Cancer Res 1990 Wainscoat 1355 60

7/28/2019 Cancer Res 1990 Wainscoat 1355 60

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1990;50:1355-1360.Cancer ResJ. S. Wainscoat and M. F. FeyAssessment of Clonality in Human Tumors: A Review

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[CANCER RESEARCH 50, 1 35 5- 13 60 , Ma rc h 1 , 1 99 0 ]

ReviewAssessment of Clonality in Human Tumors: A ReviewJ. S. W ainscoat1 and M . F. FeyD epartm ent of H aem atology, John R adcliffe H ospital. O xford, U nited K ingdom [J.S.W .], a nd Institut fur M ed izinische O nkologie, Inselspital, C H-3010,B er n, Sw it ze rl an d [M . F . F .]

AbstractThe v ar io us me th od s o f d e te rm ina ti on o f t he c lo na li ) y o f h uman tumo rsa re d es cr ib ed . Th er e a re t hr ee ma jo r a pp ro ache s b as ed on X-ch romosome

ina ct iv at io n ana ly si s, l ymphocy te a na ly si s, a nd soma ti c mu ta ti on ana lysis. For each of these approaches there are established m ethods andm ore re ce nt m eth od s b as ed o n DNA an aly sis. T he in crea sin g n um ber o fme th od s ava il ab le i nc re as es t he s cope o f c lo na li ty d et erm in at io n t o mo sttu mors. A ll th e m etho ds h ave in here nt ad van tag es an d d is ad van tag es ,a nd the se a re d is cu ss ed i n r el at io n t o t he ir c li ni ca l a pp li ca ti on .

T he clonality of hum an tum ors has direct relevance to theories of carcinogenesis and has practical im plications for bothdiagnosis and subsequent studies of disease progression. T hepurpose of this review is to discuss the different m ethods ofclonality determ ination, in particular those based on DNAan aly sis, an d to illustrate the ir use in h um an tumo rs.A clonal population of cells is defined as those cells arisingfrom th e m ito tic d iv isio n o f a sin gle som atic c ell (1 ). A ltho ug h

th is d efin itio n s eems s tra ig htfo rw ard , it mu st b e re co gn iz ed th atthe assessm en t o f clo nality m ay b e d ep en den t o n the tech niq ueused. For exam ple, the investigation of tw o cell populationsm ay suggest tw o independent clones by immunoglobulin geneanalysis but a single clone by X -linked D NA polym orphisma nalysis, reflectin g th e earlier o ccu rren ce in d ev elo pm en t of X -ch romo some in activatio n. A fu rthe r co nsid eratio n w hich comp licates th e an aly sis o f clo nality is th at the cells co nstitutin g as in gle c lo ne a re n ot n ec es sa rily g en etic ally id en tic al s in ce c lo na lev olutio n m ay o ccu r w ith in su ch po pu latio ns o f cells (2 ).It is now w idely agreed that m ost tum ors have a m onoclonalcom position consistent w ith the som atic m utation theory ofcarcinogenesis w hich assum es that a tum or results from theprogeny of a single cell having acquired one or m ore som aticmutations (3). The methods used to determ ine clonality ofhum an tum ors have been m ost readily applicable to leukem iasand lymphomas, although some techniques, for example,G 6PD 2 isoenzym e analysis, have been used on solid tum ors.H ow ever, several m ethods have been developed over recentyears which enable a higher proportion of a wide variety oftu mors to b e an aly zed for clo nality . T he an aly sis o f clo nality intum ors can provide clonal m arkers w hich are valuable in assessing disease progression and in providing insight into then at ur e o f d is ea se r em i ss io n.T he m ethods of clonality determ ination can be broadly categ oriz ed in to th e fo llowin g g ro up s: X -c hromosome in ac tiv atio n,lym ph ocy te analysis, d etection o f somatic m uta tio ns, an d v iralintegration analysis. Table 1 divides these approaches intoRec ei ve d 5 /2 /8 9; r ev is ed 10/ 20 /8 9; a cc ep te d 11/ 27 /8 9.T he co sts o f p ub lic atio n o f th is article w ere d efray ed in p art b y th e p ay men to f p ag e c ha rg es . T his a rti cle m us t t he re fo re b e h ere by m ark ed a dv erti se me nt ina cc or da nc e w ith 1 8 U .S .C . S ec tio n 1 73 4 s ole ly t o in di ca te t his f ac t.' T o w hom c or re sp on de nc e s ho uld b e a dd re ss ed .2 T he a bb re via ti on s u se d a re : G 6PD. g lu co se -6 -p ho sp ha te d eh yd ro ge na se ;

ANLL , a cu te n on lymp ho cy tic l eu kemia ; TCR , T -c ell re ce pto r; VNTR s, v ari ab len um be rs o f ta nd em re pe at s; R FLP, r es tric ti on fra gm en t le ng th p olymo rp hi sm ;EBV, Epst ein-Bar r v i ru s .

