 4). Spread beyondlymphoid tissue is much more frequent and extranodal lymphoma is common; almost invariably the case in small non-

cleaved cell lymphomas, for example. This may reflect in partthe stage of differentiation of the predominant tumor cells and

in part a greater departure from the controlling influence ofgrowth factors and homing receptors. It seems likely that thebasic lesion of the more aggressive lymphomas is retention ofthe ability to proliferate regardless of the differentiation stageand development of a greater degree of autonomy than the lowgrade lymphomas. Autonomy, however, is relative. Clinical evidence suggests that growth factors may influence the likelihood that a lymphoma will grow in certain anatomical locations. In Burkitt's lymphoma, for example, there is a very high

propensity of the tumor to grow in the developing molar toothbuds of young children in Africa and to involve the breasts ofpubertÃ ! and lactating females (63).

Interaction of Environmental Factors with the Cell Genome inLymphomagenesis, as Exemplified by Burkitt's Lymphoma

Variations in the incidence of specific types of lymphomathroughout the world, or even in different populations within asingle country, demonstrate that the development of a malignant lymphoma is not due to chance alone. There is little doubtthat both inherited and environmental factors are important,although the latter may be the major determinant of incidencein a given geographical region. Perhaps the genetic backgroundof a subset of individuals in any population leads to heightenedsusceptibility to lymphoma when exposed to relevant environmental factors. Relevant environmental factors include microorganisms, mitogens (e.g., phorbols), DNA damaging agents(e.g., chemicals), and immunosuppressants (e.g., malaria, virusinfections, or drugs). Microorganisms may be important byvirtue of their immunogenic or mutagenic properties (e.g.,through causing inflammation and free radical generation) or,in the case of viruses, via direct action on cellular genes or theirprotein products.

The variations in the incidence of different types of non-Hodgkin's lymphoma with age provide a number of insights

into lymphomagenesis. For example, low grade lymphomas,the essential characteristic of which is the slow accumulation ofabnormal cells, essentially never occur in young children andare rarely observed before the age of 25 yr. On the other hand,children may be more likely to develop highly proliferative neoplasms that originate in lymphoid precursor cells, perhaps because the total number of such cells is higher in the first decadeof life (as evidenced by, for example, the size of the thymus).The late appearance of follicular lymphomas may simply be aconsequence of the time required for a sufficient number ofgenetic lesions to accumulate (a likely probability in view of thefinding of rearranged bcl-2 genes in normal tonsils in children),for sufficient numbers of lymphoma cells to accumulate, orboth. Follicular lymphomas are also quite uncommon in developing countries (19). Whether the risk of transformation intoan aggressive tumor (i.e., as a consequence of additional geneticchanges) is higher in the setting of a developing country, suchthat a clinical phase of a low grade lymphoma is rarely seen, orwhether the bcl-2 translocation (or one of the additional geneticlesions required for the development of low grade lymphoma)rarely occurs in the developing countries remains unknown.The various factors that influence the development of a lymphoma are summarized in Fig. 6.

It is reasonable to assume that, while the degree of exposureto environmental factors may vary markedly, even in the samegeographical region, the stochastic element in lymphomagenesis is the major explanation as to why not all individuals withthe same environmental exposure develop a tumor. Burkitt's

lymphoma provides an excellent paradigm to explore further5536s




Â®/ * *

Â« Â«I 4

Â®â€¢¿�* * *Â®Â®Â®

Inherited Genetic Factors

Oncogene mutations

Ability to repair DNA

â€”¿� â€¢¿� Â® A' > â€¢¿� ^^^ ^â€”â€¢â€¢

Hyperplasia Environmental Factors

CfwmiCJk

â€¢¿� Neoplasia

Fig. 6. Diagrammatic depiction of the factors that influence the risk of developing lymphoid neoplasia. These include inherited genetic factors which mayinclude mutations in genes relevant to the pathogenesis of cancer particularly inantioncogenes, spontaneous chromosomal breakage and defects in DNA repair,or immunodeficiency syndromes. Age presumably influences the absolute numbers of target cells for pathogenctic events, and various environmental factors mayinduce hyperplasia of specific cell populations and/or genetic abnormalities.

breakpoints close to or within the c-myc gene. South Americantumors appear to be a mixture of these two types, but manytumors in Argentina have breakpoints in an intermediate position (Fig. 7). Similarly, EBV association is 95% in Africa, 20to 30% in the USA, and some 50 to 60% in South America(60,64). These relative figures do not do justice to the very largedifferences in incidence of Burkitt's lymphoma subtypes in

different world regions. The subtype most common in Africa(approximately 75% of cases) has an incidence some 70 to 100times higher in Ghana than in the USA. In contrast, the molecular variants common in the USA may have a similar or onlyslightly increased incidence in Africa.

