Originl Articles Correltions Between Cytogenetic nd Moleculr Monitoring Among Ptients With Newly Dignosed Chronic Myeloid Leukemi in Chronic Phse Post Hoc Anlyses of the Rtionle nd Insight for Gleevec High-Dose Therpy Study Luke P. Akrd, MD; Jorge E. Cortes, MD; Mher Albitr, MD; Sturt L. Goldberg, MD; Ghulm Wrsi, PhD; Meir Wetzler, MD; Solveig G. Ericson, MD; Jerld P. Rdich, MD Accepted for publiction October 23, 2013. Published s n Erly Online Relese December 5, 2013. From the Indin Blood nd Mrrow Trnsplnttion, Stem Cell Trnsplnttion Progrm, St. Frncis Hospitl nd Helth Centers, Indinpolis, Indin (Dr Akrd); the Deprtment of Leukemi, The University of Texs MD Anderson Cncer Center, Houston (Dr Cortes); the Deprtments of Oncology/Hemtopthology nd Reserch nd Development, NeoGenomics Lbortories, Irvine, Cliforni (Dr Albitr); the Division of Leukemi, John Theurer Cncer Center t Hckensck University Medicl Center, Hckensck, New Jersey (Dr Goldberg); Biosttistics (Dr Wrsi) nd Oncology Clinicl Development (Dr Ericson), Novrtis Phrmceuticls Corportion, Est Hnover, New Jersey; the Deprtment of Medicine, Roswell Prk Cncer Institute, Bufflo, New York (Dr Wetzler); nd the Clinicl Reserch Division, Fred Hutchinson Cncer Reserch Center, Settle, Wshington (Dr Rdich). Dr Akrd received reserch support from ARIAD Phrmceuticls, Inc (Cmbridge, Msschusetts), Pfizer Lbs (New York, New York), Tev Phrmceuticl Industries Ltd (North Wles, Pennsylvni), nd Novrtis Phrmceuticls Corportion nd is on the speker bureus for ARIAD Phrmceuticls, Inc, Bristol Myers Squibb (Princeton, New Jersey), Pfizer Lbs, Novrtis Phrmceuticls Corportion, nd Tev Phrmceuticl Industries Ltd. Dr Albitr ws n employee of Quest Dignostics t the time of the study in the Oncology/Hemtopthology nd Reserch nd Development Deprtments, Nichols Institute, Quest Dignostics, Sn Jun Cpistrno, Cliforni, nd is currently employed by Neogenomics Lbortories, Irvine, Cliforni, in the Reserch nd Development Deprtment. Dr Goldberg is on the speker bureus for ARIAD Phrmceuticls, Inc, Bristol Myers Squibb, nd Novrtis Phrmceuticls Corportion. Dr Wrsi is n employee nd stockholder of Novrtis Phrmceuticls Corportion. Dr Ericson is n employee nd stockholder of Novrtis Phrmceuticls Corportion. Dr Rdich is reserch contrctor with Novrtis Phrmceuticls Corportion nd does consulting for Novrtis Phrmceuticls Corportion, Bristol Myers Squibb, Pfizer Lbs, nd ARIAD Phrmceuticls, Inc. The other uthors, Dr Cortes nd Dr Wetzler, hve no relevnt finncil interest in the products or compnies described in this rticle. The results of the post hoc nlysis were previously presented t the 14th Interntionl Conference of the Europen School of Hemtology Interntionl Chronic Myeloid Leukemi Foundtion on September 21, 2012, in Bltimore, Mrylnd, s poster presenttion nd on September 22, 2012, s brief orl communiction. Funding for this work ws provided by Novrtis Phrmceuticls Corportion (Est Hnover, New Jersey). Reprints: Luke P. Akrd, MD, Indin Blood nd Mrrow Trnsplnttion, Stem Cell Trnsplnttion Progrm, St Frncis Hospitl nd Helth Centers, Indinpolis, IN 46237 (e-mil: lkrd@ibmtindy.com). Context. Although bone mrrow (BM) kryotyping hs been the stndrd in monitoring ptients with chronic myeloid leukemi, peripherl blood (PB) monitoring methods my be more convenient. Objective. To conduct post hoc nlyses of the Rtionle nd Insight for Gleevec High-Dose Therpy study to evlute correltions between results of cytogenetic testing nd moleculr monitoring from BM nd PB during the first 18 months of high-dose imtinib therpy, nd between erly nd lte moleculr responses. Design. Newly dignosed ptients with chronic-phse chronic myeloid leukemi received imtinib 400 mg twice dily nd were monitored qurterly for up to 18 months. Cytogenetic testing ws performed by kryotyping using BM or by fluorescence in situ hybridiztion using PB. Moleculr testing ws performed by quntittive reverse trnscriptse polymerse chin rection using BM nd PB. Results. Significnt pirwise correltions were found between results obtined by kryotyping, fluorescence in situ hybridiztion, nd quntittive reverse trnscriptse polymerse chin rection using PB or BM (ll pirwise correltions.0.8; P,.001). At 12 months, cytogenetic response by kryotyping correlted well with response by fluorescence in situ hybridiztion. A medin 2.579-log reduction in BCR-ABL1 level from stndrdized bseline correlted with fluorescence in situ hybridiztion negtive sttus. Ptients with greter thn 2-log reduction in BCR- ABL1 level t 3, 6, nd 9 months were more likely to chieve mjor moleculr response t 18 months thn those with 2-log reduction or less. Conclusions. Our findings support the fesibility of moleculr monitoring using PB nd suggest tht moleculr monitoring conducted t single relible reference lbortory cn dequtely trck response without invsive BM testing. Our findings re consistent with other work indicting tht erly response to imtinib predicts fvorble long-term outcome. (Arch Pthol Lb Med. 2014;138:1186 1192; doi: 10.5858/rp.2013-0584-OA) 1186 Arch Pthol Lb Med Vol 138, September 2014 Cytogenetic nd Moleculr Monitoring for CML Akrd et l
Stndrd tretment for chronic myeloid leukemi (CML) consists primrily of tyrosine kinse inhibitor (TKI) therpy tht trgets the BCR-ABL1 kinse, 1 the ctivity of which underlies the pthogenesis of this cncer. The BCR- ABL1 kinse is fusion protein expressed from n berrnt gene tht is formed through reciprocl trnsloction between chromosomes 9 nd 22 (the Phildelphi chromosome [Ph]). 2 Imtinib ws the first BCR-ABL1 TKI pproved for first- nd second-line tretment of CML. 3 Since then, nilotinib 4 nd dstinib 5 hve been pproved for first- nd second-line tretment of CML, nd bosutinib 6 nd pontinib 7 for second- nd subsequent-line tretment of CML. Adequte monitoring of ptient response to BCR-ABL1 TKI therpy is mndtory component of the successful mngement of CML. A growing body of evidence demonstrtes significnt positive correltion between the chievement of erly response to TKI therpy nd fvorble long-term outcomes, prticulrly in ptients who chieve cytogenetic nd moleculr response 3 months fter the initition of TKI therpy. 8 13 The Ntionl Comprehensive Cncer Network (NCCN), in its clinicl prctice guidelines for CML, nd Europen LeukemiNet both recommend monitoring response to TKI therpy every 3 months. 1 Both the NCCN nd Europen LeukemiNet mke recommendtions for inititing nd chnging TKI therpy bsed on cytogenetic response. Cytogenetic testing is typiclly done by kryotyping using bone mrrow (BM) smple obtined vi n invsive procedure. Cytogenetic testing cn lso be ssessed by fluorescence in situ hybridiztion (FISH) using BM or peripherl blood (PB) smple, but this method hs not been dequtely studied s method for monitoring response to TKI therpy. Thus, the NCCN guidelines do not recommend this pproch. 1,14 There is gret interest in identifying nd vlidting new methods to monitor response to TKI therpy tht re less invsive thn kryotyping. Moleculr testing of BCR-ABL1 trnscript levels is typiclly done by highly sensitive quntittive reverse trnscriptse polymerse chin rection (qrt-pcr) using either PB or BM smples. An interntionl scle (IS) hs been estblished tht enbles direct comprison of moleculr testing dt cross lbortories. Although the NCCN guidelines generlly support moleculr monitoring over BM cytogenetic testing, they recommend BM cytogenetic testing when ccess to IS-stndrdized qrt- PCR is not vilble nd do not recommend chnges in therpy bsed on moleculr studies lone. 1 In principle, comprison of qrt-pcr results mong lbortories not using the IS cn be mde, s long s the lbortories ech estblish bseline bsed on set of smples from newly dignosed ptients with CML prior to tretment nd the mgnitudes of log reduction from these lbortory-specific stndrdized bselines re compred. Such n pproch, however, would only llow comprison of reltive BCR- ABL1/ABL1 levels cross lbortories, wheres dopting the IS would llow comprison of bsolute trnscript levels. To estblish the fesibility of methods tht use PB smples for monitoring response to therpy, studies hve compred test results obtined by multiple methods, including kryotyping versus FISH 15 17 nd kryotyping versus qrt- PCR. 17 20 Although overll findings suggest high level of concordnce between results obtined with methods using PB versus BM, the concordnce of results obtined by different testing methods hs been inconsistent. 