The molecular analysis of chromosomal translocations as a diagnostic, epidemiological and potentially prognostic tool in lymphoid neoplasia

Journal of Virological Methods, 2 1 (1988) 275-289 Elsevier

275

JVM 00782

The molecular analysis of chromosomal translocations as a diagnostic, epidemiological and potentially prognostic tool in lymphoid neoplasia Ian Magrath, Pediatric Branch.

Fran&co

Barriga, Mary McManaway Shiramizu

and Bruce

Division of Cancer Treatment. National Cancer Institute, NIH, Bethesda, Maryland, U.S.A.

_ Summary Neoplasms have been shown to be associated with specific non-random chromosomal abnormalities. The present paper summarizes molecular consequences of chromosomal translocations in lymphoid malignancies, especially in Burkitt’s lymphoma. Among such consequences are derangements of the regulation of oncogene transcription. Besides their possible importance for lymphoma pathogenesis, chromosomal translocations are associated in some lymphoid neoplasms with a worse prognosis. Lymphoid

neoplasia;

Chromosomal

translocation;

Oncogene;

Prognosis

Introduction As the techniques of cytogenetic analysis have improved, more and more neoplasms have been shown to be associated with specific or non-random chromosomal abnormalities. It seems probable that all true lymphoid neoplasms result from genetic changes which cause abnormalities of proliferation and differentiation, in contradistinction to non-neoplastic lymphoproliferative syndromes which arise because of a host immunological deficiency and an inability, therefore, to control the

Correspondence stitute, N.I.H..

to: I. Magrath, Pediatric Branch. Bethesda, MD 20892, U.S.A.

Division

of Cancer

Treatment,

National

Cancer

In-

TABLE

1

Specific and non-random kin’s lymphomas

chromosomal

abnormalities

in acute lymphoblastic

leukemia

and non-Hodg-

Translocation

Oncogene

Disease

t(1; 19) (q7.1; q13) t(1; 19) (q23; p13.3) t(9: 22) (434; ql1) t(4; i 1) (q21: q23) t(X: 14) (q24; qil) t(lO; 14) (~24: qil) t(ll: 14) (p13: ql1) t(14: 14) (qll; q32) t(8: 14) (q24: q32) t(8: 22) (q24; qll) t(2; 8) (pll; q24) t(14; 18) (y32: q21) t( 11: 14) (q13: q32)

?

ALL (Ll) ALL (pre B) ALL (Ll and L2) ALL (iymphoid/mon~)cytic) ALL (T-cell) ALL (T-cell) ALL (T-celi) ALL (T-cell) ALL (L3)lSNCC Iymphoma ALL (L3)ISNCC lymphoma ALL (L3)iSNCC lymphoma FL. DLCL. SNCC lymphoma CLL. DLCL lymph. myeloma

c-my

c-m_vc 9

association

proliferation of virus-infected cells, In addition, pathologic entities defined on the basis of histological or even, in the case of the lymphoid neoplasms, immunophenotypic homogeneity, have been shown to be divisible into several subtypes containing different cytogene~ic abnormalities, frequently chromosomal translocations (Table 1). Only a handful of these cytogenetic abnormalities have been analyzed at a molecular level, but those which have, provide convincing evidence that the genetic abnormality is a critical component of the pathogenesis of the disease. In fact, the presence of specific, non-random genetic abnormality may provide the most precise means of identifying a pathologic entity, and as such, becomes an invaluable tool to the clinician and epidemiologist. Possibly of even greater potential significance is the understanding of the biochemical consequences of the genetic abnormalities which has resulted from molecular analysis of the chromosomal abnormalities. This has provided insights into the mechanism of malignant transformation, and could lead to the development of novel therapeutic approaches. We shah confine this paper to a brief review of the molecular consequences of some of the best studied translocations occurring in the lymphoid malignancies. and attempt to demonstrate how these provide not only the beginnings of a true understanding of the pathogenesis of the disease. but also permit the further subdivison of a histologic entity into subgroups with epidemiological, and potentially prognostic significance. Molecular analysis provides a tool of considerably greater sensitivity than cytogenetic analysis, since not only can the presence of a specific chromosomal translocation be inferred, but, by providing more precise analysis of the genetic abnormality, can often distinguish between individual tumors with the same cytogenetic abnormality. Since the chromosomal translocations are not present in normal cells, the resultant structural genetic changes provide specific markers for the tumor cell clone which should prove to be of value in the follow up of patients. and can permit the distinction between true recurrence and reinduction

of tumor in high risk patients - a point which may be relevant to the planned treatment approach. Because molecular analysis provides an approach to the understanding of the pathogenesis of lymphoid neoplasms, we also believe that it is probable that it will provide definitive answers to the question of the significance of the association af viruses with human neoplasia. We have used the association of Epstein-Barr virus (EBV) with endemic Burkitt’s lymphoma as an example to illustrate possible approaches to this problem. Finally, the molecular genetic abnormalities represent a potential and highly specific target for therapeutic endeavors, and it may be possible to develop molecules or drugs which react with abnormal mRNA or proteins present in tumor cells but not normal cells.

Molecular analysis of chromosomal translocations in hemopoietic neoplasia As more chromosomal translocations are analyzed at a molecular levef, a common pattern is beginning to emerge. The translocations are always reciprocal and involve: (a), a gene necessary for cellular differentiation or proliferation, or both - i.e. a protooncogene - and (b), a gene that is obligatorily expressed in the cell lineage from which the neoplasm has arisen. Lineage associated or presumptively lineage associated genes which have been identified in chromosomal translocations include the immunoglobu~in genes. the T-cell antigen receptor genes, and the socalled ‘bcr’ region of chromosome 22 (a gene which may be expressed in a variety of hemopoietic cells). Oncogenes, or presumptive oncogenes participating in the translocations, include c-myc, c-abl, and bcl-2. In each case, it appears that the expression and/or functional properties of the oncogene are altered as a consequence of its juxtaposition to lineage associated genes. Sometimes, as in the case of c-&l, the translocation results in the fusion of the major part of the oncogene to the promoters and part of the 5’ end of the lineage associated gene. In this circumstance the oncogene is regulated and expressed as if it were the lineage associated gene. In translocations involving the T- and B-cell receptor genes which have been studied so far, the mechanism whereby the oncogene is expressed inappropriately does not involve the promoters of the lineage associated genes, and derangement of the regulation of transcription of the oncogene appears to be the primary consequence of the translocation. It remains unclear as to whether positive regulatory signals derived from the lineage associated genes play an important or essential part in the deregulation process. Whatever the mechanism, the ultimate consequence of all of these translocations is a qualitative or quantitative alteration in oncogene function, and hence, in cell proliferation or differentiation. It is this which, probably in concert with additional genetic changes, ultimately gives rise to ncoptastic behavior. Techniques

of characterizing chromosomal

abormalities at a molecular level

If molecular analysis is to be of value as a diagnostic tool, it must be possible to complete investigations within a reasonable timeframe - usually measured in days. At the present time this, in essence, confines such investigations to Southern or

278

Northern blots. A prerequisite of such studies is the availability of appropriate probes capable of detecting or binding to the gene in question. Thus it is necessary that the genes involved in the translocation are identified in order that structural changes can be detected by these methods. The identification of genes involved in translocations can only be accomplished by molecular cloning. i.e. purification of the gene or a part of the gene. The cloning of a gene provides, at the same time, a probe which can be used to detect structural changes in the gene or in messenger RNA (mRNA) derived from it. In the case of lymphoid neoplasia, the frequent participation of immunoglobulin or T-cell antigen receptor genes (which have been previously cloned) in translocations, has provided readily available probes for isolating fragments of DNA which contain the chromosomal breakpoints, and hence the presumptive oncogene which has come to lie adjacent to the antigen receptor gene by virtue of the translocation. Unfortunately, not all breakpoints lie within genes - they may be some distance away, and still exert an effect on the gene itself. To determine whether the cloned DNA is part of a functional unit, it can be used to determine whether a corresponding mRNA is expressed. Sometimes the chromosomal location itself suggests the participation of a specific oncogene which has already been cloned and located to a specific chromosomal band, so that structural changes in this gene can be sought in cells bearing the translocation. Translocations which have been characterized in detail usually fall into one or other of these categories. Southern and Northern blots are means of identifying structural abnormalities in DNA and RNA respectively. In the Southern blot, DNA is digested with restriction endonucleases which cleave both DNA strands at precise locations determined by the nucleotide sequence. Thus any given gene or chromosomal region will be divided, by such an enzyme, into a specific number of fragments of specific sizes which can be separated by gel electrophoresis. The separated fragments are transferred, after denaturation, to a solid phase (filter paper) of nitrocellulose or nylon by capillarity or vacuum. and fixed in place, usually by baking. Finally. appropriate radiolabelled (sometimes biotinylated) probes derived from the cloned gene and known to react with specific fragments or regions of the gene to be examined are hybridized to the filter and detected by autoradiography. The technique of the Northern blot is similar in principle. although experimental conditions differ. Since mRNA consists of single stranded lengths of RNA, different sized transcripts are separated, transferred to filters and hybridized as in a Southern blot. Variations in the expected size of a message, in the quantity of a message. or in the presence of specific sequences can be detected by use of appropriate probes derived from the gene in question. Translocations involving bcr and c-ah1 A 9;22 (q34; qll) translocation appears to be the primary pathogenetic event in at least 90% of cases of chronic myeloid leukemia (CML), a disease which involves stem cells of multipotentiality and frequently gives rise to lymphoid blast crises (1). Some 16% of cases of acute lymphoblastic leukemia (ALL) contain the same translocation at a cytogenetic level, although the expression of the translo-

