Monitoring of BCR-ABL expression using real-time RT-PCR in CML after bone marrow or peripheral blood stem cell transplantation

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1 Leukemi (1999) 13, Stockton Press All rights reserved /99 $ Monitoring of BCR-ABL expression using rel-time RT-PCR in CML fter bone mrrow or peripherl blood stem cell trnsplnttion M Eder, K Bttmer, S Kfert, A Stucki, A Gnser nd B Hertenstein Medizinische Hochschule Hnnover, Abteilung Hämtologie und Onkologie, Zentrum der Inneren Medizin, Crl-Neuberg Strsse 1,1jyD Hnnover, Germny To nlyze the vlue of rel time RT-PCR for monitoring of bcrbl expression in CML ptients fter llogeneic or utologous stem cell trnsplnttion (SCT), we generted pirs of PCR-primers nd TqMn probes specific for either the b22- or the b32-vrint of bcr-bl. Either vrint could be detected specificlly from cdna from single K562 (b32) nd BV173 (b22) cell with the respective TqMn probe. Bcr-bl expression ws normlized by comprison with GAPDH expression, nd smples were quntitted using stndrd cdna dilutions from K562 or BV173 cells. In retrospective nlysis 13 ptients with CML fter llogeneic (n = 10) or utologous (n = 3) SCT including ptients with relpsed or persistent CML were nlyzed by both rel-time nd conventionl nested RT-PCR. In ddition chimerism ws monitored by FISH nlysis of sex chromosomes in three ptients with relpsed disese. The bcr-bl/gapdh rtio dropped t lest 1000-fold in ll seven ptients evluble prior to nd fter llogeneic SCT s estimted by rel-time RT-PCR, nd conventionl RT-PCR becme negtive in 6/7 ptients. In five ptients with relpsed or persistent disese fter llogeneic SCT the bcr-bl/gapdh rtio eventully incresed gin, nd rel-time RT-PCR ws s sensitive s conventionl RT-PCR for detection of bcr-bl. Donor lymphocyte infusions (DLI) were given to ll five ptients, nd the bcr-bl/gapdh rtio dropped to undetectble levels in two ptients both remining in continuing moleculr remission. In contrst, in three other ptients the bcrbl/gapdh rtio decresed only or did not chnge significntly fter DLI. In three ptients undergoing utologous SCT the bcrbl/gapdh rtio dropped only 1.1 to 30-fold, nd the ptients were tested positive with rel-time RT-PCR t ll time points. These dt demonstrte tht rel-time RT-PCR is vluble to quntitte bcr-bl expression in CML ptients fter trnsplnttion. Keywords: rel-time RT-PCR; chronic myeloid leukemi; miniml residul disese; stem cell trnsplnttion Introduction Chronic myeloid leukemi (CML) is myeloprolifertive disorder of pluripotent hemtopoietic stem cell chrcterized by the chimeric bcr-bl gene resulting from the juxtposition of bl-sequences from chromosome 9 to the bcr-gene on chromosome 22 in more thn 95% of ll ptients. 1 The corresponding cytogenetic hllmrk is the Ph-chromosome with t(9;22)(q34;q11). CML is bi- or triphsic disese with n initil stble or chronic phse followed ultimtely by either n ccelerted phse or directly by blst crisis terminting ftlly. Actul therpeutic options include busulfn, hydroxyure (HU), IFN-, Ar-C, nd llogeneic or utologous bone mrrow (BMT) or peripherl blood stem cell trnsplnttion (PBSCT). 2 9 Wheres HU nd IFN- given in chronic phse cn prolong survivl, llogeneic stem cell trnsplnttion (SCT) is believed to be the only curtive therpy so fr. For ptients relpsed fter llogeneic SCT donor lymphocyte Correspondence: M Eder; Fx: Received 9 Februry 1999; ccepted 19 My 1999 infusions (DLI) inititing grft-versus-leukemi (GVL) effect cn induce long lsting moleculr remission nd probbly cure. 10 To monitor the disese nd to evlute the efficcy of therpeutic regimens, hemtologic prmeters, cytogenetics of Ph+ cells, nd expression nlysis of bcr-bl using RT-PCR including competitive semiquntittive RT-PCR ssys re currently performed. The increse of bcr-bl expression fter llogeneic SCT hs especilly been shown to be predictive for relpse. 