It is not known precisely how the geographical location caninfluence both the site of the chromosomal breakpoints and thepresence or absence of EBV DNA in the tumor cells. As moreinformation accumulates, however, an overall picture is beginning to emerge, even though the molecular events relevant topathogenesis will, of necessity, always remain hypothetical (37).

Percentage

the interaction between the environment and the cells of theimmune system.

Burkitt and his colleagues (63) demonstrated that, in equatorial Africa, where the incidence of the tumor is relatively high,the distribution of Burkitt's lymphoma is climatically deter

mined; an early observation was the coincidence of its distribution with that of mosquito-vectored diseases such as yellowfever, O'Nyong Nyong, and malaria. This led to the hypothesisthat Burkitt's lymphoma may be caused by a virus transmitted

by insects, a hypothesis that led directly to the discovery of EBVin cell lines derived from tumor samples obtained from Africanpatients. Until recently, in spite of the subsequent demonstration that some 95% of African Burkitt's lymphomas contain

EBV DNA, there was no evidence that the virus was causallyassociated with the pathogenesis of the disease. Indeed, therecognition that Burkitt's lymphoma in temperate countries is

much less often associated with EBV (20 to 30%) gave rise tosome confusion, since it demonstrated that EBV is clearly notalways essential to tumorigenesis.

Chromosomal Breakpoint Locations Differ in DifferentWorld Regions. To a large degree, this confusion has beenresolved by the finding that, although Burkitt's lymphoma

throughout the world contains the same translocations (8; 14,8;22, or 2;8, all of which result in c-myc deregulation), thebreakpoints on chromosome 8, and the combination of breakpoints on the two partner chromosomes in the translocation,differ in different world regions (60). In other words, thereappear to be molecular subtypes of Burkitt's lymphoma. It is

probable that EBV contributes to some of these subtypes, but isnot required in others. The observation that there are differences in the incidence and clinical features of the tumors intropical and temperate zones suggests that the molecular differences result in clinical differences (63). Remarkably, thereappear to be two "gradients" that can be discerned with respect

to EBV association and chromosomal breakpoint location. Thisis exemplified by the characteristics of the tumor in Africa(Ghana), North America (the USA), and South America(Argentina, Chile, and southern Brazil). Most equatorial African tumors have breakpoints far away from the c-myc gene onchromosome 8, while most North American tumors have

5537s

Regulatoryregion

Breakpoint region

Trintcfiptiortal unit

Percentage

Regulatory region

Breakpoint locationChromosome 8

En1

P2Â«-P3

.,,

PsPvS

Fig. 7. Diagrammatic depiction of the patterns of chromosome 8 breakpointlocations in a series of over 90 Burkitt's lymphomas from various world regions.

1. breakpoints in tumors from Ghana and the United States; lÃ¬.breakpoints intumors from Brazil and Argentina: and C. diagrammatic depiction of the upstream region of the c-myc gene in which most of the breakpoints occur. Farupstream indicates a breakpoint outside the upstream ///mill I restriction enzymesite bounding c-myc. breakpoints within the regulatory region are between this////Â»II II site and the .S/////1site immediately upstream of the first c-myc exon, whilebreakpoints designated as within the transcriptional unit are downstream of theSmut site, usually within the first exon or intron, downstream of the predominantly used c-myc promoters, PI and Pi. While the majority of these tumors have8:14 translocations, the possibility that some of those included as "far upstream"have a "far downstream" breakpoint, as occurs in the variant (2;8 or 8;22) trans-

locations, cannot be excluded. H. ///mil 11:Ps. Pst\; P\: Pvu\i; S. Sma\. PO and Piare minor promoters but Pi is used when the breakpoint is within the transcriptional unit.