c-abl

A.

I

El

I 1

5’A

Ill Ill

El

VI VII

vm

lx

x

XI

3

TEL

I

bcr I_ I I 111111 ; IV nntrlnn n I I i i Sk&

B.x

v

IV

bcric- abl

I II Ill

111Ill

H lkb 3 TEL

Ph’ Genomic DNA v VI VII

IV

VIII

IX x

x1

3

TEL

t 1

Dcric-abl chimeric mRNA

D.

E.

-

5’

Nf%

chimeric fusion proteirf __I

3

AAA

COOH

Tyrosine kinase activity

Fig. 1. Diagrammatic depiction of the structural and f~lnctionai consequences of the 93 transiocation in chronic myeloid leukemia (CML). (A) The normal c-&f gene, showing the common breakpoint location. (B) The normal bcr region. showing the 5.8 kb region in which the majority of breakpoints occur in CML. (C) The derivative chromosome 22. showing the structural consequences of the 9:22 chromosomal translocation, with fusion between her and cab/. (D) The transcript of the fusion gene. (E) The fusion protein of 210 kDa which is believed to have enhanced tyrosine kinase activity.

cation in hemopoietic cell lineages differs from that of CML as do the findings at a molecular level (2-8). In both CML and ALL, however, 9;22 translocations result in the major part of the c-a61 oncogcne being moved from its normal position on chromosome 9 into a gene, known as ‘bcr‘ (breakpoint region). situated on chromosome 22 (Fig. 1). This gene is so named because of the limited region (5.8 kb) of DNA on chromosome 22 in which chromosomal breaks are detected in CML. Although the same genes are involved in both CML and ALL, the breakpoint region on chromosome 22 differs in the two diseases, being further upstream (5’) in ALL (2,3,5,6). The reason for this difference and its functional consequences have not been elucidated. Nevertheless, in both diseases the breakpoint on chromosome 9 is within c-abl, so that the normal promoter region of this gene is not translocated. Instead, the protein coding region of c-abl is fused to the 5’ sequences (including the promoters) of bcr. This results in the c-abl gene being regulated as if it were bcr, and in addition appears to cause enhancement of the tyrosine kinase activity of c-&I [9]. The precise function of c-abl has not been de-

fined, but tyrosine kinase activity is present in the cytoplasmic portion of a number of cell surface receptors. including growth factor receptors. suggesting a similar function for c-abl. The CML 9;22 translocation can be detected by Southern blotting, which permits the demonstration of a rearrangement of both c-abl and hcv. The rearrangement also results in the expression, detectable by Northern blot, of a novel 8 kb mRNA produced by the hcriuhl fusion gene in addition to the normal 6 and 7 kh transcripts [ 101. The 8 kb transcript is remarkably constant in CML because even though the precise location of the breakpoints may vary (within limits). the splicing out of the long intron segments in her results in a relatively invariant gene product. In addition. a fusion protein with a molecular weight of approximately 210 kDa is synthesized (Fig. I). This product is present even in a high proportion of CMLs which have been called Ph’ chromosome-negative in the past [Ill, and the presence of the 210 kDa fusion protein therefore defines a specific pathological entity in which several slightly different genetic changes have the same biochemical consequence. In ALL with Y;22 translocations, the difference in breakpoint location frequently creates a novel mRNA species of 7 kb, and a fusion protein of approximately 190 kDa (3,6.7). This differs from CML in lymphoid blast crisis in which the same 210 kDa protein is present as can be seen in the chronic phase of the disease. Thus, analysis of DNA or RNA by Southern or Northern biots, using appropriate c-&l probes. can be used to confirm a diagnosis of CML or to differentiate between CML in lymphoid blast crisis and ALL with a 9;22 transiocation.