11,12 However, sensitive, quntittive, nd routine setting RT-PCR is not currently estblished for monitoring the course of CML but could be helpful in individul decisionmking concerning the best therpeutic option. We therefore nlyzed the vlue of rel-time TqMn RT-PCR in CML ptients fter llogeneic or utologous SCT. Rel-time TqMn PCR technology uses the bility of Tq-polymerse to displce nd hydrolyze fluorescence-lbelled reporter oligonucleotide nneled to trget sequence during PCR mplifiction (5 nuclese PCR ssy, for review see Ref. 16). This enbles the genertion of specific fluorescence signl upon excittion tht cn be followed rel-time on per cycle bse llowing the quntittion of the initil copy number per smple. We show here in retrospective nlysis of 13 ptients fter either llogeneic or utologous SCT tht the normlized bcrbl expression cn be quntitted over time with pproprite pirs of PCR primers nd TqMn probes in CML fter SCT. Both llogeneic nd utologous SCT nd DLI cn lower the bcr-bl/gapdh rtio, nd rel-time RT-PCR is t lest s sensitive s conventionl RT-PCR in detecting bcr-bl expression. Mterils nd methods Ptients cdna smples from 18 ptients referred to our institution for SCT or DLI tretment were nlyzed for bcr-bl expression by both conventionl RT-PCR nd rel-time RT-PCR. For 13/18 ptients probes were nlyzed fter SCT, wheres for five ptients no smples were vilble fter SCT. Ptients BF nd BH were not trnsplnted t our institution. From the 13 ptients evluble fter trnsplnttion eight received llogeneic trnsplnts (PBSCT n = 7, BMT n = 1) from HLA-identicl siblings fter conditioning with totl body irrdition (TBI)/cyclophosphmide (n = 2) or busulfn/cyclophosphmide (n = 5) or busulfn/cyclophosphmide/thiotep (n = 1). One ptient ws treted with busulfn/ cyclophosphmide nd underwent BMT from n unrelted HLA-identicl donor, nd one obtined PBSCT from her syngeneic twin fter conditioning with TBI/cyclophosphmide. Three ptients received utologous PBSCT fter conditioning with busulfn only.

2 1384 From ll 18 ptients nlyzed t ny time prior to or fter SCT, 15 ptients expressed the b22-vrint, nd three the b32-vrint of bcr-bl. None of the ptients expressed both vrints simultneously s determined by conventionl nested RT-PCR shown to be cpble of detecting the stimultneous presence of both vrints. All smples nlyzed were from peripherl blood mononucler cells if not stted otherwise. RNA isoltion, cdna synthesis Totl RNA ws extrcted with Trizol (Gibco BRL, Life Technologies, Krlsruhe, Germny) from 10 6 to 10 7 peripherl blood or bone mrrow mononucler cells fter density centrifugtion using Ficoll Hypque. RNA ws trnscribed into cdna in 20 l rection initilly with AMV-RT nd lter, becuse of higher yields, with MMLV-reverse trnscriptse using rndom hexmer primers under stndrd conditions. Conventionl nested RT-PCR The conventionl nested RT-PCR ws performed s described by Murer et l. 17 The mplifiction from the b22 vrint nd the b32 vrint result in mplic of 320 bp nd 395 bp, respectively. Genertion of PCR primers nd TqMn probes for rel-time RT-PCR The PCR-primers nd TqMn probes to mplify nd detect the b22 nd the b32 vrint of bcr-bl were designed using the Primer Express softwre version 1.0 (Perkin Elmer/Applied Biosystems, Foster City, USA) s follows: b22 sense: tgtgctccgctgtccc (Tm 59 C), b22 ntisense: gtcgtgctctggccg (Tm 58 C), b32 sense: tccctcgccctggttt (Tm 58 C), b32 ntisense: tgggctcgtcgtgctct (Tm 58 C). The PCR primers were purchsed from Phrmci (Uppsl, Sweden). The TqMn probes were plced to cover the fusion region of the b22 nd b32 vrint, respectively. The probes b22: tgcctctggggcccttcgc (Tm 69 C) nd b32: cggttcgcccttcgcggc (Tm 69 C) were purchsed lbeled with 6-crboxy-fluorescein (FAM) s the