Detecting minimal residual leukemia

Detecting minimal residual leukemia

Detecting Minimal Residual Leukemia Anna Butturini and Robert Peter Gale INTRODUCTION hHensiv(; (:hemotherapy [)ro(h](:es (:omplete r e m i s s i o ...

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Detecting Minimal Residual Leukemia Anna Butturini and Robert Peter Gale


hHensiv(; (:hemotherapy [)ro(h](:es (:omplete r e m i s s i o n s in a high prol)ortioll of subjects with acute lymphoblasti(: leukemia (ALl,) and acute mvelobhlsli(: leukemia (AML) I1,2 I. However. m a n y of these patients u l t i m a t e l y relapse. Complete r e m i s s i o n s are c o n s i d e r a b l y less c o m m ( m following (:hemotherapy for (:hroni(: m y e l o g e n o u s leukemia (CML). c h r o n i c lyml)hO(:yti(: leukemia (CLL), and hairy (:ell leukemia ([ICL). Nevertheless, those a(:hieving c o m p l e t e r e m i s s i o n s with i n n o w i t i v e therat)ies like bone marrow t r a n s p l a n t a t i o n , biologi(: ~.|g,elltS like interferon, or drugs that mo(lifv l)yrimidine meta[)olism, often relapse I3-6]. Thus, leukemia re(:urre, tl(:e is a major prol)lem even after suc(:essful inilia] treatment t)f leukemia. Most data suggest that relat)ses arise from residual leukemia (:ells s u r v i v i n g initial treatment. These (:ells are referred to as m i n i m a l residual leukemia {MRI,). The disl)arity b e t w e e n the (:lini(:al definition of (:omt)lete remissi(m imd the t)resem:e of residual leukemia (:ells results from the inability to (tete(:t small n u m b e r s of leukemia (:ells. Because (tete(:ting m i n i m a l residual leuke, mi;i might have iml)()rtanl I)i()logi(: and c:linic:al implit:ations, there is (:onsi(tera[)le interest in (leveloping m()re sensitive dete(:tion te(:hni(:s, tlere, we review data regar(linM MR1, and (tis(:uss its (:lini(:,l and bioh)gi

Identification of leukemia (:ells is based on (teterminiIlg qualitative, or (luuutitatixe differen(:es b e t w e e n these ~|lld n o r m a l (:ells. Typical apl)roa(:hes fo(:us on morphi,logi(:, phenotypi(:, meta|)oli(:, proliferative, and geneti(: features. The




to identify



(:ells i n v o l v e s


of bone marrow and blood (:ell morl)h(dogy. Ex(:el)l for rare [:ases ()1 leukemia with sl)e(:ifi(: morl)hologi(: feature, s, like Auer bodies in A M I , it is not possible to k n m v with certainty if a (:ell is leukemi(:. Thus, m o r p h o l o g y is ()nly~ useful if lhere is an increased n u m b e r or l)roportion of blasts in the blood a n d / o r bone marrow. In a~:ute leukemia, the n o r m a l u p p e r limit for these (:ells is typi(:ally 1% in the I)1oo(t and 5% ill boll.e marrow 17]. For examl)le, persons with AML and 5% myeloblasts in the bone marrow are (:onsi(tered to have residual leukemia. However, w h e n there are " 5% myeloblasts, the tmrson is regarded as being in relnission regardless (ff w h e t h e r (:ells are n o r m a l or leukemi(:. Clearly, these s i t u a t i o n s have entirely different iml)lica-

From the l)el)~lrtm(mt o[ I).diMri(:s I:\. ILL [ hli\,eFxily ()t I)~Hnla, I);irm~l. [|lily, iltld lhl! Division .t l Jem~dul ogy-()n(:ldilg},' (R. P. (',.). I)ell~H'tmitnl iiJ Medi~:in~. 1.1(]1.,\ S( h . o l . t l',,|ltdii:ine, I.iis ;\lW, l!lcs, ( : . l i h w . i . .