"trad itio nal" m eth od s and tho se b ased on v ario us ty pes o f DNAanalysis.Both DN A and traditional m ethods depend upon the demonstration that a cell population is hom ogeneous w ith respectto a pa rticu lar m ark er. S ince at th e tim e o f presen tatio n tumorsm ay h av e un derg on e ex ten siv e g enetic ch an ge an d co ntin uingselectio n o f p articu lar su bclo nes m ay h av e occu rred , clo nalityas assessed by any of the currently available m ethods m ay stillnot necessarily reflect the earliest events in tum origenesis. Atum or m ay have originated from several cells, the progeny ofone of these cells (bearing the m arker) eventually having outgrow n all the others (4).X-Chromosome Inac tivat ionIn fem ales inactivation of one X chrom osom e occurs in each

somatic cell in early em bry on ic dev elopmen t an d is p assed o ntothe progeny of the cell in a stable fashion (5-7). Femalesh etero zy go us for p olymorp hic X -chromo some g en es are th erefore m osaics with respect to X-chrom osom e activity (8, 9).T here is ev id en ce th at X -chromo some inac tiv atio n is related tod iffere ntial m eth ylatio n o f cytosin e in the DNA o f X -chromosome genes (10). The exact role of gene methylation in thev ario us asp ects o f X -ch romo some inac tiv atio n h as been ex tensiv ely re viewe d e lsewh ere a nd a d eta ile d d is cu ss io n o f th e mec hanism s of X -chrom osom e inactivation is beyond the scope ofthis review (5, 7, 11-13). From the point of view of clonalityassessment in tumors it is sufficient to recognize that theinactivation or m ethylation patterns of X -chrom osom e genescan be used for the detection of clonality of tum ors in fem alesh ete ro zy go us fo r a p artic ula r X -lin ke d p olymo rp hism .G 6PD Iso en zyme A naly sis. T he first stu dies of h um an tumo rclonality using the G 6P D isoenzym e system w ere reported by

Linder and G artler (14, 15) and Beutler et al. (9) in uterineleiom yom as and m alignant tum ors, respectively. T his form ofclonality assessm ent w as extended to the analysis of a largevariety of hum an tum ors in the now classic work of Fialkow(16). This form of analysis is based upon the fact that a fem alepatient heterozygous for a polym orphism of the X -linked enzyme G6PD will express both the normal type of GdB and avariant type of GdA or GdA~ in her norm al tissues but only asingle G 6PD isoenzym e in each individual cell. Therefore, aneoplasm arising from a single cell w ill show a single G 6PDis oe nz yme p he no ty pe , whe re as a p oly clo na l n eo pla sm w ill h av ea double G 6P D enzym e phenotype (17).The m ajority of hum an cancers analyzed by this techniquehave been show n to be m onoclonal (including breast cancer,c arc in oma o f th e c olo n, c arc in oma o f th e u te rin e c erv ix , o va ria nteratom as, and m any hem atological neoplasm s (16, 18-20).G 6PD stu die s h av e b ee n p artic ula rly u se fu l in th e in ve stig atio nof chronic m yeloid leukem ia since R BC s and platelets cannotbe investigated by cytogenetic or DNA analysis as they have non uc le i. A few e xc ep tio na l c as es o f malig na nt tumors w ith d ou bleen zyme p hen oty pes in clu din g co lo nie carcin om as, b reast cancers, and a hepatom a have been reported. T hese rare cases m ay

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CLONALITY IN HUMAN TUMORSTab le 1 Me thod s o f clonal ity d ete rmination

MethodsGeneral approach Traditional DN AX-chromosomeinactivationLymphocyte analysis