15 20 The Rtionle nd Insight for Gleevec High-Dose Therpy (RIGHT) study evluted the effect of imtinib 400 mg twice dily on the chievement of moleculr nd cytogenetic responses in the first 18 months of therpy in 115 ptients with newly dignosed CML in chronic phse. 21 Ptients in this study hd rpid response to tretment; by 18 months, rtes of complete cytogenetic response (CCyR), mjor moleculr response (MMR; 3-log reduction in BCR-ABL1 trnscript level from stndrdized bseline), nd complete moleculr response (undetectble BCR-ABL1 level using n ssy with 4.5-log sensitivity) were 83%, 63%, nd 55%, respectively. Given the limited number of studies compring different methods of CML monitoring, the post hoc nlyses of the RIGHT study described herein sought to determine the degree of concordnce between moleculr nd cytogenetic testing methods using PB or BM, nd the usefulness of erly response to predict future response to TKI therpy. METHODS Study Design nd Objectives The RIGHT study design hs been described previously. 21 Briefly, this ws single-rm, open-lbel, phse 2 study of high-dose imtinib (400 mg twice dily) in ptients 18 yers of ge or older with CML in chronic phse who were dignosed within 6 months of study entry nd hd received tretment for CML for no more thn 1 month, with the exception of hydroxyure nd/or ngrelide. Ptients hd Estern Coopertive Oncology Group performnce sttus of 0 to 2, nd dequte crdic nd liver function. All ptients were tken off the study t 18 months. Ptients could hve continued on imtinib, nd the dose used fter 18 months ws selected by the treting investigtor. The objectives of these post hoc nlyses of the RIGHT study were to determine the correltion between cytogenetic response s ssessed by FISH versus kryotyping, nd between cytogenetic response s determined by FISH versus moleculr response s ssessed by qrt-pcr. An dditionl objective ws to determine the correltion between BCR-ABL1 log reduction t 3, 6, nd 9 months nd rte of MMR t 18 months (1 month ¼ 4 weeks). The RIGHT study ws conducted in ccordnce with the Declrtion of Helsinki nd guidelines on good clinicl prctices. The study protocol ws pproved by the pproprite ethics committees, institutionl review bords, nd regultory orgniztions. All ptients provided written informed consent ccording to institutionl regultions. Hemtologic nd Cytogenetic Testing Hemtologic testing (neutrophil nd pltelet counts) nd cytogenetic testing (kryotyping nd FISH) were crried out t single commercil reference lbortory (Quest Dignostics, Sn Jun Cpistrno, Cliforni). Hemtologic testing ws done t screening; bseline; weeks 1, 2, nd 4; nd months 2, 3, 4, 6, 9, 12, 15, nd 18. Complete hemtologic response ws defined s bsolute neutrophil count greter thn 1500/lL, pltelet count greter thn 100 3 10 3 /ll, nd no circulting blsts or extrmedullry involvement. Kryotyping ws done t screening nd month 12; FISH ws done t bseline nd months 3, 6, 9, 12, 15, nd 18 (Figure 1). At lest 20 metphse cells were nlyzed for kryotype nd t lest 500 cells for FISH. If the number of metphse cells ws insufficient for kryotyping, FISH ws used insted. The Vysis BCR/ABL1/ASS1 Tri-Color Dul Fusion FISH Probe Kit (Abbott Lbortories, Abbott Prk, Illinois) ws used; the sensitivity of the FISH probe ws 0.5%. Cytogenetic responses (for both kryotyping nd FISH) were clssified bsed on the percentge of Ph-positive metphses: CCyR (0%), prtil (PCyR; 1% 35%), mjor (0% 35%), minor (36% 65%), or miniml (66% 95%). Moleculr Testing Moleculr testing by qrt-pcr ws lso crried out by Quest Dignostics. Testing using BM cells ws done t screening nd months 12 nd 18; testing using PB cells ws done t bseline nd Arch Pthol Lb Med Vol 138, September 2014 Cytogenetic nd Moleculr Monitoring for CML Akrd et l 1187