A(/(/r~!ss r(tpz'ixlt retpxcsts It): l~ot)erl l~e,tcr (,(ll(t, ~\11). Phi), l)ivisi(m (Jt lhml(~tol(J(,4y-()m;(~loey. Dcp(zrtment of Medichm. (ICI,A S(:hool o/,\le(ti(:hl~, l('),'~3;:tl,c Con/(( Av~mue. Los An,-~qes. CA 90f}24-1 l~78. l~ec(dved lttne 1, 1990: a(:(:eptcd [tree 11. 1990. 19

(c}1991 Elsevier S(:i(,qlc:ePublishing Co.. Inc. 655 Avenue of the Americ:as.Ne,w York, NY 10010

Cancer Genet Cyh)genet 52:19 26 (1(,]91) [)165-4(;08/91/$03.50


A. Butturini and R. P. (;ale,

lions. A similar limitation applies to morph(flogi(: attempts to detect persisting4 A1,L (:ells. Clearly. more sensitive and specific te(:hni(:s are needed to detect residual leukemia (:ells. Many approaches are used. In some, instances, studies ot (:ell p(qmlations enri(:hed for potential leukemia (:ells arc used to increase sensitivity of (lete(:ti(m 18, 9]. Also, different techniques are sometimes combined to incre~sc' specifh:ily IlI}, 11 IO n e approa(:h to identifying, residual leukemia (:ells involves stud ies of cell phelmlype. This strategy is based on the notion fllat leukemia (:ells ext)ress (tither spe(:ifi(: antigens (,r (:ombinations of normal antigens not Iyl)i(:al ot normal (:ells. Examples in(:lude terminal deoxynu(:h,'()ti(le transferase (T(IT) and (]D5 or ()ther mature T- and B-cell determinants in ALL[12 14],(3)22 and t-(;P prot(fin, C D l l t : o r t t C 2 in hairy (:ell leukemia [15[, CD5 and B-(:ell determinants in B-(:ell CLI, [lli[. (:ell phen(,tyl)e studies are less common in AML [17I. Analysis of (:ell tfl~elmtype ofhm uses fluores(:ein- or rhodamine-lal)eled monochm~d antibodies with thmble, or multiple st~fining techniques. Sensitivity of this method using fhmresc~;n(:e micros,:opy or a fluores(:ence activated (:ell sorter (I:ACS) is about 0.1 -5%. rl'he sensitivity of immun(,hist()logic technics, such as PAIL APAAP, immuno-ooht ~md others, is ~,reater, ,bout 0.01 0.1% in most studies. Sensitivity of these t e c h n i c s i s l i m i t e ( l b y a n t i g e n h e t e r o g e nelly and antigen modulati,m on leukemia ctdls. Also. no leukemia-specific anti,4ens are as yet identified 118 21I. A second apl)roa(:h to detect residual h;ukemia cells involves hist()<:hemit:~d studies of intra(:ellular enzymes. Ex(:ept for TdT, these (ire rarely used. Serum h~vels of enzymes (lire(:tly or indire(:tly li(Iked to leukemia m'e rep(Hte(t to pre(tic! re.lapse in some studies ]22I; this ix (:ontroversial. Also, this appr(m(:h has h~w spe(:ifi(:ity and sensitivity, and ix not qmmtitalive. A third approa(:h to detect resi(h,Jl leukemia cells is based on a leukemia-specific pattern of growth in vitro usually using chmogeni(: assays in semi-solid malri(:es IlO, l l , 231. The sensilivity and spe(:ifi(:ity of these techniques is (:(mh-oversial; they are un(:ommonly used. Sometimes these studies are ccm~tflemente(I by (:ylo~enetic analyses of (:ells from the ¢:olonies. This d l o w s spe¢:ifi(: dete(:tion of leukemia (:ells when a (:ytogene3i(: marker ix present (vide infra). In role stu(tv of persons with ALL in remission, in vitro gr()wth of h;ukemi~t (:ells, (:(mfirmed by (:yt(),'4eneti(: analyses, failed to predict relapse [10[. A fourth apt)roa(:h to detect residual leukemia (:ells involves mlalysis of (:ytogeneti(: or leukemia-spe(:ifi(: gene abnormalities. Consistent (:hr~m/osome abnormalities o(:(:ur in virtually all cases of CMl, and in 5() 80% of cases of acute leukemias lZ4]. Because