Somatic mutation

Viral integrationanalysis

G6PD isoenzymes X-linked RFLPsImmunogl obul in l ig ht Immunogl obul in and TCRchain analysisCytogene l ic analys is gene analysisD e te cti on o f c hromo some l os sby RFLP ana ly s isDetec tion of chromosometranslocations by breakpoint

cluster probesDe te cti on o f somati c mutati on s by DNA f inge rprintingDe te cti on o f spe ci fi c pointmutationsAnal ys is o f g enomi c EBV te rmini

Polyclona l

B am HI Bam HIHpa II

Monoc lona l

Bam HI B am HIHpa II

Pa te rna l

Ma te rna lbe explained by an admixture of normal cells in the tumors ample anal yz ed (17, 21). This m ight be a po ss ible e xplanationfo r c onflic ting re sults o f c lo nality s tudie s in parathy ro id adenoma; the demons tration o f a po ly clonal o rig in o f parathyro idadenomas by G6PD isoenzyme studies (22) has not been conf irmed by more re cent work us ing mole cular analy si s w ith DNAmarkers (23 ). N ev ertheless some hereditary tumo rs such astric ho epithe liomas (2 4) and ne uro fibromas (2 5) do hav e a double enzyme pheno ty pe. The po ly clo nal composition o f the setumors is pre sumably re late d to the diffe re nt tim e c ours e andmec hanism o f tumorig ene sis. S im ilarly a multic lo nal o rig in o fc ol ore ctal adenomas in Gardne r's s yndrome ( fam il ial adenom-atous polyposis syndrome) has been proposed based on theanaly sis o f G6PD mos aic -ism (2 6). Howe ve r, more re ce nt dataon the clonality of colorectal adenomas collected by DNApo lymo rphism analy sis sug gest that at least some adenomasare clo nal tumo rs (2 7). These div ergent results may be due tothe f ac t that in the s tudy us ing re combinant te chnique s c ontaminating no nneo plastic cells w ere remo ved from the tumo r byhi sto log ical analys is o f cryostat s ec ti ons.The G6PD approach has until recently been limited to theGdA and GdA~ vari ants in blacks which are eas ily di stinguis ha

ble from the no rmal B enzyme by starch g el electro pho resis.M ore recently , it has bee n possible to ex tend this appro ach toindividuals heterozygous for the Mediterranean variant ofG6PD by v irtue o f its dif fe rential utili zation o f 2 -deoxyg lucos e6 -pho sphate as c ompare d to the no rmal is oe nz yme (2 8-3 0).X -linke d RFLP Analy sis. Mole cular probe s fo r X -linke d polymo rphic g ene s now make it po ss ible to de te ct c lo nal marke rsin tumor cells in a manner analogous to the G6PD isoenzymes tudie s de sc ribed above ( 31 , 32) . Es sential ly ac tiv e and inac tiv ec opie s o f po lymorphi c X -chromosome gene s are dif fe rentiatedthrough differences of gene methylation patterns. This appro ac h g re atly w ide ns the s co pe o f c lo nality s tudie s to inc ludeall females in w hom a suitable X -linked DNA po lymo rphismis present.Both normal and tumor DNA are first digested with theappropriate re stric ti on endonuc le as e to dis tinguis h the mate rnal and pate rnal c opie s o f the g ene thro ugh an X -linke d RFLP.A seco nd endo nuclease sensitiv e to methylatio n o f cy to sinere sidue s in its re co gnitio n s eque nc e dis ting uishe s ac tiv e frominactiv e co pies o f the g ene throug h chang es o f the DNA methy latio n patte rn (3 3, 3 4). In a po ly clo nal c ell po pulatio n whe reX -chromosome inac tiv ati on occurs randomly the pate rnal andmate rnal al e le sare c le av ed to some e xte nt by this e nz yme s othat tw o fragments o f re duced intensity remain v isible on au-toradio graphy . In DNA ex tracted from a tumor w ith a mo no -