The primary molecular genetic abnormality is the chromosomal translocation which results in the juxtaposition of c-mycto immunoglobulin sequences and the consequent expression ofthe c-myc gene as if it were an immunoglobulin gene. In a cell

of the B lineage, this effectively means continued expression ofc-myc and maintenance of the cell in a proliferative phase.There is evidence that the translocations occur in immatureB-cells, close to the time of (27) or even before immunoglobulingene rearrangement. It seems probable, from the epidemiolog-ical evidence, that the chromosomal breakpoint location is determined largely by environmental factors. While there is noevidence that EBV can influence the breakpoint location, otherenvironmental agents (e.g., microorganisms or plant productssuch as phorbol esters) may be able to do so, perhaps by influencing the numbers of B-cells at various stages of differentiation. Malaria, in fact, has been shown, in mice, to cause anincrease in the rate of production of B-cell precursors, particularly early and intermediate pro-B-cells, and increased precursor B-cell loss (65). Purely on a statistical basis, this couldincrease the risk of a genetic abnormality occurring in suchcells, but if the increased cell loss indicates an increase in therecombinase error rate, as has been suggested, then the risk ofdeveloping a genetic abnormality may be greater than would besuggested by the increase in precursor cell numbers alone.Moreover, the stage specificity of the effects of an environmental agent may be important both with regard to the fragility ofvarious regions of the involved genetic loci (because of differentpatterns of proteins bound to the DNA of these regions) andwith respect to the functional consequences (on c-myc transcription) of the breakpoint locations. Cells at different stagesof differentiation, for example, may express different proteinfactors relevant to c-myc or immunoglobulin regulation.

EBV Can Cooperate with the c-wyc/Immunoglobulin Trans-

location. Recently, we have been able to demonstrate that oneof the EBV latent genes (i.e., genes expressed only in the nonreplicative phase of EBV infection), EBNA1, can collaboratewith the chromosomal translocation (66) in affecting the transcription of c-myc, and it seems likely that in some circumstances the presence of EBV is essential to the deregulation ofc-myc. Factors which influence the number of EBV-containingcells in the body and the rate of infection of cells which have thepotential to undergo transformation into tumor cells (e.g., immature B-cells in the bone marrow, particularly the bone marrow of the jaw in African children) probably influence the riskof the development of Burkitt's lymphoma. Factors that have

been shown to increase the level of EBV-infected B-cells inperipheral blood by impairing EBV-specific immunity includemalaria (or inherited or acquired immunosuppression, including AIDS), while phorbol esters, derived from a plant, Euphorbia tirucalli, widely used for medicinal purposes in Africa, appear able to increase the efficiency of infection of EBV as wellas to increase the likelihood of translocations (67, 68). Malariaand Euphorbia in combination, therefore, may increase the levels of specific B-cell precursors in bone marrow, increase thelikelihood that they will be infected by EBV, and perhaps alsoincrease the risk of a genetic error. The additive effect of theseeffects may well be enough to explain the greatly increasedfrequency of a particular molecular subtype of EBV-associatedBurkitt's lymphoma in equatorial Africa. The early age of EBV

infection (100% by 3 years of age) in Africa presumably alsocontributes to the epidemiolÃ³gica! picture.

In other world regions there is no evidence that climaticfactors influence distribution, but environmental agents not en

countered in Africa, possibly other infectious agents, may stillinfluence the frequency of EBV-associated Burkitt's lymphoma

as well as the overall incidence of the tumor and the chromosomal breakpoint locations in any given geographical region.Interestingly, EBV can be associated with all molecular subtypes of Burkitt's lymphoma (60, 64), suggesting that cooper

ation with wyc/immunoglobulin translocations does not involve a direct interaction with c-myc regulatory elements.