chromosome analyses typically (lete(:t only (lividiug (:ells, aml .s ntmfl)ers of mitoses examine(l are usually limited, sensitivity of tiffs method is relatively low. Different techniques are use(t to ()vert:ome these limitations; examples in¢:lu(h: flow (:ytolnetry of interphase (:ells or use of growth fa(:tors though t() stimulate leukemi~ (:ells to divide [25 I. We recently show(.'(t this approach can also mask hmkemia-re.lated abnormalities by preferenlially stimulating normal (:ells [261. It is also possible to use hmkemia-specifi(: ~ene markers, such as gene a[mormalities or T-cell receptor or immtmoglobulin (lg) gene rearrang,ements, to detect residual leukemia cells, The most commonly used (m(:ogene abnormalities are the BCR/AB1, fusion gene in virtually all cases of (]M1, and NRAS point mutations in about 25% of cases of AML and in a smaller pr(q)ortion of ALL. In these instances, (:orrespon(ling mRNAs and proteiils are also abnormal. MY(; and I(;H rearrangement o(:(:ur in B-AI,L with the t(8:14), t(2:8), t(8:22) translo(:ations and the, B(]I,2/I(; fusion gene o(:(:urs in (:ommon ALL and lymt)homas with the t(14:18) Iransh),:ation. In these cases. correspon(ling InRNAs and proteins may [)e (tuantitatively but not qualitatively almorlllal (for a review, see [27]). Other Ieukenlia-spet:ifi(: ,qelle alterations possil)ly useful to detect MRL are the E 1 2 / E 4 7 gene rearrangement in pre (/-ALl, with the t(l:19)

Dete(:ting Minilnal Residual l,eukemia


translot:ation [281, the CAN proto-oncogene rearrangement in AMI~ with the t(6:9) translot:ation [291, and interferon gene deletions in ALL 130]. l)elta an(t/or gamma T(:ell receptor and/or Ig heavy and light-t:bain gene rearrangements occur in virtmdlv all cases of ALL 131 I, in many hybrid or undifferentiated leukemias, and in a subset of (:ases of AML 132 341. Metho(ls used to study DNA rearrangements or t)oint mutations, su(:h as Si)uthern or northern blots or hybri(tizatilm to svnthetic oligmmcleotide t)rl)bes, usually detect -- 1 5'51, leukemia (:ells. In some stulties, these assays were i)erformell folh)wing enrit:hment of leukemia (:ells by gradient set)aration and/or i l n m u n e selel:til)n 18, 9[. Another potentially sensitive technique involves aml)lificatitm ()t abnl)rmal I)NA fragments or mRNAs by the l)olymerase chain real:lion (P(]R) 1351. This at)proa(:h i:an inl:rease sensitivity to 0.002 2%. but there are considerable prol)lems with false positive reactions. Also, use of these approaches is limite,d to hmkemias with (:onsistent rearrangements or mutational sites [36 391. When the genetic rearrangement characterizing the leukemia clone is variable, such as with MY(], the T-cell rel:eptor, ()r lg genes, st)ecifi(: DNA sequem:es must I)e determined in each (:ase to devell)p appropriate P(]R primers [7.8, 40[. Because this is laborious, different strategies have been attempted. Because in 70(}:oof (:ases o[ ALL, re,arrange, merits of T-cell re(:eptor delta and galnma genes o(:(:ur within a limite(t number of DNA seglnents, and a similar situatil)n is described for the V-region ()f Ig heavv-chain gene ill l:l-ce]l AI,L 131l, stu(ties have been fo(:usell im these genes 141 441. Sometimes these approaches are less spe(:ific because, they (tetect similar r e a r r a n g e m e n t s ill llorlllal lymphocytes. Another technique to detect MRL involves leukemia-specific DNA or RNA probes for in situ hybri(tization 1431. It is also i)ossible to detect exl)ression of on(:ogenerelate(t abnormal proteins t)7' i m m u n e histology or i m m u n e fluorescence. For example, antibo(ties to mutated RAS proteins and to the BCR/ABI~ fusion protein are rel)orte(I 145 471 . Quantitative analyses ot RNA or t)roteiu expression are less sl)e(:ifit: 1481.


It is not yet k n o w n if detecting MRI, is important in treating leukemia. Presumably, persons with MRL might be more likely to relapse than those in whom leukemia is completely eradicated. However, this is not certain. For example, it may not be necessary to eradicate leukemia tot cure 149]. It is also possible that different cases wtry: some with MRL might relapse, others not. This may' reflect qualitative or quantitative differences, or both. Table 1 summarizes approaches to detect MRI, in different leukcqnias. (:NIL can be studied by PCR amplification of the BCR/ABL fusion mRNA and possibly bv studies of the BCR/ABL fusion protein. Acute leukemias are more complex. In most cases of ALL, cytogenetic analyses and studies of T-cell receptor and Ig gene rearrangement are potentially useful. PCR amplification of BCL2/IGH gene is important in rare cases w i t h the t(14;18) translocation. The importance of other gene markers, such as MYC, E12/E47, or other rearrangements, is not yet established. The value of other atlproaches, such as studies of cell phenotype or in vitro growth assays, is questionable. In AML, diagnosis of MRL can be based almost exclusively on studies of RAS mutations or c h r o m o s o m e abnormalities. One major problem in detecting MRL is the hcqerogeneity of leukemia cells. We discussed the limitation of teclmology based on cell phenotype. Because leukem()genesis is likely a multi-step process [50], even leukenlia-specific genetic markers may be late events and might be absent ill residual leukemia (:ells. An examph~ is NRAS mutations, which nlay be present at (Iiagnosis but at)sent at relapse (and presumably in residual leukemia cells) 138, 51 I. This is also described for chromosome abnormali-