reporter dye nd 6-crboxy-tetrmethyl-rhodmine (TAMRA) s the quencher fluorescent (Perkin Elmer/Applied Biosystems, Weiterstdt, Germny). The TqMn probes were 3 phosphorylted in order to prevent elongtion during PCR. The TqMn-GAPDH Control Regents (Perkin Elmer/Applied Biosystems) were used to mplify nd detect GAPDH s recommended by the mnufcturer. Rel-time PCR Rel-time PCR ws performed on n ABI PRISM 7700 Sequence Detector, using the ABI PRISM 7700 Sequence Detector Softwre 1.6 (Perkin Elmer/Applied Biosystems). Optiml rection conditions for mplifiction of both GAPDH nd bcr-bl were s follows: 40 cycles of two-step PCR (95 C 15 s, 60 C 60 s) fter initil denturtion (95 C for 10 min) with 2 l of cdna rection, 1 TqMn PCR rection buffer, datp, dctp, dgtp, dttp: 200 m, MgCl mm, ech primer: 0.9 m, TqMn probe: 200 nm, AmpliTq Gold: U/ l. Normliztion nd quntittion According to n initil conventionl RT-PCR nlysis for the presence of either bcr-bl vrint the pproprite TqMn probe/pcr primer set ws chosen for rel-time RT-PCR nlysis for ll ptients. For quntittion bcr-bl expression ws normlized by comprison with GAPDH expression. Bcr-bl nd GAPDH expression were quntitted using stndrd cdna dilutions freshly prepred for every nlysis from stock solution corresponding to 10 4 BV173 (b22) nd K562 (b32) cells, respectively. The stndrd dilutions included t lest five vlues spnning five orders of mgnitude with r 0.97 in ll cses. Ech stndrd nd smple vlue ws determined in triplictes in ll but one ptient (duplictes). If cdna smple expressed GAPDH corresponding to fewer thn 100 BV173 nd K562 cells, respectively, the smple ws excluded from further nlysis (n = 8 fter reverse trnscription with AMV-RT). The bcr-bl/gapdh rtio ws clculted s number of reference cells expressing equl mounts of the corresponding bcr-bl vrint divided by the number of reference cells expressing equl mounts of GAPDH. Therefore the expected vlue for the bcr-bl/gapdh rtio for smple of the reference cells lines BV173 nd K562 is one. FISH nlysis of sex chromosomes Fluorescence in situ hybridiztion ws performed fter fixing in methnol/glcil cetic cid (4:1) nd hybridiztion in Cep Hybrid buffer (Vysis, Stuttgrt, Germny) t 37 C overnight with the X- nd Y-chromosome specific probes Spectrum Green nd Spectrum Ornge, respectively (Vysis). Results Specific nd sensitive detection of bcr-bl vrints using rel-time RT-PCR To detect specificlly the b22 nd the b32 vrint of bcrbl we generted TqMn probes covering the b22 nd the b32 fusion sequence, respectively. Using the corresponding pirs of PCR primers mplifiction from the b22 nd b32 templtes result in mplic of 82 nd 73 bp, respectively. When cdnas from BV 173 (b22) nd K 562 (b32) cells were prepred nd serilly diluted, bcr-bl ws detectble in the cdna corresponding to single cell for both vrints by both rel-time nd conventionl nested RT-PCR (Figure 1). Further dilution corresponding to the mount of cdna of 0.5 nd 0.1 BV173 cells resulted in specific detection of bcr-bl in 8/9 nd 5/18 nlyses by rel-time RT-PCR, respectively (dt not shown). The sme cdna smples were tested positive in 2/2 nd 0/2 experiments by conventionl RT-PCR, respectively (Figure 1 nd dt not shown). It should be noted tht the b22 pir of PCR primers lso enbles the mplifiction of sequences from the b32 templte. However, specific detection of the b32 vrint is obtined by the specificity of the TqMn probe. To ssure specific detection of either vrint, cdna from both BV173 nd K562 cells ws nlyzed using the primer pirs/tqmn probes designed to detect either the b22 or b32 vrint. As expected specific detection ws only chieved for BV173 cdna with the b22 (but not the b32), nd for K562 cdna with the b32 (but not the b22) TqMn probe t the highest cdna concentrtion tested (cdna from 10 5 cells, dt not shown).