Other Genetic Lesions. There is experimental evidence frommice transgenic for c-myc that, although the myc/immuno-globulin translocations can lead to continued proliferation ofprecursor B-cells, such that they do not enter a resting phase

as they differentiate, additional genetic lesions (which can substitute for each other) are required to generate a neoplasm(69, 70). The infrequency of occurrence of these additionallesions presumably accounts for the monoclonality of the resultant tumors. In Burkitt's lymphoma, several genetic abnor

malities in addition to the c-w.lv/immiinoj4lobulin transloca

tions have been identified, the most prominent being p53mutations that occur in some 30% of freshly biopsied tumors(49, 50). Occasional ras and Rb mutations have also been observed (71). No correlations between these additional geneticlesions and geographical origin, the presence or absence ofEBV, or the chromosomal breakpoint location have been discerned to date, and it appears that a number of alternativelesions may be able to contribute to pathogenesis, as is the casein Ep-myc transgenic mice (69, 70). One of these is presumablyable to substitute for EBV in EBV-negative tumors.

Hypothesis

These observations can be assembled into a hypothesis toaccount for the environmental interactions that determine theincidence of Burkitt's lymphoma. While it is highly probable

that several genetic lesions are necessary to induce the neoplasm, a central requirement is myc deregulation. This is accomplished by the wyc/immunoglobulin translocations whichoccur early in B-cell differentiation. The functional result ofderegulated myc expression is maintenance of the cell in a perpetually proliferative state, even though it can mature to thestage of an immunoglobulin-expressing and even secreting cell.The perpetual proliferation of cells bearing a translocation, andpresumably the expansion of the cell clone, increases the likelihood of the development of additional genetic abnormalities,although it appears that the nature of these additional lesionsvaries from one tumor to another, suggesting that several different mutations are able to interact with the chromosomaltranslocation during oncogenesis. EBV can also cooperate withthe translocation, and whether or not it is an absolute requirement is probably dependent upon either the specific stage ofprecursor cell in which the translocation occurs or perhaps thepresence or absence of other, environmentally determined genetic lesions. Factors that increase the numbers of circulatingEBV-infected cells (which may thus affect the number of B-cellprecursors infected with EBV), including malaria, AIDS, andperhaps early age of EBV infection increase the risk of thedevelopment of EBV-associated Burkitt's lymphoma. Finally,

the likelihood that a translocation will occur is dependent uponthe number of potential target cells present in an individual, afactor that is influenced by age, the environment, and life-style,while the relative proportions of cells at different levels of differentiation probably influence the molecular characteristics(chromosomal breakpoints) of the translocation.

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Clearly major strides have been made in recent years in understanding the pathogenesis of lymphomas at a molecular level. It is to be hoped that this new information, and informationyet to come, will be of practical as well as theoretical value, i.e.,that it will lead to improved diagnosis and classification, tomeasures designed to prevent the continuing increase in theincidence of non-Hodgkin's lymphomas and, perhaps also, to

new approaches to treatment.

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phomas. pp. 15-28. London: Arnold, 1990.2. Magrath, I. Infectious mononucleosis and malignant neoplasia. In: D.

Schlossberg (ed.). Infectious Mononucleosis, Second Ed., pp. 142-193. NewYork: Springer Verlag.

3. Filipovich, A. H., Shapiro. R., Robison, L., Mertens, A., and FrizzerÃ , G.Lymphoproliferative disorders associated with immunodeficiency. In: I. T.Magrath (ed.). The Non-Hodgkin's Lymphomas. pp. 135-154. London: Ar

nold. 1990.4. Fialkow, P. .1.. Klein, E., Klein, G.. el al. Immunoglobulin and glucose-6-

phosphate dehydrogenasc as markers of cellular origin in Burkitt's Kmphoma. J. Exp. Med.. 138: 89-102, 1973.

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13. Zelenetz. A. D., Campbell, M. I., Bahler, D. W.. Takahashi, S.. et al. Follicular lymphoma: a model of lymphoid tumor progression in man. Ann.Oncol., 2(Suppl. 2): 115-122. 1991.

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18. Price, C. G., Meerabux, J.. Murtagh. S.. Cotter, F. E., Rohatiner. A. Z.,Young, B. D., and Lister, T. A. The significance of circulating cells carryingt(14;18) in long remission from follicular lymphoma. J. Clin. Oncol., 9:1527-1532.1991.

19. Bressac. B.. Kcw, M.. Wands, J., and Ozturk, M. Selective G to T mutationsof p53 gene in hepatocellular carcinoma from southern Africa (see comments]. Nature (Lond.). 350: 429-431. 1991.

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1992;52:5529s-5540s. Cancer Res Ian Magrath Molecular Basis of Lymphomagenesis

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