A. lhJllurini an(t R. P. (;ale

Table 1

Methods for detection of MRL in different l e u k e m i a s ( r e t e r e n ( : e s [7 48])



('.rowth (:MI,


Ph(;notvl)e Not rel)orte(I

assa3s N(~I


l(9;22J I'

I'(![)(ll'{(~(] '~


"Fcs ~'

T I ) T + (:1)5 j' TI)T ~ (]I)IW'



M ( d . ( : u h . ' Markers

t:R;R:AtlL RNA by P(:R I' p210 B(W,,AFll, I)v Mo:\l~ slaining TCR. 1(; I)N/\ rc.rral~g.m(mi b\" ,qoLHhcrtl

()r I ' ( ] R I'

H,'rN ~(]ll(~ F(!iiFl'ii/l~(!H](HIt'


B(]I,2 1(; I)NA by l'(]N"





l)\: Southern

tla:221 l(1:191






Mul,ted I)21 RAS by MeAl'; staining MY(L MYtL SIS RNA by in silll hybrid. '1




(3)5 + BAg (]1)22 ~ 1 (;I)

Not rlq)orh!(I

E12,1'A7 ~etm rearrangem(!rd' RA,"; I)N,'\ b \ t'(]R h

I((Lg] NI)I

( ]/'~N

I'( !il I'I'H I I g(!l/1 ( H 1I

1!4 rl~arrangimu!lli hv ,'4(mthern or PCR


CI)22 t H(:2 C1)22 + C I ) I I ( : " RHrl!lv /Ibl!(t ill a~sl)(ialiHIi kxilh (:]lroltl(~s(Hm~ ailal ~sis (d
I . ~ s s i h V .setul to (l.t(~(l M R I ,

ties a n d T-(:e, ll

recept(." ~ene rearrangement [52-551.

Hm'lherm(m~, the dislribution


residual l e u k e m i a cells in tim b()dv is like, Iv he(erogenous 151i]. Also. t h e numbe, r o f residual cells may be so small as to be u n d e t e c t a b l e by any technique. A second issue is w h e t h e r detecting MRI, pr(;dR:ls h m k e m i a relapse. Studies el the Ph ~c h r o m o s o m e and BCR/ABL m R N A in patients with CML in c o n t i n u o u s r e m i s s i o n 1 to 8 }'ears after b o n e m a r r o w t r a n s p l a n t a t i o n

s h o w p e r s i s t i n g cells w i t h t h e s e

a b n o r m a l i t i e s [57-61 ]. S i m i l a r results are reported in persons with ALL using leukemia growth assays or studies of T-cell receptor gone r e a r r a n g e m e n t s [8. 10]. O n e possible e,xphmaliol] (1t these findings is that these, persons will s u b s e q u e n t l y relapse. Data from the il[()lllic bOllll) survivors dm,'ehqfing CMI, showed a lalem:v interval o t 2 to 31 years [(i21. Alternatively. imrllaF.S ii1]I .ll residual leukemia ~:ells can cause leukemia relapse. There might b{~ sm,'eral reasons for this. ()he is that these cells were damaged by prior therapy and c a n n o t pr(Aiferat(~ in rive. This seems u n l i k e h ' be,cause these cells pr(fliferate adequately in vitro. A se(:ond p[)ssibility is that residual cells with these leukemia associated a l m o r m a l i t i e s c m m o t proliferate,. For (~xample, it is hypothesized that rp,sidual BCR/ABI, positive
l)ete(:ting Minimal Residual Leukemia