Figure 1. Schedule of cytogenetic nd moleculr testing performed in the Rtionle nd Insight for Gleevec High-Dose Therpy study. Abbrevitions: BID, twice dily; BM, bone mrrow; FISH, fluorescence in situ hybridiztion; PB, peripherl blood; qrt-pcr, quntittive reverse trnscriptse polymerse chin rection. months 3, 6, 9, 12, 15, nd 18 (Figure 1). The Quest qrt-pcr ssy ws vlidted by the Rdich lbortory t the Fred Hutchinson Cncer Center, Settle, Wshington, lbortory tht is stndrdized to the IS. The Quest qrt-pcr ssy used ABL1 s the control gene; the Rdich lbortory ssy used b 2 - microglobulin. The men bseline rtio of BCR-ABL1 to ABL1 ws clculted from 113 prestudy smples tken from newly dignosed ptients enrolled in the RIGHT study in ddition to other smples ssyed in the Quest lbortory. A stndrdized bseline BCR-ABL1:ABL1 rtio of 3.797 ws used to clculte log reductions of individul smples following therpy. The Quest qrt-pcr ssy ws cpble of detecting 4.5-log reduction in BCR-ABL1 trnscript level below this bseline. In this study, ptients who chieved undetectble BCR-ABL1 trnscript levels on study were either considered to hve n undefined log reduction or rbitrrily recorded s hving 10-log reduction. Correltion Between Cytogenetic nd Moleculr Testing Results The Spermn rnk correltion nlysis showed highly significnt correltion between ll pirwise comprisons of testing methods (Tble 2). The highest correltion coefficients were observed between results obtined by qrt-pcr using BM versus PB (Figure 2) nd by FISH (PB) versus kryotyping (BM). At 12 months, 83 ptients were evluble for both kryotype (BM) nd FISH (PB) testing. A comprison between the FISH results nd the kryotyping for these ptients t 12 months showed n overll concordnce in Sttisticl Methods Spermn rnk correltion ws used for ll correltions. A Spermn correltion coefficient of 1 indictes perfect correltion between results obtined by the 2 methods being compred. The P vlues re from test of the null hypothesis tht the correltion is zero ginst the lterntive hypothesis tht it is nonzero. Tble 1. Bseline Ptient Demogrphics nd Disese Chrcteristics (N ¼ 115) Medin ge (rnge), y 50 (19 81),65 yers, No. (%) 91 (79) 65 yers, No. (%) 24 (21) Sokl risk score, No. (%) Low 80 (70) Intermedite 20 (17) RESULTS High 14 (12) Ptients Missing 1 (1) Bseline ptient demogrphics nd disese chrcteristics Prior imtinib, No. (%) 19 (17) Men durtion of prior imtinib hve been described previously 21 ; selected chrcteristics re (rnge), d 19 (1 38) shown in Tble 1. Of 115 ptients enrolled, 83 (72%) Medin time from dignosis to first study dose (rnge), mo 0.96 (0.13 9.69) completed the RIGHT study. The remining 32 ptients Clonl evolution, No. (%) (28%) discontinued the study becuse of dverse events (n ¼ 3 (3) Received imtinib for less thn 1 month, per protocol inclusion 10; 9%), withdrwl of consent (n ¼ 10; 9%), unstisfctory criteri. therpeutic effect (n ¼ 6; 5%), protocol violtion (n ¼ 4; 3%), b Defined s the presence of chromosoml bnormlities in ddition to deth (n ¼ 1; 1%), nd loss to follow-up (n ¼ 1; 1%). 21 the Phildelphi chromosome. 1188 Arch Pthol Lb Med Vol 138, September 2014 Cytogenetic nd Moleculr Monitoring for CML Akrd et l
Tble 2. Pirwise Overll Correltion Between Cytogenetic nd Moleculr Testing Results Pirwise Comprtor 1 Pirwise Comprtor 2 No. of Ptients No. of Smples Correltion Coefficient P Vlue FISH (PB) Kryotyping (BM) 99 151 0.9198,.001 FISH (PB) qrt-pcr (BM) 96 181 0.8583,.001 FISH (PB) qrt-pcr (PB) 113 352 0.8729,.001 qrt-pcr (BM) Kryotyping (BM) 116 193 0.8054,.001 qrt-pcr (PB) Kryotyping (BM) 87 126 0.8193,.001 qrt-pcr (BM) qrt-pcr (PB) 94 170 0.9256,.001 Abbrevitions: BM, bone mrrow; FISH, fluorescence in situ hybridiztion; PB, peripherl blood; qrt-pcr, quntittive reverse trnscriptse polymerse chin rection. Kryotyping: percentge of Phildelphi chromosome positive cells of 20 or more cells nlyzed; FISH: percentge of positive cells of 500 or more cells nlyzed; qrt-pcr: log reduction of BCR-ABL1 from stndrdized bseline of 3.7970, clculted from ll prestudy smples in the Rtionle nd Insight for Gleevec High-Dose Therpy study nd other lbortory dignostic smples. 