Fi g. 1 . Princ iple o f a S outhe rn bl ot X -li nke d RFLP c lo nal ity s tudy . B ox es .al el es ;s hade d are as , m ethy late d f ra cti on. L ef t, DNA f rom a po ly cl onal c el lpopul ati on o f a f emal e he te ro zy gous f or an X -l inke d RFLP . B oth the pate rnaland the mate rnal a l e le so f the g ene a re methy late d to s ome e xte nt. T he RFLP i sd ete cted by BamHI; th e nonme thylated parts o f e ach a l el eare cut by th e enzymeHpa \\ a nd , th er efo re , t he in te ns it y o f b oth fr a gme nt s i s r ed uc ed o n a uto ra d io g-raphy . R ight, DNA f rom a mono cl ona l c el l popu lati on o f a f emal e he te ro zy gousf or the s ame X -l inke d RFLP. In thi s parti cul ar c as e onl y the p ate rnal al el eo fthe g ene i s m ethy late d and is . the re fo re , no t c le av ed by // // ILHowe ve r, thenonme thylated mate rnal a l el e s d ige sted comp le te ly by th is enzyme and hencei s no t d ete ctabl e. T he f ac t that i n thi s c el l popul ati on onl y one o f the two al el esis methylated is evidence ofc lonal ity.clonal composition one of the two allelic fragments is comple te ly dig este d re sulting in its dis appe aranc e while the o the rremai ns unalte red. The princ iple o f this approach is illus tratedin Fig. 1.X -linked gene po lymorphisms us ef ul fo r such s tudie s inc ludeRFLPs o f the hypo xanthine pho spho ribo sy ltransfe ras e g ene(3 5) and the pho spho gly ce rate kinas e g ene (3 2). The he te ro zy -gosity rate of these genes is approximately 30% which is al im iti ng f ac to r in the ir prac tic al appli cation. It is po ss ible thatothe r X -linked probes w ith hig her hetero zy go sity rates maypro ve useful.3 The neo plasms fo und to be clonal by this technique inc lude acute and chroni c l eukemias , uterine l ei omyomas ,W ilm s' tumors, and parathy ro id ade nomas (2 3, 3 2).Bone Marrow Recons ti tu ti on after Treatment for Leukemias .S ome o f the mos t inte re sting re po rts re ce ntly publis he d us ingboth G6PD isoenzyme studies and X -linked RFLPs concernthe nature of clinical remission in A NLL. There is good evidence for a monoclonal origin of leukemic cells from bothkaryotypi c and G6PD i soenzyme s tudi es . It would be reasonableto assume that w hen a patient enters a clinical remissio n thiswould be ac companied by a re turn to a normal ( i. e., po ly clonal)patte rn o f hemato po ie sis . Howe ve r, bo th G6PD and X -linke dRFLP studies hav e pro vided evide nce sug gesting that this isno t alw ay s the case. U sing the X -linked RFLP approac h, Fea-ron et al. (35) found that 3 of 13 patients w ith ANLL inremissio n had mature g ranulo cy tes o f a mo no clo nal o rigin,presumably (although not proven) of the same clone as theiro riginal blasts. Fialkow (36 ) using G6PD isoenzyme studie sshowed 5 of 13 patients with ANLL in remission to have3 G. A brahamson, N . J. Fraser, Y . Bo yd, I. Craig, and J. S. Wainscoat. Ahi ghl y i nf ormati ve X -c hromo some probe , M27 fi , c an be us ed f or the de te rm inati on o f tumor c ol onal ity . B r. J. H aemato l. , i n p re ss .

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C LO NA LIT Y IN H UM AN T UMOR Sp artially o r c om p le te ly c lo nal m arro w s te m c ells . E x pe rim e ntsin cats hav e show n that, follow ing chem otherapy w ith dim e-thy lbusulfan, hem atopoiesis m ay return w ith a m onoclonalp atte rn , p re su m ab ly re fle ctin g d am ag e to o r d ep le tio n o f n orm alstem cells (37). In tw o studies of allogeneic bone m arrowtransplant recipients treated f or leuk em ia, the m ajority of thep atie nts sh ow ed n o ev id ence o f clon al reco nstitutio n o f g ranu -locy tes and m ononuclear cells f rom their graf ts as assessed bythe X -link ed R FL P m ethod (38, 39). How ev er, tw o patientsh ad m o noc lo nal o r o lig oc lo nal d on or-d eriv ed hem atop oiesisaf te r allo ge ne ic tran sp lan tatio n su gg estin g th at in th es e c ase s asingle or a v ery lim ited num bers of stem cells w ere suf f icient todom inate the hem atopoietic sy stem (39). Further studies ofA N L L are required to conf irm that clonal granulocy te populations in in patients in rem ission are indeed part of the sam eleu kem ic clo ne as seen in th e p resen tation m arro w .Lympho cy te Anal ys is