may also operate. For example, there may t)e (:ompetition between donor stem (:ells and leukemia (',ells for preferential sites in the mi(:roenvironment that permit (:hmal e x l ) a n s i o n [67 I. A l t e r n a t i v e l y , regu l a t o r y m o l e ( : u l e s m a y i n h i b i t p r o l i f e r a t i o n of l e u k e m i a (:ells. A n t i - l e u k e l n i a m e ( : h a n i s m s are prol)a[)lv d i f f e r e n t in (Iifferent l e u k e m i a s a n d t)()ssiblv in d i f f e r e n t p e r s o n s . S o m e m e ( : h a n i s m s m a y (:(mtrol r a t h e r t h a n e r a d i t : a t e h , ' u k e m i a (:ells. P r e s e n t l y , it is t m c e r t a i n w h e t h e r ( t i a ~ n o s i s of MRI~ woul(I be u s e f u l for (h;signing l e u k e m i a t h e r a p y , l tow(',ver, t h e s e s t u d i e s are, likely h) t)e i m p o r t a n t in u n d e r s t a n d ing h o w l e u k e m i a is o r c a n be c u r e d .

Sul)porh~d in part I)v grant (;A 385(~!) h'l}ln thr, N(II, NIIt. USP|tS. I)HHS. and a grant D()m lhc (:enter for A d v a n c e d Studies in I.eukemia. Roberl Peter (;ale is Iht~ Wahl t:oundalion Sl:holar in Biomedil:al (]()lllllltlllil ~tliOll itlld ()resident of the Armand Halnln(~r (:enter for Adwml:ed Studies ill Nuclear Energy and Health. We thank l,:ma]mol M.i{hmbm'g for h!l:hni{al assishume and Katharine Fry for Ivl)ing the manus(:ripI.

REFERENCES 1. (]hanlplin R, (;ale RP (198,q): z'\c:ute lymphohlastit: h:ukemia: Recent a(Ivancl!s ill hiology ~llld therapy. Bh)od 73:2051 206(i. 2. Champlin R, (;ale RP (1987): Al:uh: myelogt!llous leukl!mia: Rel:ent ildvanl:l~s in therapy. t~lood B!):155 1- 151i2. 3. (;oI(hnan IM. (;ale RP, Horowilz MM. Biggs J(;, Champlin RE. (]lm:klnan 1",, Hoffmall R(;. Jacobsen S], Marlnonl AM. Mc(;lave PB, Messlmr HA. Rimm AA. Rl~zmall C. Speck B. Tura S. Weim!r RS. Borlin MM (1,988): Bone marrow lraIlsplanlatioll h)r (:hronic myelog(mous leukl~mia in chroni< t)hase: hn:reasod risk ot i'elapse associated with T (:,ll depletion. Ann [IHern Mod 108:80(}-814. 4. Talpaz M, Kantarjian H. Mc,(;redie K, Trujilh~ IM, Kealing MI, (',utlerman ][! ( 1986): ltmnato logic rmnissilm and cytogeneti< improvelnent induced h?< ri!t:Oilll)inaiMh u m a n interl'eroll alpha in chronil: myelogonous leukemia. N Engl I M e d 314: IIIli5-i()7(). 5, Quesada ]R, Reuben I, M a i m i n g rl'.Hersh, EM. (;uth,~rmall ]I.I(19~H]: Alpha hm!Mi!rlm for induclhm of rl:misshm in hairy c:ell hmkemia. N I:me,l I M e d 31(I:15 19. 6. l~andini (;. Mauri(:ette M, Rosti (;, Tura S. Bone m a r r o w Iransl)laiItati(m in
8. Zohldmuer BA. PaI'del I)M. Burkt, PJ. (;raham Nil,. Vogelstein B (l!l~l(~): hnmul,)glolnllill g,t!ll¢! re~trritllgtHIIP, IltS in remission bone marrow spet:ilnens h'oln patienls with alCIIl(~ lymph oblastic lmlkemia, l~lood f17:~153 .~5~L ,9. Bregni M, Siena S. Neri A, Bassan R. Barbui T, Delia f). I~;om.hmna (',, I)alhul:evera R. (;ialmi AM ( 1,~)~;!)]: Minimal residual disease in al:ute 1~,mphldJlasli~: leukomi, d(d(~l:l(~d t1~.' immulm selection and ~,ene rearrangement analysis. ] Clin ()m:ol 7:388 343. 10. F:sh'l)v Z. (;runbt~rgcr R, lJube IlL Wang YP. Freedman MIi {1(,)86]: l)etet:litm ()t r(~silhml al:uh; lymphobhlsli(: leukemia

A. B u t t u r i n i an{t R. P. ( ; a l e

14. P i h a n {;, M c F a d d e n M I . Savas 1,, Woda BA {1989}: A s e n s i t i v e le{:hnique lot the dele

36. l~ee MS, {;hang KS, {]abanillas F. tq'eirei{:h t';S. Trujilh} IM, Stass SA [1987): l)ele(:liol] of minilnal residual {:ells {:aI'l'Vitlg the t{I4;lS} bv I}NA sequen{:{~ amplifi{:ation. S{:iem;e 237:175 178.