A translocation in which a part of a transcriptional unit, referred to as hcl-2, on chromosome 18 is moved into the heavy chain locus on chromosome 14 occurs in most nodular lymphomas as well as in a small proportion of large cell lymphomas (LC) and small non-cleaved cell (SNCC) lymphomas (12.13). SNCC lymphomas containing 14:18 translocations occur in patients over the age of 40 years and represent a separate pathological entity from the SNCC lymphomas containing 8:14 or variant transiocations which occur predominantly in the first two decades of life (see below).

MOLECULAR

CONSEQUENCES

Ig

bcl-2

Chromosome

Fig. 2. Diagrammatic

depiction

OF 14;18

18

I

gene

Chromowme 14

of the derivative

The breakpoint appears to be at a DJ

TRANSLOCATION

junction.

chromosome

14

and results in a

coding region of hcl-2.

rcsuiting from a 14:li: translocation. fusion gene which lacks the 3’ non-

281

The 14;18 translocation results in the production of a fusion gene composed of the 5’ (upstream) region of bcl-2 and an immunoglobulin heavy chain, consisting of sequences extending downstream from J, - the joining region of the immunoglobulin gene (13,14) (Fig. 2). Interestingly, the immunoglobulin diversity segment (Dn) mediates the junction between the distal region of chromosome 14 and chromosome 18, while the distal portion of chromosome 18, containing hcl-2, is fused to a joining (Jn) segment on chromosome 14. This strongly suggests that the translocation occurs during DJ joining - the first part of the molecular rearrangements which result in the assembly of a complete immunoglobulin molecule from the various segments which are initially separated on chromosome 14 (16,17). This is consistent with the finding that immature B cell lines, especially pre-B cells, express considerably more bcf-2 than mature cell lines which do not contain a 14;lS translocation (18,19). More than 70% of the breakpoints on chromosome 18 occur within the 3’ untranslated region of bd-2 (20). The influence of the Ig juxtaposition is unclear, but increased levels of bcl-2 are likely to be produced through a combination of increased transcription and decreased post transcriptional regulation. In this context, since the Ig portion of the fusion gene replaces the large untranslated 3’ portion of bcl-2, it could have effects on post-transcriptional processing, or simply replace a region involved in transcriptional regulation. Additionally, the Ig transcriptional enhancer element of the heavy chain region is juxtaposed to bcl-2, although it may be too far from the promoter region of the latter gene to influence its transcription. The presence of a 14;18 translocation can be detected by Southern blotting to demonstrate the presence of both Ig gene rearrangements and rearrangements of bcl-2. When appropriate probes are used, rearrangements of bcf-2 are detectable in more than 80% of follicular lymphomas, even, on occasion, when cytogenetics fails to reveal a translocation (20,21). By Northern blot analysis, an abnormal hybrid message of variable size, ranging from approximately 5.8 kb to 7.6 kb and containing both Ig and bcl-2 sequences is observed (14,1.5). The absence of the 3’ untranslated region from the hybrid message (which is the only bcl-2 message expressed by cells bearing 14;18 translocations) is readily shown by using a probe derived from this region of the gene. Trans~ocatio~ ~nvoIving c-myc and the T-cell receptor gene In T-cell ALL and lymphoblastic lymphomas, a number of translocations have been described in which the Tcr gene situated on chromosome 14 is involved in a reciprocal translocation with another chromosome, usually 8. 10, 11 or even 14 itself (l&17,22,24). Few of these translocations have been fully characterized, and the nature of the non-lineage associated gene participating in the translocation is a matter for speculation. The 8;14 translocations, however, bear strong similarities in B- and T-cell tumors, excepting that the lineage associated gene differs. In one well characterized tumor, a part of the Tel gene was translocated to the chromosome 8 region 3’ of c-myc (24). It seems likely that the mechanisms of deregulation of c-myc are analogous to the c-pnyc/Ig translocations involving light chain im-