3 1385 Figure 1 The mplifiction blots for detection of the b32 () nd the b22 (b) vrint of bcr-bl re shown in the upper pnels. The curves correspond from left to right to the mount of templte cdna from 10 4,10 3,10 2, 10, 1,0.5, nd 0.25 K562 () nd BV173 (b) cells, respectively. The x-xis shows the mplifiction cycle, nd the y-xis the normlized reporter signl minus the bckground noise determined during PCR cycles 3 15 ( Rn). The horizontl lne mrks the threshold of Rn to be considered positive. The lower pnels show ethidium bromide-stined grose gels from PCR products fter conventionl nested RT-PCR. PCR ws performed with cdna templtes corresponding 10 4 (lne 1), 10 3 (lne 2), 10 2 lne 3), 10 (lne 4), 1 (lne 5), 0.5 (lne 6), 0.25 (lne 7), nd 0.1 (lne 8) K562 (c) nd BV173 (d) cells, respectively. Lne 9, no templte, control. Accurcy of rel-time RT-PCR quntittion nd correltion with cytogenic nlysis To determine the ccurcy of quntittion depending on the mount of cdna per smple, we nlyzed the bcrbl/gapdh rtio in the cdna from 10 2 to 10 4 BV173 cells. In five independent experiments the men bcr-bl/gapdh rtio ws 0.90 (10 2 cells, coefficient of vrition (cv) = 0.254), 0.98 (10 3 cells, cv = 0.292), nd 1.06 (10 4 cells, n = 4, cv = 0.276), respectively. In ddition to cdna from BV173 cells, cdna smples from two CML ptients were quntitted twice s follows: identicl cdna smples obtined t different time points during the disese were prepred for nd nlyzed by rel time RT-PCR in two independent experiments. For the first ptient the difference of the bcr-bl/gapdh rtio in eight smples quntitted in two independent TqMn nlyses ws between 6.0% nd 71.1% (men difference 29.6%). For the second ptient the bcr-bl/gapdh rtios differed from 10.8% to 61.0% (men difference 30.9%) between two independent rel-time quntitions of cdna smples from five different time points (dt not shown). Smples from five ptients were vilble for both nlysis of Ph+ metphses nd rel-time RT-PCR quntittion. As shown in Tble 1, Ph-positive metphses were detected in seven out of nine smples with miniml bcr-bl/gapdh rtio of In contrst only Ph-negtive metphses were found in two smples with bcr-bl/gapdh rtios of nd 0.003, respectively. Bcr-bl expression prior to trnsplnttion Prior to SCT, smples from 15 ptients were vilble for quntittion of bcr-bl expression (Tble 2 nd b). For ptients with more thn one smple vilble prior to SCT the quntittion of the first probe tken t our institution is given Tble 1 Correltion of bcr-bl/gapdh rtio with cytogenetic nlysis in CML ptients Ptient Source bcr-bl/ Ph+ of cells GAPDH metphses UPN 554 BM /30 UPN 612 BM /20 UPN 378 PB /10 UPN 408 PB /20 BM /18 PB/BM /13 UPN 403 BM /15 PB /11 PB /4 Smples prior to trnsplnttion. PB, peripherl blood; BM, bone mrrow; PB/BM bcr-bl/gapdh from PB, cytogenetic nlysis from BM. in the Tble. The bcr-bl/gapdh rtio ws between nd (men 2.506). These dt include three smples from ptients without (men: 3.886, rnge ), nd from 12 ptients under or immeditely fter different tretments (men: 2.161, rnge: ). The bcrbl/gapdh rtio did not obviously correlte with the number of peripherl blood leukocytes. In ddition, ll ptients were lso tested positive with conventionl RT-PCR. Bcr-bl expression fter llogeneic trnsplnttion From 10 ptients nlyzed fter llogeneic SCT, five becme negtive for bcr-bl expression fter SCT s determined by both conventionl nd rel-time RT-PCR. All nlyses from these ptient smples t different time points were tested negtive by both PCR methods (n = 25), nd ll ptients remined