I)etecting Minimal




37. Ste.tJ(!r ~tevem.;()n M, Raffah] IN,,],(:()hon P, ( : a ~ m a n I (1988): I)eh!(:thm cfl O(:CHII flflli(:ul.r ] y m p h o m a by sl)e(:i[i(: D N A .mplifi(:ali(m. lflood 72: 1~'122-]825. 38. Farr CJ, S.iki RK. t':dich tIA, Mc(:ormi(:k V, M . r s h a l l CJ (1988): ,'\rod\sis of r.s gem~ mutalion.'-; in ac:uh~ mveloid leukemia by chaiu rea(:liou . m l olig(mu(:h~)Wde prol)es. Proc Nail A(:a(I .';(:i [1SA 85:1(i29 1(i33. 39. l.e~ MS, la~ Maistre S, Kan(arjian tIM. T . I p a z N1. lq'eirei(:h El. T r u j i l l o ]NI, Slass t-;A ( 1.q891: I)et¢~(:timl (fl' l w o ~dternalive B(:R:ABI, mRNA ium:thms . m l minimal resichlal ( in lflfihMelphia ¢:hromos(mle I)()siti\'~ ¢:hrmfic: im,~do?lmmls h,ukemia [)\' l~(dylm.'.se ( r e . d iota Ifl()od 73:2165--2170. 4(I. Katz V, Ball l,, (;ibb(m H. ( : h e s s e l l s ] (1989/: T h e us(~ of I)NA I)r/)l)es to nionihlr m i n i m ; d residuM (liseast~ in i:hildh()(~d .c:ute Ivln[)h()[flaslil: llmkelnim B r ] I h m m . t M 73:173 114().

41. lhmsen-Hagge TE. Y(ik(ita ,% Barlrmn CR (1!)89): l)eh!ction (it miniulal re.'-;idual di.'-;em-;~ in at:ule lympilobhlsli(: l e u k e m i , by iu vitro alnl)lilic:ati(m of rem'rmlqt!(n T (:ell ret:el)l(}r (I (:h.iu ~4i!(]ll(',ll(:(~,q, }{]()t)d 74:i 762 1767. 42. I ) ' A u r i o l 1+, Ma(:illtVl't~ E. (;alibert F, ,';igaux 1;{ 19~'~91: Ill vitro ililll/[iii(;at]Otl (it T (:t+ll gilllltlili ~(!ll(! i'earraugenlent:-;: a ill!~.v tool tot the a,'-;ses,'-;ulent ¢11 l/iiniuia] rt+,'-;idu,I di,,-;t!as+! in acute Ivulllhol)lasti(: hmkemi~l, l,eukemia 3:155 15fi,

43. I)t~al~e M, Norton 1I) (199(11: I)eteditm ()t inununl)gh)lmli]~ gt!nt! re.rr.nMemcmt in tT lymldl(fid malignlmcies by p()lylll(!l'ilSt! d l . i u rt!ildioll ~(!IH! .mplifit:.lim~. Br 1 H.l!mah)l 74:251 256. 44. Sibel NI., Kirsch IR (19~19): 'l'uulor delectimi through Ihe use ot i n u n u u c ~ h d n d i n rearrlmgenient.s combil~ed w i l h tissue in situ h v l M d i z a t i l m , l'llood 74:17!11 1795.