Im m unoglobulin L ight C hain A n aly sis. O ne of the standardm ethods f or def ining m onoclonality in B -cell neoplasm s is thedem onstration of a single light chain isoty pe, either K o r A , o nthe cell surf ace of neoplastic ly m phoid cells; this technique isread ily ap plicable to B -c ell ch ro nic ly m ph ocy tic leu kem ia andso m e cases o f B -cell n on -H od gk in 's ly m ph om as (4 0, 4 1). T hereis n o an alo go us sy stem o f su rf ace m ark ers ind ic atin g clo nalityin T -cell tum ors. H ow ev er, m onoclonal antibodies w hich arespecif ic f or fam ilies of related v ariable regions w hich use thes am e v ariab le re gio n g en es c an ac t as in dic ato rs o f th e c lo nalityo f som e T -cell po pu latio ns (4 2).Im m unoglobulin and T CR G ene A naly sis. T he m ore recentuse of m olecular probes to detect im m unoglobulin and T CRgene rearrangem ents ov ercom es m any of the lim itations ofim m unoglobulin light chain analy sis (43-45). E ssentially allty pes of ly mphoid m alignancies are am enable to this ty pe ofanaly sis. Im m unoglobulin and T CR gene rearrangem ents giv erise to D NA m ark ers unique to each indiv idual ly m phoid celland its progeny . In a poly clonal ly m phoid cell prolif erationth ese rearrang em e nts are n ot d etectab le by S ou th ern b lo ttingsince they are w ell below the threshold of the m ethod's sensitiv ity . H ow ev er, all cells in a m onoclonal population hav e thes am e g en etic re arran ge m en t w h ic h is re ad ily d ete cte d b y S o uthern b lo ttin g. Im m u no glo bu lin an d T C R g en e rearrang em e ntsm ay als o p ro v id e in fo rm atio n ab ou t th e c ell lin eag e o f ly m p ho idn eo plasm s . In g en eral, im m u n og lo bu lin g en e re arran ge m en tsare indicativ e of B -cell neoplasm s, w hereas T CR gene rearrangem ents are regularly found in T -cell ly mphom as andle uk em i as . U sin g th es e te ch niq ue s it h as b ee n p os sib le to p ro v e,f or ex am ple, a clonal B -cell origin of hairy cell leuk em ia and ac lo nal T -c ell o rig in o f m y c osis f un go id es .A sig nif ic an t lim i tatio n o f th e c on v en tio nal S o uth ern b lo ttin gtech niq ue is its inab ility to d ete ct g en e rearran gem e nts at m u chbelow the 5% lev el. T he poly m erase chain reaction techniquenow of fers the possibility of detecting clonal m ark ers at m uchlow er lev els. T here are m ajor problem s in the am plif ication ofim m u nog lo bu lin o r T C R fe arran ge m en ts b ec au se o f th e v erylarge num ber of v ariable and joining segm ents w hich canu nd erg o rearran gem e nt. H ow ev er, th e T C R -y g en es hav e a m o relim ited germ line repertoire and a m ethod has been describedw hich detects T CR 7 rearrangem ents by poly merase chain reaction in leuk em ic sam ples (46). It is hoped that generallyap plic ab le m e th od s w i ll b e d ev elo pe d to d ete ct im m u n og lo bu linan d T C R re arran gem e nts p resen t at v ery lo w le vels sin ce th esew ould be ex trem ely useful clonal m ark ers in the study ofm in im al residu al d isease in ly m ph om a an d leu kem ia.