94% of cses (Tble 3). Ninety-five percent of smples tht were negtive by kryotyping were negtive by FISH, nd 99% of smples tht were negtive by FISH were negtive by kryotyping. Ten smples were positive by FISH, but only 6 of these smples were lso positive by kryotyping. Seven smples were positive by kryotyping, nd 6 of these smples were lso positive by FISH. Correltion Between Moleculr Response nd Cytogenetic Response At 3, 6, 9, 12, nd 18 months, 21, 39, 51, 90, nd 82 ptients, respectively, were evluble for both FISH (PB) nd qrt-pcr (PB) testing. Of these evluble ptients, 18 of 21 (86%), 25 of 39 (64%), 36 of 51 (71%), 66 of 90 (73%), nd 76 of 82 ptients (93%) hd detectble BCR-ABL1 trnscript levels t the respective time points; the remining ptients hd undetectble BCR-ABL1 levels. A totl of 55 ptients with detectble trnscript levels chieved CCyR during the study, including 7 ptients with first documenttion of FISH-negtive sttus t 3 months, 12 ptients t 6 months, 14 ptients t 9 months, 21 ptients t 12 months, nd 1 ptient t 18 months (Figure 3). The medin log reductions from bseline in BCR-ABL1 level mong ptients with documented FISH-negtive sttus nd detectble BCR-ABL1 levels were 2.237 t 3 months, 2.807 t 6 months, 2.471 t 9 months, nd 2.801 t 12 months; overll medin log reduction in BCR-ABL1 ssocited with FISH negtivity ws 2.579. (The ptient who chieved FISH negtivity t 18 months hd 3.579-log reduction in BCR-ABL1 level t tht time.) Correltion Between Erly Moleculr Response nd Cytogenetic nd Moleculr Response t 18 Months For the nlysis of log reduction in BCR-ABL1 level nd chievement of CCyR t 18 months, 83 ptients t 3 months, 79 ptients t 6 months, nd 81 ptients t 9 months hd cytogenetic response dt t 18 months nd were evluble t ech time point (Tble 4). The likelihood of chieving CCyR t 18 months incresed with incresing mgnitude of log reduction in BCR-ABL1 level, regrdless of the time point t which erly response ws ssessed. Of the 9 ptients with less thn 1 log reduction in BCR-ABL1 level t 3 months, 5 remined t less thn 1 log reduction t 6 months, 1 hd 1- to 2-log reduction t 6 months, nd 3 hd no disese progression t 6 months. For the nlysis of log reduction in BCR-ABL1 level nd chievement of MMR t 18 months, 81 ptients t 3 months, 78 ptients t 6 months, nd 76 ptients t 9 months hd moleculr response dt t 18 months (Tble 5). The chievement of MMR t 18 months ws more likely in ptients with greter log reduction in BCR-ABL1 level, regrdless of the time point t which erly response ws ssessed. Medin log reduction in BCR-ABL1 level t 3 months ws 1.94 log for ptients who never chieved CCyR on study (n ¼ 15), 2.257 log for ptients with CCyR (s determined by FISH-negtive sttus) t 3 months (n ¼ 7), 2.50 log for ptients who lter chieved CCyR (n ¼ 84), 2.73 log for ptients who lter chieved MMR (n ¼ 77), nd 2.80 log for ptients who lter chieved complete moleculr response (n ¼ 67). This pttern suggests tht log reduction in BCR-ABL1 Figure 2. Sctterplot of results of quntittive reverse trnscriptse polymerse chin rection (PCR) conducted using bone mrrow (BM) versus peripherl blood (PB). Horizontl nd verticl dshed blck lines indicte log reductions of 0 log nd 2 log in the qrt-pcr ssy. The solid blck line denotes ctul correltion. Arch Pthol Lb Med Vol 138, September 2014 Cytogenetic nd Moleculr Monitoring for CML Akrd et l 1189
Tble 3. Correltion Between Kryotyping nd FISH Testing t 12 Months (N ¼ 83) Kryotyping, % Phþ FISH, % þ 0 1 35.35 0 72 1 0 1 5 4 2 0 6 35 0 0 2.35 0 1 1 Abbrevitions: þ, positive; FISH, fluorescence in situ hybridiztion; Ph, Phildelphi chromosome. Bold vlues represent negtive-negtive nd positive-positive correspondent results. level t 3 months my correlte with best response to imtinib tretment. COMMENT Becuse estblished clinicl prctice guidelines recommend tht response monitoring be done qurterly, interest in the vlidtion of less invsive modes of monitoring response to TKI therpy in ptients with CML is considerble. To our knowledge, the nlyses of the RIGHT study we hve conducted re the first