45. \,Vong (., A r n h e i m N, (:lark R, Mc(' P. Innis M. A h l w i n 1. Nilecki 13. M~:(;ormi(:k 1"{ 1!1861: I)etei:ticm ()J' activated Mr 21.0(10 pr(itein, the ])rolhll:l ()1 12`AS (mc(~gmlt~.'-;. uMn~ antihc)dh~s ,.vilh :-;pec;ifh;ity [(ir anlill(lac:i(I ]2.. (;anl:(!r RI!.'-; 4(i:6029 fi(13',L 4(i. ~1OO1"(!IP, Evan (;l {19871: hnmut'~t)assa\'s [()r onl:Olm)((!ins. Nalul'(! 327:733


47. V()II ])en(lt~ren 1. t h ! r n l a n s A, i"vlt!(rtlwst!ll '1'. 'l'rt)eistra (:. /.(!g(!l'S N. ]{()(!l'.qlil/i '~', (:i'osvl~ld (;, Villi I:,vvijk W (1989): Antihl)(Iv rec:i)gllilic)ll o[ lhc IIIlnor Sl)t~(:ifi(: B(3~/ABI, ioinin?- rt!~ion iu /:hrllnii myehlid h!ukelnia. ] I'J×I3 Mell 11i9:87 !18. 48.

l)i(:ke KA, I)i(:ke l~JvinRl!r M]. ,":,l/ino]()J,'\. ,";lfilzer (;S 119891: l)mlbh~ inlensific.ti(m incliMin~ .utoh/~()us 1)()11(! 11161rr()~A' lranslllanlati(m (AF~MT) in a(:ute nlyel()~UllOUS [(~uk~mfia {AMI,): term folly.iv,: tll) iill(] d~.~tet:timt ()t i n i n i m . I residual d i s e . s e . I}lo()d 7 4 ( s u p p l 11: 283a.

4!1. ('.idl! RP, Butturini A. H o w do b c m . nmrrov,: t l ' a n s p h m l s c u r e hmkelni~." In: N e w ,'-;(rate~ies ill l}onl! ~.,lill'l'(p,v T r a n s p l a l f l a t i t m , (;ah~ t2,1'. ( ; h a n l p l i n R. ells. W i h w l,iss. N e w Y()rk, in press. 50. Klein (;. Kh;in E (19/G): t",vcfluti(m cfl' t u m o P , , m l iml)m:t ol m(dc~cuhu' (m(:(fl~igy. Nilhll'e 115:190 195. 51. FI,rtram CR, L u d w i g WI), H i d ( t e m a n n W. l~vons 1, l~uschle M, Rilh~r I. llarboll 1, l"r~)hlich A. I , n s s e n IW 1198.9/: Al:ule mwqcfid h~ukenfi,: , n , l v s i s c~t I'~iSg(rlH! m u h f l i o n s mid ~:hmlllitv (]el'ille(] by llolimorlfldl: X linked h)l:i. [,(!tlkl!tllilt 3:247 256, 52. I)(! Cuia MR, A l i m e n a (;. (;astahli R. Si)irili NI,\. (;i(ma 1:. Nhml:ini N1. Nl.n(h~lli 1: ( 198~3): Al:uh~ lnvl~lol/laslic l e u k m n i a w i t h 1{8:21] f()lh~wing Phi h l d i d p h i , posit ixl! ~u:ute uwehfllh~sti*: h m k e m i . . I,eukemia 3:3 Ill 313. 53. Pui CH. Raimon(li S(:, lhdun l:(;, O(:hs l. F u r m a n WI~. B u n i n Nl.12`ililfir()12`(LTinshw P;\, Mirr(i ] (1986]: Shill in [)[aM cell tlhen//lype mill k . r y o t y p e lit relal)sl~ o[ (:hihlh(~(M lvnv)h(dll.sti(: leuketnill, lih)od 68:1306 1310. 54. (]-a.'4tll)n (;A, C h i h l s C(], l,e M , i s l r e A. Kelding N1, (]()rk ,,\, Truiilh) IN1. N~fllis K. I:rlfireic:h K. .';laSS S,'\ (19fitl): Moh~(:uhu" hehu'ogeneily in a(:ule leuk~m~ia line.g~ switch. BIo(M 74:2{}88-2(}95. 55. ( ; a r d i n a SL, S c h r o f f RW, W o o d h o u s e CS, (;olde, I)W, O l d h a m RK, Cle, ary NIL, Sklar I, Pritikiia N, F o o n KA {1985): l)etection of l w o distinct m a l i g n a n t 13 cell c l o n e s in a single l)atierd tlSillg a n t i - i d i o t y p e IllOllOCl(illa[ alatibody alld i n u n u n o g l o b u l i l a gene rt~alTallgeln(31at. Bill(ill 66:'1017-172i. 56. MartullS ACM, ,'qi:hullz FW. Hagenlleek A (19fi71: NOli htllllt),U,(HltHl,'q (iisll'ibutil)n o[ h!ukeluia ill till [)on(~ I/llil'l'tl%v (hiring ininilnal reMdual 131ood 7(1:1(173 I(178.