It s ho uld b e n ote d th at im m u n og lo bu lin g en e re arran ge m en tsare n ot c on siste ntly s tab le c lo nal m ark ers sin ce th ey are su bje ctto v ariability as a result of ongoing som atic m utation. D if f eren ces in im m u nog lo bu lin g en e rearran gem e nt p attern s am o ngv arious specim ens f rom a giv en case do not necessarily ref lecttrue bi- or m ulticlonality in ly m phoid tum ors. T his has beenclearly sh ow n in tw o stud ies o f f ollic ular ly m ph om a b earin g th ec harac te ri sti c c hro m o so m al tran slo cat io n t( 14 ;1 8) . I n th e s tu dyof R af feld et al. (47) 6 of 16 cases show ed v ariation in im m un og lo bu lin p atte rn s o v er tim e , alth ou gh th e t(1 4;1 8) b re ak p oin tw as co nserv ed in all in div id uals. S im ilarly th e stu dy b y C learyet a l. (48 ) o f five b ig en otyp ic a nd b ip hen otyp ic fo llicu la r lymphom as show ed that f our of these cases had identical t(14;18)rearrang em e nts co nf irm ing th e sing le cell o rig in f or th ese neoplasm s. T he single ex ceptional case w as further studied byn uc leo tid e seq uen ce an aly sis of clon ed b reak po in t D N A w h ichs ho w e d id en tic al t( 14 ;1 8) c ro ss ov e rs in th e t w o s ub po pu lat io ns .T he translocated chrom osom e 18 DN A rearrangem ents arem ore reliable clonal m ark ers, although these m ay also be aff ec te d b y s om atic m u tatio n.Soma ti c Mu ta tio ns

C ytogenetics. M any hum an tum ors hav e now been found tohav e co nsisten t, n on ran dom chro m oso m e ab norm alities w h ichbehav e as clonal m ark ers (49). T he classic ex am ple is thePhiladelphia chrom osom e in chronic m y eloid leuk em ia (50).Increasingly , chrom osom e studies are being used not only f orth e d etec tio n of clo nal m ark ers b ut also to p ro vide in form atio nre le v an t f o r d iag no sis , c las sif ic atio n, an d p ro gn os is ( 51 -5 3) .T he lim itatio ns of cy to gen etics are that o nly cells in m ito siscan b e stu died an d th at th ese cells can no t be id en tif ied m o rp holo gic ally . T h is latte r p ro ble m h as b ee n at le as t p artly o ve rc om ein a recen t stu dy o f clo nal ch ro m oso m al ab norm alities in acu teleuk em ia by a new technique using the detection of lineage-sp ecif ic an tig en s in d iv id in g leu kem ic cells by m o no clo nal antib od ies (5 4). In solid tu m ors cy tog en etic stu dies are d if ficu ltas cell cultures are of ten unsuccessful. D NA analy sis nowp ro vid es a co m plem e ntary ap pro ach f or th e d etectio n o f c lo nalc hro m oso m e ab no rm alitie s in s olid tu m ors.C hro m oso m e L osses D etected by M o lecu lar Pro bes. T he lo ssof chrom osom al m aterial is one of the m ost com m on abnorm alitie s in m alig nan t c ells. D N A an aly sis d ep en ds o n th e ab ilityto distinguish the tw o chrom osom al hom ologues by the detection of R FL Ps. T he analy sis is inf orm ativ e if constitutionalD NA display s heteroz ygosity for a particular R FL P; loss ofo ne o f these al elesn tum o r D N A in dicates ch rom o som al lo ss.T his approach has dem onstrated chrom osom al loss in m anytum ors. T he use of probes detecting poly m orphism s due toV N TR s m ak es this m ethod feasible in m ost cases on accountof their high heteroz ygosity rates (55-57). T his approach hasle d to th e d isc ov ery o f tu m or-su pp ressin g g en es o r "an tion co -g en es" as d em o nstrated b y th e ex am p le of th e retin ob lasto m agene and its protein product (58-60). How ev er, it should benoted that DN A analy sis is a relativ ely crude technique todetect chrom osom al loss in a m ix ed population of norm al andab no rm al c ells as c om p are d to c yto ge ne tic s.C hro m oso m e T ran slo catio ns D ete cte d b y B re ak po in t C lu ste rR egion Probes. C hrom osom al translocations can now also bedetected in hum an neoplasm s by D N A analy sis. T he principleof this analy sis is that a D NA probe is used w hich hy bridiz esto the region of the break point on one of the chrom osom esinv olv ed in the translocation. B reak points m ust be clusteredw ithin a def ined DN A sequence otherw ise a large num ber ofprobes w ould hav e to be used f or the detection of translocated1357