282

munoglobulin regions in Burkitt’s lymphoma (which involve translocations of Ig gene regions to chromosome 8, 3’ of c-myc) (17,25), although in neither the B-cell nor the T-cell translocations has a precise mechanism of deregulation been proposed on the basis of the molecular findings. In these tumors there will be rearrangements of Ta genes. but c-myc is not necessarily rearranged. It remains to be seen whether mutations will be detectable in the regulatory region of c-myc as in the c-myc/Ig translocations. although this seems highly likely (25-27). No consistent abnormalities have been described in Northern blots to date. Translocations involving c-myc and Ig genes Translocations between chromosomes 8 and 14 are the second commonest translocation in the non-Hodgkin’s lymphomas [12]. They occur in some 75% of SNCC lymphomas and so-called L3 ALL (almost certainly a different clinical phase of SNCC lymphoma) and a small proportion of large cell lymphomas. This translocation results in the relocation of the c-myc gene to the heavy chain immunoglobulin locus on chromosome 14. The SNCC lymphomas which do not contain 8;14 translocations contain translocations which involve c-myc and one of the immunoglobulin light chain loci on chromosome 22 (lambda) or chromosome 2 (kappa) (25). Although different chromosomes are involved, and light chain sequences are translocated downstream of c-myc which remains on chromosome 8. the latter ‘variant’ translocations probably give rise to similar molecular conseBREAKPOINTS IN RELATIONSHIP