to ssess pirwise correltions mong 4 testing methods: kryotyping (BM), FISH (PB), qrt-pcr (BM), nd qrt-pcr (PB). Pirwise comprisons of these tests show strong nd sttisticlly significnt correltion between ll pirs of comprtors, prticulrly qrt-pcr (PB versus BM) nd FISH (PB) versus kryotyping (BM) (Tble 2). In this study, PB qrt-pcr log reduction of 2.579 log from study-specific stndrdized bseline correlted with FISH-negtive sttus, suggesting tht moleculr monitoring by PB might obvite the need for invsive BM testing. These findings corroborte other work showing tht qrt-pcr cn be relibly performed using either PB or BM. 17,19 They lso support the use of FISH (PB) to confirm dignosis when kryotyping (BM) is not fesible, s recommended in the NCCN guidelines. 1 Our nlysis of the concordnce between results by FISH (PB) versus kryotyping (BM) t 12 months (Tble 3) supports the concept tht FISH using 500-cell preprtion is more sensitive ssy thn kryotyping. Smples tht were wekly FISH positive (1% 5%) were more often found to be kryotype negtive thn positive, which my reflect degree of nonspecific FISH stining or suggest tht residul disese tht is detectble by FISH my be flsely chrcterized s undetectble by kryotyping. Of note, ll smples tht were modertely to strongly FISH positive (.5%) were scored positively by kryotyping. These observtions suggest threshold level of residul disese below which kryotyping is not suitble for response monitoring nd t which other, more sensitive methods should be used. Our findings support the vlue of using FISH (PB) during the initil 18 months of tretment to ssess tretment response, becuse it is s sensitive s kryotyping (BM). Becuse the reltionship between cytogenetic response nd qrt-pcr response ws lso correlted, our dt lso support the vlue of qrt-pcr s n lterntive to kryotyping (BM) for monitoring response. Thus, 2 PB monitoring techniques, FISH nd qrt-pcr, pper to offer n lterntive method to ccurtely ssess tretment response during the initil 18 months of tretment of CML without the need for BM exmintion. With respect to qrt-pcr testing, this study used commercil lbortory tht hd its methodology vlidted by reference lbortory ligned to the IS. Not ll reference lbortories report qrt- PCR test results on the IS, so the widespred pplicbility of our results to those lbortories is not cler. For lbortories not ligned to the IS, our dt support the use of FISH (PB), rther thn kryotyping (BM), in combintion with qrt- PCR (PB) to monitor response. For deeper levels of response, such s MMR, FISH (PB) is unlikely to be of vlue; in those circumstnces, qrt-pcr testing idelly ligned to the IS will prove most prcticl. Although qrt- PCR ligned to the IS, the preferred pproch, ws not used in this study, the qrt-pcr ssy ws vlidted by n ISstndrdized lbortory. Nevertheless, this spect remins limittion of this study, in the context of modern pproches to moleculr monitoring in ccordnce to the IS. We determined tht the medin log reduction in BCR- ABL1 level corresponding to FISH negtivity ws 2.579 log (Figure 2). Becuse our findings lso show high degree of correltion between FISH (PB) nd kryotyping (BM), we consider FISH negtivity to represent CCyR. Work of others 8,19 hs shown correspondence between greter thn 2-log reduction in BCR-ABL1 level nd CCyR. Becuse FISH testing in the RIGHT study ws done t 3-month Figure 3. Log reduction in BCR-ABL1 level t the time of first documenttion of fluorescence in situ hybridiztion (FISH) negtive sttus mong ptients with detectble trnscript levels. Log reductions (circles) nd medin log reduction (br) of ptients with FISH-negtive sttus t 3, 6, 9, nd 12 months re shown. One ptient hd first documenttion of FISH-negtive sttus t 18 months (dt not shown); log reduction in BCR-ABL1 level for this ptient ws 3.5794 t tht time. 1190 Arch Pthol Lb Med Vol 138, September 2014 Cytogenetic nd Moleculr Monitoring for CML Akrd et l