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C LO NA LIT Y IN H UM AN T UMOR Sseq uenc es. S tu dies o f the Ph ilad elp hia ch rom o som e illu stratethis approach; the break points on chrom osom e 9 are v ariablebut those on chrom osom e 22 are clustered in m ost cases ov era re gio n o f 5 .8 k ilo bas es, k no w n as th e b re ak p oin t c lu ste r re gio n(6 1, 6 2). D N A pro bes w h ich hy brid iz e to th e break po in t clu sterreg io n o n ch ro m oso m e 2 2 w ill d etect m o st cases o f Ph 1-po sitiv ec hro nic m y e lo id l eu k em i a.Other translocations w hich m ay be detected directly byS outhern blotting include the t(ll;14) found in som e B -celln eo pl as m s ( 63 ), th e t (1 4;1 8) c harac te ris tic o f f o lli cu lar l ym p h o-m as (64-66), and the t(8;14) as found in sporadic Hurk in'sly m phom a (67). In principle these translocations m ay now bedetectable at v ery low cell concentrations by the poly m erasec hain re ac tio n te ch niq ue , p ro vid ed th e b re ak po in ts o n th e c hrom o so m es are clustered w ith in d ef in ed reg io ns (6 8, 6 9).Point M utations. A n ex citing recent discov ery in cancer research is that specif ic point m utations occur in the oncogenesof som e hum an tum ors. T he m utations best characteriz ed areth ose in the ras o nco ge nes o ccurrin g p red om inan tly at cod on s1 2, 1 3, or 6 1; ef ficien t scre ening m e th od s b ased o n g en e am p lif ication h av e n ow b een d ev elop ed f or th eir detectio n (70 -7 2).T here has been interest in the use of such m utations in theclonal analy sis of A N L L . U nf ortunately the present data indicate a disparity in the incidence of these m utations at presentation and relapse (73). It has been reported that ras genem u tation s o ccu r in o ver o ne-th ird o f h um an co lo rectal can cersand that m ost of the m utations are at codon 12 of the c-K i-raigene (74-76). Furtherm ore it w as show n that in f iv e of sixcancers that the sam e ras m utation w as present in the adenom -atous (benign) regions of the tum or, suggesting that the m utation p reced ed the d ev elo pm e nt o f th e m align an cy (7 4).A further ex am ple of the use of a ras gene m utation is thereport of the pluripotent stem cell origin of idiopathic m y elo-f ib rosis (7 7). T he p erip heral b lo od cells o f o ne p atien t sh ow eda point m utation at codon 12 of the N -ras oncogene. T hism utation w as then used as a m ark er to inv estigate the clonalre lati on sh ip o f d if f ere nt c ell lin eag es b y c ell s ep aratio n an aly s is .T he presence of this specif ic m utation in granulocy tes, m ono-c ytes, ery th ro blasts, B -ly m ph ocy te s, and T -ly m ph ocy tes in dic ate d a p lu rip ote nt ste m c ell o rig in f or id io path ic m y e lo fib ro sisin th is p atie nt .D N A Fin ge rp rin tin g. C om p arativ e D N A f in ge rp rin t an aly sisof a patient's tum or and constitutional D NA represents a newm e th od f or th e detectio n of clo nal m ark ers (7 8). T he f easib ilityo f th is ap pro ach d ep en ds o n th e pro perties o f th e m in isatelliteD NA probes w hich detect a large num ber of V N T Rs scatteredthroughout the genom e (79, 80). A study of gastrointestinaltum ors show ed dif ferences betw een the tum or D NA and constitutional (peripheral blood and m ucosa) D N A in about tw o-th ird s of patien ts w h en screen ed w ith th ree f in ge rp rin t p ro bes(81). M any of the dif ferences resulted f rom the loss of bands inthe tum or D NA ; w hether these correspond to m ajor chrom osom al deletions or to subm icroscopic changes is not k now n.M utant bands w ere also observ ed in tum or D NA sam ples; theprecise m echanism responsible for their generation is notk now n, although som e V N TR loci hav e been show n to hav e ahigh m utation rate (82). D N A f ingerprinting is a m ethod w ithp ote ntial f or f ollo w in g se rial g en etic c han ge s in tu m ors, p articularly in tho se cases lack in g an o bv io us cy tog en etic abn orm ality.V i ral I nt egrat ion A nal y si s