TO RECOGNIZED REGULATORY ELEMENTS ST486 CA36

MC116

Daudi

I Sm

Hd

PS

-1257

~~~~Bishop

and

sevens.

-353

-101

+510

Genes and Development.

1:659. 198’

Fig. 3. Diagrammatic depiction of some of the known regulatory elements in the c-m~~ gene. In this figure. the 5’ prime region from the Hind111 site and the 1st and 2nd exam of the c-myc are shown: + . a positive regulatory element for the promoter shown (Pl or P2) and -, a negative regulatory; element. It can be seen that. for example, a mutation in the immediate 5’ region of the gene could result in failure of initiation at the PI site because of deletion of the positive regulators, mutation of the’negative regulator for P2 and mutation in the region required for PI expression in the absence of the positive regulatory elements.

2x3

quences to the 8;14 translocations, namely deregulation of the c-myc gene. In each of these three translocations, the c-myc gene. or at least its protein coding sequences, become juxtaposed to immunoglobulin sequences which are likely, in some way, to help bring about the deregulation of the c-tnyc gene. This could be mediated through enhancer regions present within the immunoglobulin locus. through the induction of mutations within the c-myc regulatory regions or simply by the maintenance of an open chromatin structure around c-myc, permitting access to RNA polymerase. The c-myc gene is not mutated in its protein-coding region, and it would appear that the gene product is normal. but inappropriately expressed (25). The c-myc protein runs in polyacrylamide gels with an apparent molecular weight of 64 kDa, and is a nucleic acid binding protein which is necessary for cellular proliferation in a wide variety of cell types, so that it is easy to envisage that inappropriate expression will result in aberrant cellular proliferation. The breakpoint locations on chromosome 8 vary greatly. They can be far 5’ of the gene, immediately adjacent to the gene, within the non-protein coding region of the gene itself, or far 3’ of the gene (as occurs in the variant translocations) (25). When the breakpoint is outside the gene, mutations are detectable within the 5’ regulatory portion of the gene (usually within the first exon) (27). It would seem highly probable that several different mechanisms can give rise to altered expresGENETIC REARRANGEMENTS IN BURKITT’S LY~~HO~A The derivative 14q+ in switch and 3~ breakpoints 1. lntroniswitch

H

breakpoints

E

E

Chromosome

8

H

Chromosome

I

14

Breakpoint

2. I~~KxI/JH breakpoints

Chromosome

8

I

Chromosome

14

Breakpoint

Fig. 4. Diagrammatic depiction of alterations in restriction endonuclease sites when the breakpoint on chromosome 8 is in the 1st intron while that on chromosome l-1 is in the switch-b region or JH region. E = EcoR1. H = HirzdIII. II and III are the second and third exons of c-myc respectively and Sp and Cp are the switch and constant regions of the p gene. For further explanations, see text.

sion of c-myc. These include deletion of the normal promoters and regulatory elements which control transcription (28), deletion or mutation of some of the regulatory elements (Fig. 3) or participation of a positive transcriptional element within the immunoglobulin locus, or of non-cellular origin (e.g. of viral origin). SNCC lymphomas which contain one of these translocations represent a specific pathological entity which might be most accurately defined as a B-cell tumor induced by deregulation of c-myc (the participation of other factors is not excluded). Such tumors can therefore be diagnosed at a molecular level by the presence of Ig gene rearrangements and either a rearrangement of c-myc, or the presence of a mutation in c-myc. which is frequently detectable by Southern blot. When the breakpoint is in or close to the c-myc gene, it is often a simple matter to localize the breakpoints on both chromosomes 8 and 14 to specific fragments of the c-myc and immunoglobulin gene, simply by the appropriate choice of restriction enzymes and probes. For example, intron breakpoints are readily detectable by the fact that the 1st and 3rd exons are present in different sized EcoRl and Hind111 fragments. In this circumstance it is often quite simple to determine whether the breakpoint is on chromosome 14, as long as additional DNA rearrangements have not occurred. For example, when associated with breakpoints in the switch region, the rearranged EcoRl fragment containing the 3rd exon of c-myc is markedly smaller te germline fragment and always smaller. by a fixed amount, than the Hind111 tf

Pofy-A FWA Prabed with 1st Exon c-myc

KK

MC

ST

JD

MO

2.4 kbs

1.35 kb*

Fig. 5. Northern blot showing separate sence of transcripts of normal size (2.4 in c-my indicates that the normal allele lanes) show the position of the normal

migration of 1st exon sequences as a 0.9 kb message. The abkb) in ST486 and JD38, both cell lines with intron breakpoints is not transcribed. Ceil lines without a break in c-myc (all other sized transcript and the absence of a small 1st exon transcript.

fragment detected with the same probe (Fig. 4). Breakpoints within the gene also result in the ‘comigration’ of the 3rd exon of c-myc with either S,. J, or another immunoglobulin gene region, while the first exon, remaining on chromosome 8 should also show comigration with one or other of these fragments. In Northern blots, there may or may not be detectable abnor’malities, depending upon the location of the breakpoint. If the latter resides 3’ of the normal promoters of the gene, then first exon sequences will not be present in the same transcript as second and third exon sequences. The latter migrate in a message of approximately 2.4 kb, whereas first exon sequences are either not detectable. or migrate as a separate transcript of approximately 0.9 kb (Fig. 5).

Molecular differences lymphomas)

between endemic

and sporadic SNCC lymphomas

(Burkitt’s