Tble 4. Log in BCR-ABL1 Level From Bseline t 3, 6, nd 9 Months nd Complete Cytogenetic Response (CCyR) t 18 Months Time on Imtinib, mo intervls, the exct time of conversion to FISH negtivity is not known. It is likely tht the log reduction in BCR-ABL1 level would be lower thn 2.579 log if the exct time of conversion to FISH-negtive sttus were known. Current NCCN guidelines consider 1-log reduction in BCR-ABL1 level to be the moleculr equivlent of PCyR 1 ; however, the NCCN hs not yet estblished n equivlency between moleculr response nd CCyR. Therefore, s recommended in the NCCN guidelines, 1 ptients who do not demonstrte either prior BM-bsed CCyR or BM-/PB-bsed MMR t 12 months should undergo cytogenetic testing to determine the level of cytogenetic response. Our findings tht chievement of erly moleculr response correltes with chievement of CCyR nd MMR t 18 months (Tbles 4 nd 5, respectively) re consistent with lrge body of work showing tht erly cytogenetic or moleculr response to TKI therpy predicts significntly improved long-term outcomes. Severl studies hve shown tht chieving cytogenetic or moleculr response to TKI therpy t erlier time points confers n dvntge over Tble 5. Log in BCR-ABL1 Level From Bseline t 3, 6, nd 9 Months nd Mjor Moleculr Response (MMR) t 18 Months Time on Imtinib, mo Log Log No. of Ptients With Log No. of Ptients With Log No. (%) of Ptients With CCyR t 18 mo 3(n¼ 83 b ),1 9 4 (44) 1 2 24 20 (83).2 50 45 (90) 6(n¼ 79 b ),1 6 2 (33) 1 2 3 2 (67).2 70 62 (89) 9(n¼ 81 b ),1 5 0 (0) 1 2 8 4 (50).2 68 63 (93) CCyR is defined s 0% Phildelphi chromosome positive. b Number of ptients t ech time point who hd corresponding cytogenetic response dt t 18 months; ptients not included in this nlysis were considered nonevluble becuse smples were either not collected or not suitble for nlysis. No. (%) of Ptients With MMR t 18 mo 3(n¼ 81) b,1 9 3 (33) 1 2 22 12 (55).2 50 40 (80) 6(n¼ 78) b,1 6 0 (0) 1 2 3 2 (67).2 69 52 (75) 9(n¼ 76) b,1 5 0 (0) 1 2 8 3 (38).2 66 50 (76) MMR is defined s 3-log reduction in BCR-ABL1 level from stndrdized bseline of 3.797, clculted from ll prestudy smples in the Rtionle nd Insight for Gleevec High-Dose Therpy study nd other lbortory dignostic smples. b Number of ptients t ech time point who hd corresponding moleculr response dt t 18 months; ptients not included in this nlysis were considered nonevluble becuse smples were either not collected or not suitble for nlysis. chievement t lter time points. 22,23 In our nlyses, however, mong ptients who chieved greter thn 2-log reduction in BCR-ABL1 t 3, 6, or 9 months, similr proportion of ptients lter chieved CCyR or MMR t 18 months, suggesting tht the chievement of greter thn 2- log reduction t n erlier time point does not increse the likelihood of future CCyR or MMR. Other studies hve shown significnt improvements in long-term overll survivl in ptients who chieve cytogenetic or moleculr response t 3 months. 9 11,13 Becuse RIGHT ws n 18- month study, the cpcity of our nlyses to ssess the effect of erly response on long-term outcome nd survivl is limited. In conclusion, the findings of these post hoc nlyses of the RIGHT study suggest tht for mesuring response to TKI therpy in ptients with CML in chronic phse, kryotyping (BM) nd FISH (PB) re suitble methods for monitoring cytogenetic response, nd qrt-pcr using BM or PB is relible method for monitoring moleculr response. The wider use of methods tht rely on PB rther thn BM smples should limit the frequency t which ptients must undergo invsive BM biopsies. Ann Lu, PhD, nd Cludette Knight, PhrmD, of Percoltion Communictions LLC provided editoril ssistnce tht ws funded by Novrtis Phrmceuticls Corportion. References 1. Ntionl Comprehensive Cncer Network. NCCN clinicl prctice guidelines in oncology: chronic myelogenous leukemi. Version 1. 2014. http://www.nccn.org/professionls/physicin_gls/pdf/cml.pdf. Accessed September 25, 2013. 2. Nowell PC, Hungerford DA. Chromosome studies on norml nd leukemic humn leukocytes. J Ntl Cncer Inst. 1960;25:85 109. 3. Gleevec (imtinib mesylte) [US prescribing informtion]. Est Hnover, NJ: Novrtis Phrmceuticls Corportion; 2012. 4. Tsign (nilotinib) [US prescribing informtion]. Est Hnover, NJ: Novrtis Phrmceuticls Corportion; 2012. 