57. /,m:t:ari;.t A, Rosti (;, S e s s a r e g o M, I"rassoni V, ['vl/trnlout A, Galimllerti M, liaront 1). lqlipt)etti A. I)i ]}arh)h)lneo P, l)i (;irtdamo (; ( 1988]: 12`(dal/Se after allogenei(: b m m IIIOI'F()~V (ransl)hmtation for PhihMe]i)hi. (:hronmsome positive chrcmi(: myeh)id hmkelni.: cytogelwlit: a n a l y s i s of 24 l)a{it~lll,'-;. []Ollt~ Nlal'l'(Iw '['l'illlslllllll[ 3:4 13-423.


A. Butturil~i (m(t R. P. (;ah,'

58. A r t h u r CK, Apperh!y ]1", (;uo AlL Rtlss¢)~l F, (;ao I~M, ( ; o l d m a n ]M (1988): Cytogenetic (!vents after bone m a r r o w transp[anhdicm t'¢~i"c h r o n i c lll~,'l!lo'4I!llOIIS I(!llkei/lia ill c:hrol/[c: [)base. Blood 71:1179. 59. R¢~th MS. Antill ]H. Bil~gham El.. ( ; i n s b u r g I) (1989): I)~te¢:iioll of Pbil.delt)hia ¢:hrolnOSOm(! positive (:(dis by the t)olymeras(! c h a i n reaction following bone~ Marl'otv trallsi)lalltali¢m for c h r o n i c Illv(~logellt)tls l e . k ( m d a . Blood 74:~/I:{2-~'I~{5. [10. (;abert I, Tburet 1, LCdag¢~ M, (]~trt:asS()llll(! x(, l'vlttl'anill(:bi I), N:Iallll()ll[ P {19,~i9l: I)¢~t~c:tion of B(]R-ABI, trallsloc:alit)ll I)y p(dvlm~ras, c h a i n rear:lion hi (:hr(mic in~(doid h!ukemia ])al[(!lll% ~dh~r bon(~ lll}lrro~A.z transphmtatioll. [.ail(;et ii:1125 112~i. 61. }tu,gll(h'-; '[', Martial P. NiOFI4all G, Sav,.'vt!r S(;. "~,"ilh! ()N, (;()hllall }M (1991)): .%i,qnilh:all( (! ot residual letlk(!l/li~l tFallSt:l'J])t5 af|(~l' I~()ll(~ III~IFI't)IA' lFallsIll~llll for (]NIL. ]~all(;cl 335:51). 62. M o h m e v W(~ (1987]: Rattle)genii: h~uk(~mia revisih~d. Bh)od 70:9(15 908. 63. I:hltturill[ A, (]a](~ RP. A r(!vi(!w c)] tl.~ i'(!Iill]oll~;hi[)b(!lv.,'(!l!ll (:lolHlilv at.I trallslorlllatioll ill ~](! hmk¢m~i., l,t~txk Rt~s, {ill I)l'(~;~,]. 64. TruilI I~.L, (;ah~ RP, Bortill MM [1987): C(!lluhlr immtmoth(~rt~i)v oI (:~m(:(~r. A h m R. I,iss. N(~w York. 65. BiilIill'illi A, (;ah~ RP [ 19117]: The role c>tT (:(~ll.~ it] pl'(~V(',lltillg ~'(~l~I)S(~i~] chl'(mi(: I(!/lk(!ll]ia. [~on{! Marrow 'J'u'anSl)hmt Z:351 354. (i(i. tlorowilz MM, (;ah~ RP. SOllf](!I PM, (;i)Idlllan II~vl. k(~l'Sey 1, kolb HI, Rinun Ai\. Rillgd~m (), Rozlllall (~. Si)(!(:k I{. Truilt RI,, Swarm FE, Borlill M (1~:)9(I): (;l'it]'l v(!rg/IS Ietlk(unia r(~action a f t ( ~ r h o n t ~ m a r r o w t r ~ m s p h m l ~ t i t m . Bh)od 75:555 5~i2. (i7. tiah~ (L (:obbold S. W a h l m a m ~ It { 19[/}I]: T (:(~ll depletiol~ wilh (:alnpath-1 in alhlglm(!i(: I)cm~! ll/ttrro'w trailsl)lalllal[oll. Tr~lll,'-;l)lalltalic)ll 45:75;I 75~'i,