4 1386 Tble 2 Bcr-bl/GAPDH rtio in CML ptients prior to trnsplnttion Ptient Source of cells bcr-bl vrint bcr-bl/gapdh Therpy Leukocytes (/nl) UPN 378 PB b HU 16.9 UPN 408 PB b HU 10.0 UPN 379 PB b HU/IFN 2.8 UPN 503 PB b HU 5.5 UPN 554 PB b IFN 6.0 UPN 543 PB b UPN 399 BM b HU 46.7 UPN 462 PB b UPN 403 BM b HU 6.2 UPN 432 PB b Ptients listed in Tble 2 were nlyzed both prior to nd fter SCT. The upper seven ptients received llogeneic SCT, wheres the lower three underwent utologous PBSCT. PB, peripherl blood; BM, bone mrrow; HU, hydroxyure; IFN, interferon-. Tble 2b Ptient Source of cells bcr-bl vrint bcr-bl/gapdh Therpy Leukocytes (/nl) UPN 387 PB b IFN 5.3 UPN 402 PB b BH PB b b 2.3 UPN 448 PB b c 3.9 UPN 612 PB b HU 8.4 For ptients listed in Tble 2b, no smples were vilble fter SCT. PB, peripherl blood; BM, bone mrrow; HU, hydroxyure; IFN, interferon. After G-CSF for stedy-stte stem cell pheresis. b After therpy with Ar-C, etoposide, idrubicin for blst crisis. c After therpy with Ar-C, vincristine, mitoxntrone for blst crisis. in moleculr remission fter llogeneic SCT so fr (dt not shown). Five ptients with relpsed (n = 4) or persistent (n = 1) CML fter llogeneic SCT were selected for nlysis of bcr-bl expression t multiple time points fter SCT (Tble 3). In those ptients the bcr-bl/gapdh rtio dropped t lest 1000-fold or bcr-bl becme undetectble fter SCT. In UPN 378 (mle) the bcr-bl/gapdh rtio decresed from 8.65 to t 4 weeks fter llogeneic PBSCT from n HLA-identicl sister. At the sme time point, 3/300 peripherl blood cells hd n XY kryotype s determined by FISH nlysis. The bcrbl/gapdh rtio incresed to mximl (2770-fold increse) 17 weeks fter PBSCT with concomitnt increse of utologous hemtopoiesis (145 XY/300 cells). DLI ( nd T-lymphocytes per kg) were given 12 nd 17 weeks fter PBSCT, nd the bcr-bl/gapdh rtio decresed from t 22 weeks to become 0 t 32 weeks fter SCT. In prllel, chimerism turned to donor origin with 100% XX mononucler cells t the sme time. The ptient hs remined in continuing moleculr remission without therpy s determined by both rel-time nd conventionl RT-PCR (four consecutive time points, 32 months fter PBSCT, dt not shown). Ptient BF presented t our institution 3 yers fter llogeneic BMT with /nl leukocytes under therpy with hydroxyure with bcr-bl/gapdh rtio of A totl of seven DLI in incresing doses ( to T-lymphocytes per kg) were given in 6 months, nd the bcr-bl/gapdh rtio decresed from mximl to 0 t 2 months fter the lst DLI. In prllel the ptient developed GVHD nd the peripherl blood leukocytes dropped to 0.800/nl, but finlly recovered, nd the ptient is in continuing moleculr remission s determined by both conventionl nd rel-time RT-PCR 59 months fter trnsplnttion (four consecutive time points, dt not shown). In UPN 408 with persisting disese the bcr-bl/gapdh rtio dropped from 3.93 to t bout 7 weeks fter llogeneic PBSCT from n HLA-identicl sister. At the sme timepoint 54/300 cells exhibited XY-kryotype s determined by FISH nlysis, nd conventionl RT-PCR ws positive ll the time. The bcr-bl/gapdh rtio incresed to mximl , nd four DLI in incresing doses ( to T-lymphocytes per kg) were given over period of 4 months. However, the bcr-bl/gapdh rtio incresed further to mximum of , nd the ptient hd 100% utologous hemtopoiesis from bout 1 yer fter PBSCT. He hs currently stble disese 27 