5. Sprycel (dstinib) [US prescribing informtion]. Princeton, NJ: Bristol Meyers Squibb Compny; 2011. 6. Bosulif (bosutinib) [prescribing informtion]. New York, NY: Pfizer Lbs; 2012. 7. Iclusig (pontinib) [prescribing informtion]. Cmbridge, MA: ARIAD Phrmceuticls, Inc; 2012. 8. Brnford S, Rudzki Z, Hrper A, et l. Imtinib produces significntly superior moleculr responses compred to interferon lf plus cytrbine in ptients with newly dignosed chronic myeloid leukemi in chronic phse. Leukemi. 2003;17(12):2401 2409. 9. Brummendorf TH, Kntrjin HM, Gmbcorti-Psserini C, et l. Assessment of erly moleculr response s predictor of long-term clinicl outcomes in the phse 3 BELA study. Blood (ASH Annu Meet Abstr). 2012; 120(21):69. 10. Hnfstein B, Muller MC, Hehlmnn R, et l. Erly moleculr nd cytogenetic response is predictive for long-term progression-free nd overll survivl in chronic myeloid leukemi (CML). Leukemi. 2012;26(9):2096 2102. 11. Hochhus A, Hughes TP, Sglio G, et l. Outcome of ptients with chronic myeloid leukemi in chronic phse (CML-CP) bsed on erly moleculr response nd fctors ssocited with erly response: 4-yer follow-up dt from ENESTND (Evluting Nilotinib Efficcy nd Sfety in Clinicl Trils Newly Dignosed Ptients). Blood (ASH Annu Meet Abstr). 2012;120(21):167. 12. Mrin D, Hedgley C, Clrk RE, et l. Predictive vlue of erly moleculr response in ptients with chronic myeloid leukemi treted with first-line dstinib. Blood. 2012;120(2):291 294. 13. Mrin D, Ibrhim AR, Lucs C, et l. Assessment of BCR-ABL1 trnscript levels t 3 months is the only requirement for predicting outcome for ptients with chronic myeloid leukemi treted with tyrosine kinse inhibitors. J Clin Oncol. 2012;30(3):232 238. 14. Bccrni M, Deininger MW, Rosti G, et l. Europen LeukemiNet recommendtions for the mngement of chronic myeloid leukemi: 2013. Blood. 2013;122(6):872 884. 15. Le Gouill S, Tlmnt P, Milpied N, et l. Fluorescence in situ hybridiztion on peripherl-blood specimens is relible method to evlute cytogenetic response in chronic myeloid leukemi. J Clin Oncol. 2000;18(7):1533 1538. 16. Lesser ML, Dewld GW, Sison CP, Silver RT. Correltion of three methods of mesuring cytogenetic response in chronic myelocytic leukemi. Cncer Genet Cytogenet. 2002;137(2):79 84. Arch Pthol Lb Med Vol 138, September 2014 Cytogenetic nd Moleculr Monitoring for CML Akrd et l 1191
17. Lim L, Bernl-Mizrchi L, Sxe D, et l. Peripherl blood monitoring of chronic myeloid leukemi during tretment with imtinib, second-line gents, nd beyond. Cncer. 2011;117(6):1245 1252. 18. Hochhus A, Lin F, Reiter A, et l. Quntifiction of residul disese in chronic myelogenous leukemi ptients on interferon-lph therpy by competitive polymerse chin rection. Blood. 1996;87(4):1549 1555. 19. Kntrjin HM, Tlpz M, Cortes J, et l. Quntittive polymerse chin rection monitoring of BCR-ABL during therpy with imtinib mesylte (STI571; gleevec) in chronic-phse chronic myelogenous leukemi. Clin Cncer Res. 2003;9(1):160 166. 20. Wng L, Person K, Pillitteri L, Ferguson JE, Clrk RE. Seril monitoring of BCR-ABL by peripherl blood rel-time polymerse chin rection predicts the mrrow cytogenetic response to imtinib mesylte in chronic myeloid leukemi. Br J Hemtol. 2002;118(3):771 777. 21. Cortes JE, Kntrjin HM, Goldberg SL, et l. High-dose imtinib in newly dignosed chronic-phse chronic myeloid leukemi: high rtes of rpid cytogenetic nd moleculr responses. J Clin Oncol. 2009;27(28):4754 4759. 22. Mrin D, Milojkovic D, Olvrri E, et l. Europen LeukemiNet criteri for filure or suboptiml response relibly identify ptients with CML in erly chronic phse treted with imtinib whose eventul outcome is poor. Blood. 2008;112(12):4437 4444. 23. Quints-Crdm A, Kntrjin H, Jones D, et l. Delyed chievement of cytogenetic nd moleculr response is ssocited with incresed risk of progression mong ptients with chronic myeloid leukemi in erly chronic phse receiving high-dose or stndrd-dose imtinib therpy. Blood. 2009; 113(25):6315 6321. 1192 Arch Pthol Lb Med Vol 138, September 2014 Cytogenetic nd Moleculr Monitoring for CML Akrd et l