As mentioned above, the breakpoint locations on chromosome 8 vary markedly at the molecular level in X;14 transiocations. We have shown that the regions commonly involved in endemic tumors differ significantly from those in sporadic tumors, and have recently extended this work (27). By detailed Southern blot analysis of 56 tumors and cell lines, the strategy for which is shown in Fig. 6, we have been able to assign the breakpoint location in each tumor to one of 5 regions, designated by restriction enzyme sites in or close to c-myc. There is a frequency gradient in opposite directions when breakpoint locations in these 5 gene regions are plotted graphically for endemic and sporadic tumors (Fig. 7). The majority of endemic tumors have breakpoints far S’ of the gene, and none, so far detected at least, have breakpoints within the first intron. Conversely, the majority of sporadic tumors have breakpoints within the intron or immediate 5,’ region of the gene. These findings have significance for the mechanism of deregulation of c-myc, which clearly differs in the two subtypes of SNCC lymphoma. In tumors with far 5’ breakpoints, which also have mutations in the first exon, it is likely that a major SOUTHERN

1
2 I

H3-Ps

BLOT STRATEGY

3 I

Ps-Sm

5

4

Pv-Ps

I%-Pvl

I

(_..___-______) (-___-_______________) (____‘____-__-______________________----, (__________________________________-________________________ I

Ps Pv

Sm

Pv

Sm

II

PS

Fig. 6. Strategy for determining breakpoint location by Southern blot. Enzymes which cut at precise locations are used to examine the integrity of given DNA fragments which are sized on an agarose gel. By using probes for regions within each fragment, the breakpoint location can be discerned at least to within a given fragment. We have arbitrarily divided the breakpoint locations into 5 main regions as shown.

286

BREAKPOINT

DISTRIBUTION

ON CHROMOSOME

8

19 17 15 13 g (0

11

9

9 7 5 3 1 Ps-Sm Sm-Pv Pv-Ps H3-Ps BREAKPOINT REGION m SPORADICTUMORS ENDEMICTVMORS

>H3 m

Fig. 7. Graphic depiction of the location of breakpoints on chromosome 8 in endemic and sporadic Burkitt‘s Iymphoma. The regions shown in the histogram correspond to those shown in Fig. 6.

mechanism of deregulation of c-myc is abrogation of the elongation block which normally occurs at the 1st exoniintron border. In tumors with breakpoints 3’ prime of the normal promoters of the gene, Pl and P2. a cryptic promoter present in the first intron must be utilized, and it seems probable that a positive regulatory element is required for this, perhaps supplied by the juxtaposed immunoglobulin sequences. When the breakpoint is immediately 5’ of the gene, it is possible that derangement of the normal positive and negative regulatory elements for Pl and P2 (28), occasioned by deletion of some of the elements and, possibly. mutation in others, is involved in altered c-myc expression.

Correlation

between breakpoints

and EBV association

in Burkitt’s lymphoma

In the 56 tumors and cell lines examined for breakpoint location, we also sought a correlation between breakpoint location on chromosome X and the presence of EBV DNA, using a molecular hybridization technique with a probe consisting of the BamHl ‘K’ fragment of the EBV genome. There was a strong positive association of EBV with tumors which had breakpoints far 5’ of the gene. Twenty two of 23 tumors of this kind, whether of endemic or sporadic origin, contained EBV DNA (Table 2). In tumors with breakpoints in other regions, both EBV containing- and EBV-lacking tumors were found. with no obvious correlation between breakpoint location and EBV association, except that in sporadic tumors, only one of 15 with a breakpoint immediately 5’ of c-myc contained EBV, whereas about half of tumors with an intron breakpoint did so. It remains possible that if the breakpoints were to be located even more precisely, and related to c-myc regulatory regions, a pattern will emerge. Such a finding would strongly suggest that EBV has a direct or indirect influence on c-myc expression via the regulatory elements.

287 TABLE

2

Correlation

between

Within the Hind111 fragment HirldIII

geographic

origin

and EBV association

Sporadic

EBV

6

27

12

21

19

4

22

1

Endemic

Breakpoint

Outside

breakpoints,

+

EBV

-

The technique of Southern blotting permits the detection of a clone of cells representing as little as 1%-S% of the total population. The use of oncogene rearrangements rather than immunoglobulin or T-cell receptor gene rearrangements as markers of a malignant clone has the major advantage that detection of a rearrangement is essentially diagnostic of a specific malignancy in an appropriate setting, for example in a specific cell lineage. Thus oncogene rearrangements can be used to follow patients serially (e.g. by examining bone marrow samples, where relevant) and could lead to the early detection of relapse. This technique also enabled us to definitively demonstrate the development of a second, clonally discrete SNCC lymphoma 3 years after elimination of the first, in a patient with HIV infection (29). This clearly represented reinduction of a new tumor as the result of a new translocation in a high risk patient, rather than recurrence of the first tumor.

Potential

prognostic

value of molecular

analysis

It has clearly been shown in ALL that patients with chromosomal translocations have a worse prognosis than patients in whom genetic abnormalities cannot be demonstrated. Some patients, e.g. those with 4;ll translocations, have a particularly poor prognosis. However, it has not yet been shown that the prognosis varies, according to the particular structural changes present, within an individual pathologic entity defined by a chromosomal translocation. Thus, even though it is possible that the prognosis in patients with SNCC lymphomas may relate in part to the breakpoint locations, in view of the relatively small patient numbers that have been characterized in detail, this remains an unanswered question. Of special interest is the possibility that the molecular abormalities of the nonHodgkin’s lymphomas will provide a tumor specific therapeutic target. It may prove possible, for example, to develop drugs or other molecules which interact with fusion proteins but not their normal counterparts. In Burkitt’s lymphoma in which the chromosome 8 breakpoint is 3’ of the normal c-myc promoters, the presence of 1st intron sequences in the c-myc mRNA, a result of the transcription of the gene from a cryptic promoter in the first intron, provides a unique target which is absent in normal cells. The possibility of using antisense molecules to block translation of c-myc specifically in Burkitt’s lymphoma cells in which 1st intron sequences are present in the mRNA is one that we are currently exploring.

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