T he m olecular analy sis of tum ors containing v iruses of f ersan interesting and nov el approach to the study of clonality . A n

ex perim e ntal sy stem h as b een recen tly d escrib ed in m ice w h ichillu strates th e p rin cip les be hind this ap pro ach (8 3). T he clo nality o f v ario us cell po pu lation s w ith in th e hem atop oietic sy ste m w as stu die d b y re in fu sin g re tro viru s-in fe cte d b on e m arro wcells into m ice w hose bone m arrow had been depleted byirrad iatio n. R e po pu latio n o f th e b on e m arro w w as d eriv ed f ro mcells that had been clonally m ark ed by unique retrov iral integration sites. T he appearance of new clones and loss of clonesfrom som e lineages w ere then traced by S outhern blot hy bridiz ation of D N A from v arious hem atopoietic cell populationsw ith p ro be s s pe cif ic f or th e tran sd uc in g re tro v iru s.A sim ilar m e tho d f or assessin g th e clon ality of E pstein-B arrv irus-asso ciated tu m ors h as b een d ev elo ped ov er rec ent y ears(84). EB V D N A is a double-stranded linear m olecule w ithho m olo gou s tan dem rep eats at each term in us. T he lin ear D N Ate rm i ni o f th e v iru s are jo in ed in trac ellu larly to f orm c ov ale ntlyclo sed episo m al D N A f ollow in g v iral in fectio n o f su scep tibleho st cells. T he struc tu re o f th e v iru s dif fers f or each in dep en de ntly c irc ulariz ed g en om e o n ac co un t o f th e p re se nc e o f v ariab lenum bers of tandem repeated sequences present at each linearte rm i nu s; th es e d if fe re nc es c an b e re ad ily d ete cte d b y S o uth ernblotting. T he progeny of each infected cell contain m ultipleidentical v iral episom es w ith the sam e fused term ini. If theoriginal in v iv o m ultiplicity of infection is assum ed to be low(/.('., o ne v irio n/cell), th en in a m o no clo nal p op ulatio n o f c ells(the progeny of one-EB V infected ly m phoid cell) only onecircular f orm of the E BV episom e w ill be present, and this w illbe seen as a single band on S outhern blotting. In contrast apoly clonal population of B -cells w ill contain v iral episom esw ith dif f erent fused term ini w ith a m ultiple band pattern onSou the rn b lo tt ing .T his m ethod w as used and a m onoclonal EB V pattern hasb een f ou nd in carcin om as o f th e n asop hary nx , the p aro tid gland(84), and in a few cases of Hodgk in's disease (85), w hereasp oly clo nal E B V - in fe cte d c ell lin es c on tain ed m u ltip le f orm s . Ina further study of H odgk in's disease EB V genom e sequencesw ere d etected in R eed -S ternb erg cells b y in situ h yb rid iz atio nin ab out 2 0% o f cases, an d the f ragm e nt pattern w as c on siste ntw ith a m onoclonal population of cells (86). A study of transp lan t-ass oc iate d ly m p ho pro lif erativ e d is ord ers h as re po rte db oth t he c on f ig uratio n o f im m u no glo bu lin g en e re arran ge m e ntsand fused EB V term ini. T he data w ere considered to be consisten t w ith the n otio n that th e ly m ph op ro lif eratio ns in itiate asp oly clo nal e xp an sio ns o f E B V - carry in g B - ce lls , w h ic h p ro gre ssto m ulticlonal ly m phom as in m ost patients (87). In principlethis approach could be env isioned using probes f or the breakpo in ts of h um an p ap illo m a v iru s in h um an cerv ical carcin om asor hepatitis B v irus in liv er tum ors (88, 89).

ConclusionT he clonality of hum an tum ors is a central issue in theunderstanding of tum origenesis. T his is ex em plif ied by theleuk em ias in w hich clonal analy sis can be used to study thestem cell nature and the dif ferentiation potential of the neo-p lastic cells. C lo nal an aly sis of p articu lar tu m ors h as b een th estarting point f or the discov ery of im portant genes such as thetu m or-su pp re sso r g en es n ow b ein g c harac te riz ed in th e re tin o-b lasto m a m o del. Fu rther research in to th e clo nal co m po sitio nan d ev olutio n o f tu m ors w ill u nd ou bted ly p rov id e new insig htsinto tum or dev elopm ent and grow th, as w ell as prov ide clonalm ark ers relev ant to the clinical diagnosis and follow -up ofindiv idual cases.1358



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AcknowledgmentsW e are indebted to Dr. H. J. Peter (Departm ent of M edicine,In se ls pital, B e rn ) and D r. K . G atte r (D ep arm e nt o f H isto path olo gy ,John R adclif fe H ospital, O xf ord) f or critically rev iew ing the m anuscript.

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