months fter trnsplnttion. UPN 373 ws trnsplnted in second chronic phse fter lymphoid blst crisis. He tested negtive for bcr-bl expression by both conventionl nd rel-time RT-PCR for up to 8 months fter llogeneic PBSCT t seven consecutive time points. Therefter bcr-bl becme detectble, nd the bcrbl/gapdh rtio incresed from to in 6 months. The ptient relpsed with lymphoid blst crisis nd received one DLI ( T-lymphocytes per kg) 3 months fter tretment with mitoxntrone. However the ptient did not respond nd died 6 weeks lter. In UPN 379 (femle) the bcr-bl/gapdh rtio dropped from before to undetectble levels fter PBSCT from n HLA-identicl brother s determined by both conventionl nd rel-time RT-PCR for t lest 7 months (nlysis t five different time points). Therefter bcr-bl becme detectble severl times by both PCR methods but disppered gin

5 Tble 3 Bcr-bl/GAPDH rtio in CML ptients with relpsed or persistent disese fter llogeneic trnsplnttion 1387 Pt (gender recipient/donor) Time fter TX (time fter DLI) RT-PCR bcr-bl/gapdh FISH sex chromosomes UPN 378 (mle/femle) 4 neg XY/300 (8.653 prior to SCT) 9 pos XY/ pos XY/ (2) pos XY/ (5) pos XY/ (9/4) pos XY/ (10/5) pos XY/ (12/7) pos XY/ (13/8) pos XY/ (15/10) pos XY/ (20/15) neg 0 0 XY/300 B.F. (mle/mle) 159 pos (3) pos (9/6) pos (12/9/3) pos (16/12/6/2) pos (20/17/11/7/2) pos (24/20/14/10/5/4) pos (27/24/18/14/9/8/7) pos (28/25/19/15/10/9/8) neg 0 UPN 408 (mle/femle) 7 pos XY/300 (3.933 prior to SCT) 10 pos XY/ pos XY/ pos XY/ pos XY/ pos XY/ (3) pos XY/ (7/2) pos XY/ (13/8/1) pos b 300 XY/ (24/19/12/6) pos XY/ (34/29/22/16) pos ND 89 (41/36/29/23) pos XY/300 UPN 373 (mle/mle) [13 33] neg 0 40 pos pos (6) pos UPN 379 (femle/mle) [5 28] neg XX/300 (0.774 prior to SCT) 39 pos XX/ neg 0 19 XX/ neg XX/ pos XX/ pos XX/ pos XX/ (8) pos XX/ (13) pos XX/ (14) pos XX/300 The bcr-bl/gapdh rtio prior to SCT is given in prentesis for UPN 378, UPN 408, nd UPN 379. Time is given in weeks fter SCT or fter the first (nd following) DLIs in prenthesis. Time in prenthesis [ ] immeditely fter SCT for UPN 373 nd UPN 379 refers to the period of time the ptient ws tested negtive for bcr-bl expression. b Bone mrrow. ND, not done. until it ws detected by rel-time RT-PCR 58 weeks fter PBSCT. At the sme time the rtio of utologous hemtopoiesis ws rised to 33 XX/300 cells. From then the bcrbl/gapdh rtio incresed nd conventionl RT-PCR turned positive ll the time. In ddition the percentge of utologous hemtopoiesis incresed to mximum of 200XX/300 cells nlyzed. After first DLI ( T-lymphocytes per kg) bout 2 yers fter PBSCT the bcr-bl/gapdh rtio did not significntly chnge. By now (31 months fter PBSCT) the ptient is in hemtologic remission under therpy with IFN- nd fter second DLI. Bcr-bl expression fter utologous trnsplnttion Three ptients were nlyzed for quntittive bcr-bl expression fter conditioning with busulfn nd utologous PBSCT (Tble 4). The bcr-bl/gapdh rtio dropped bout 30-

6 1388 Tble 4 Bcr-bl/GAPDH rtio in CML ptients fter utologous trnsplnttion Ptient Time RT-PCR bcr-bl/ fter TX GAPDH UPN pos neg pos UPN pos pos pos UPN pos pos pos Time is given in weeks fter uto-pbsct. fold in two ptients (UPN 462, UPN 403), nd bout 1.1-fold in the third ptient (UPN 432). The leukphereses used for trnsplnttion expressed bcr-bl/gapdh rtio lower thn 0.06 in the first two ptients, but ws quntitted between 0.40 nd 0.72 for the third ptient (dt not shown). After PBSCT ll three ptients were tested positive by conventionl nd rel-time RT-PCR. From totl of nine nlyses, one conventionl RT-PCR ws negtive wheres rel-time RT-PCR gve bcr-bl/gapdh rtio of (UPN 462). In this ptient the bcr-bl/gapdh rtio remined between nd over period of 46 weeks fter uto-pbsct. He is currently in hemtologic remission under therpy with IFN- 20 months fter trnsplnttion. UPN 403 hd bcr-bl/gapdh rtios between nd in the peripherl blood up to 23 weeks fter PBSCT nd is currently in hemtologic remission 28 months fter PBSCT. The bcr-bl/gapdh rtio for UPN 432 ws between nd in the peripherl blood for up to 71 weeks fter PBSCT. The ptient now hs stble disese under therpy with hydroxyure 2 yers fter SCT. Discussion We report here the quntittion of bcr-bl expression in mononucler cells from CML ptients before nd fter llogeneic or utologous SCT using rel-time RT-PCR. We demonstrte tht this method is s sensitive s conventionl nested RT-PCR for the detection of bcr-bl in both cell lines nd ptient smples with detection limit corresponding to the cdna of single BV173 or K562 cell. In ddition the quntittion by rel-time RT-PCR is reproducible with men error in our hnds of bout 30% when identicl cdna smples were quntitted twice in two different TqMn PCR nlyses. Prior to SCT, bcr-bl ws detectble in ll cses by both PCR methods, nd the bcr-bl/gapdh rtio ws between nd in 15 ptients nlyzed. There ws no correltion between the normlized bcr-bl expression nd the totl peripherl blood leukocyte count including ptients under cytoreductive therpy. In contrst, cytogenetic nlysis demonstrted Ph-positive metphses in seven out of nine smples with miniml bcr-bl/gapdh rtio of 0.250, wheres Phnegtive metphses only were found in two smples with bcr-bl/gapdh rtios of or less. As confirmed by conventionl nested RT-PCR bcr-bl/gapdh rtios s low s represent positive signl for bcr-bl expression. These dt suggest correltion between the number of Phpositive metphses nd n incresing bcr-bl/gapdh rtio but the limited smple number hs to be considered. After llogeneic SCT the bcr-bl/gapdh rtio dropped to 0 or t lest more thn 1000-fold in ll ptients nlyzed including one with persistent disese fter llogeneic PBSCT. Interestingly, the bcr-bl/gapdh rtio decresed only fold in three ptients fter utologous PBSCT fter conditioning with busulfn. This difference in the efficcy between llogeneic nd utologous SCT is most likely due to the reconstitution of bcr-bl negtive hemtopoiesis in ptients fter llogeneic SCT. Whether different conditioning regimens contribute to the different efficcy is currently not known. Although only three ptients were nlyzed fter identicl conditioning it is interesting to note tht the two ptients with 30-fold decrese in the bcr-bl/gapdh rtio received trnsplnts with 10-fold lower bcr-bl/gapdh rtio thn the ptient with lmost no chnge of bcr-bl expression fter uto-pbsct. In the five ptients with relpse or persistent disese fter llogeneic SCT, bcr-bl remined or becme detectble by both conventionl nd rel-time RT-PCR t bout the sme time in ll cses. A shift in mixed chimerism s mesured by FISH nlysis of sex chromosomes with n increse of utologous hemtopoiesis prlleled the rise of the normlized bcr-bl expression in three evluble ptients. DLI were given to ll five ptients with persistent disese or relpse, nd the bcr-bl/gapdh rtio dropped finlly to undetectble levels in two ptients 8 nd 15 weeks fter the lst DLI, but decresed only temporrily or chnged less thn 10-fold in three other ptients. One of these ptients with bd response to DLI is still under tretment with incresing cell doses fter first DLI, nd one received DLI for relpse of lymphoid blst crisis. These dt correspond to recent study by Burmnn et l 18 who report the kinetics of GVL effects fter DLI in three ptients with relpsed CML fter llogeneic SCT. Between 9 to 18 weeks fter DLI ll three ptients exmined chieved moleculr remission. Mensink et l 19 first reported the use of rel-time RT-PCR in CML ptients fter llogeneic SCT. They lso found decrese nd finl disppernce of bcr-bl expression fter DLI in three ptients with relpsed CML. They lso demonstrte the disppernce of Ph-positive metphses with decresing bcr-bl expression. However, there re some differences to our study. We used PCR-primer/TqMn probes specific for either the b22 nd the b32 bcr-bl vrint nd TqMn probes locted close to the 5 PCR primer in order to mximize the efficcy of the TqMn PCR. In ddition we compred rel-time RT-PCR with conventionl non-quntittive nested RT-PCR nd found high correspondence between the two PCR methods. Only two smples were tested negtive by conventionl but positive by rel-time RT-PCR wheres ll smples tested positive by mens of conventionl RT-PCR were lso tested positive with rel-time RT-PCR. In contrst to GAPDH, Mensink et l 19 use PBGD for normliztion. No stndrd housekeeping gene for RNA normliztion in hemtopoietic cells hs been estblished so fr, nd some vrition of gene expression depending on exogeneous stimuli hs been described for exmple in thyroid cells. 20 In recent study 21 we hve nlyzed the differentil quntittion of similr trget sequences by TqMn PCR technology in detil by mixing cdna from lterntively spliced mrnas. We found precise quntittion of either isoform even in the presence of 100-fold excess of the lterntive vrint using isoform specific TqMn probes. Accordingly, when cdna from BV173 nd K562 cells ws mixed, either vrint could be differentilly quntitted with the pproprite Tq-

7 Mn probe even in the presence of the lterntive isoform (dt not shown). Therefore the use of seprte TqMn probes for the b22 nd the b32 vrint of bcr-bl llows the independent quntittion of either isoform nd my llow the monitoring of clonl evolution in CML ptients expressing both the b22 nd the b32 vrint. However, bcr-bl fusions different from b22 nd b32 cn not be quntitted with the TqMn probes described here. Rel-time RT-PCR is fst, sensitive nd semi-utomted method to quntitte the initil templte number when pproprite conditions re estblished. Quntittion of the normlized bcr-bl expression my not only be helpful fter llogeneic SCT for detection of erly relpse in order to strt DLIs nd to control their effects. It should be possible to monitor the dynmic of the disese nd the efficcy of ny therpeutic regimen before nd fter SCT 22 by rel-time RT-PCR. It will lso enble qulity control of leukpheresis products collected for utologous SCT. For ll these purposes it will however be necessry to evlute reltime RT-PCR in prospective setting of lrge number of ptients. Acknowledgements This work ws supported by grnt of the Deutsche Forschungsgemeinschft to ME (Ed 34/2-2). We would like to thnk E Dmmnn for excellent ssistnce with clinicl documenttion. References 1 Kurzrock R, Guttermn JU, Tlpz M. The moleculr genetics of Phildelphi chromosome-positive leukemis. 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