poor prognosis for p2ry8-crlf2 fusion but not for crlf2 over

ORIGINAL ARTICLE

Poor prognosis for P2RY8-CRLF2 fusion but not for CRLF2over-expression in children with intermediate risk B-cellprecursor acute lymphoblastic leukemiaC Palmi1,9, E Vendramini2,9, D Silvestri3,4, G Longinotti1, D Frison2, G Cario5, C Shochat6,7, M Stanulla5, V Rossi1, AM Di Meglio2, T Villa1,E Giarin2, G Fazio1, A Leszl2, M Schrappe5, G Basso2, A Biondi4, S Izraeli6, V Conter4,8, MG Valsecchi3,9, G Cazzaniga1,9 and G te Kronnie2,9

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has achieved an 80% cure rate as a result of a risk-adaptedtherapy largely based on minimal residual disease (MRD) monitoring. However, relapse is still the most frequent adverse event,occurring mainly in the patients with intermediate MRD levels (intermediate risk, IR), emphasizing the need for new prognosticmarkers. We analyzed the prognostic impact of cytokine receptor-like factor 2 (CRLF2) over-expression and P2RY8-CRLF2 fusion in464 BCP-ALL patients (not affected by Down syndrome and BCR-ABL negative) enrolled in the AIEOP-BFM ALL2000 study in Italy.In 22/464 (4.7%) samples, RQ-PCR showed CRLF2 over-expression (X20 times higher than the overall median). P2RY8-CRLF2 fusionwas detected in 22/365 (6%) cases, with 10/22 cases also showing CRLF2 over-expression. P2RY8-CRLF2 fusion was the most relevantprognostic factor independent of CRLF2 over-expression with a threefold increase in risk of relapse. Significantly, the cumulativeincidence of relapse of the P2RY8-CRLF2þ patients in the IR group was high (61.1%±12.9 vs 17.6%±2.6, Po0.0001), similar tohigh-risk patients in AIEOP-BFM ALL2000 study. These results were confirmed in a cohort of patients treated in Germany. Inconclusion, P2RY8-CRLF2 identifies a subset of BCP-ALL patients currently stratified as IR that could be considered for treatmentintensification.

Leukemia (2012) 26, 2245–2253; doi:10.1038/leu.2012.101

Keywords: CRLF2; pediatric BCP-ALL; prognosis

INTRODUCTIONThe cure rate of new diagnosed pediatric B-cell precursor acutelymphoblastic leukemia (BCP-ALL) is higher than 80%. However,the probability of survival of patients who relapse after treatmentis only 30%. In the AIEOP-BFM ALL2000 study, risk groupstratification was largely based on minimal residual disease(MRD) monitoring as a measure of early response to therapy.Approximately, 30% of patients were at standard risk (SR), 55% atintermediate risk (IR) and 15% at high risk (HR) of relapse. In spiteof the risk-adapted therapy, the majority of relapses occur in thelarge heterogeneous IR group.1 Therefore, the identification ofdisease features with prognostic values within current risk groupsremains a formidable challenge in childhood ALL.

Chromosomal translocations that were identified as key factorsin the pathogenesis of ALL and provided essential prognostichallmarks have been incorporated in patient risk stratification.There is a need to identify which of the recently discoveredgenetic alterations have the potential to improve patientstratification2 as well as for the development of targetedtherapeutic approaches.3–5

In a subset of BCP-ALL patients without known chromosomalaberrations, two genomic abnormalities have been reportedthat involve Ikaros (IKZF1)6,7 and cytokine receptor-like factor 2

(CRLF2)8–13 genes. The latter abnormality includes smalldeletions within the pseudoautosomal region (PAR1) of the sexchromosomes as well as the translocation of this region to theIGH@ locus on chromosome 14. Several studies have providedevidence for the mechanistic basis of the over-expression of CRLF2as a consequence of these chromosomal abnormalities.8,10 PAR1deletion juxtaposes the first non-coding exon of P2RY8 to thefirst exon of CRLF2, leading to CRLF2 expression driven by thepromoter of P2RY8. The translocation of PAR1 region to IGH@locus, however, brings CRLF2 under the control of IGH@ enhancerelements. Elevated CRLF2 expression was found to be associatedwith IKZF1 deletions and activating JAK2 or CRLF2 pointmutations.10–12,14

CRLF2 rearrangements were shown to be correlated with pooroutcome in BCP-ALL patients,12,14 but it is still under debatewhether this prognostic value is associated to CRLF2 over-expression or to specific CRLF2 aberrancies. Moreover, CRLF2aberrations were not proven to bear significant prognostic valuein children with ALL treated on UK protocols.13

Here, we present data on the incidence and prognostic impactof CRLF2 over-expression, specifically P2RY8-CRLF2 fusion, atdiagnosis in 464 Italian BCP-ALL children treated with the protocolof the Associazione Italiana Ematologia Oncologia Pediatrica

1Centro Ricerca Tettamanti, Clinica Pediatrica, Universita di Milano Bicocca, Ospedale San Gerardo, Monza, Italy; 2Laboratory of Oncohematology, Department of Pediatrics,Universita di Padova, Padova, Italy; 3Centro di Biostatistica per l’Epidemiologia Clinica, Universita di Milano Bicocca, Monza, Italy; 4Clinica Pediatrica, Universita di MilanoBicocca, Ospedale San Gerardo, Monza, Italy; 5Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany; 6Sheba Medical Center,Tel Hashomer, Ramat Gan and Tel Aviv University, Tel Aviv, Israel; 7Migal-Galilee Bio-Technology Center, Kiryat-Shmona, Israel. and 8Pediatria, Ospedali Riuniti, Bergamo, Italy.Correspondence: Dr G Cazzaniga, Centro Ricerca Tettamanti, Clinica Pediatrica, Universita di Milano Bicocca, Ospedale San Gerardo, Via Pergolesi, 33, 20900 Monza, Italy.E-mail: [email protected] authors contributed equally to this work.Received 14 October 2011; revised 21 March 2012; accepted 30 March 2012; accepted article preview online 9 April 2012; advance online publication, 11 May 2012

Leukemia (2012) 26, 2245–2253& 2012 Macmillan Publishers Limited All rights reserved 0887-6924/12

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(AIEOP) and Berlin-Frankfurt-Munster (BFM) group (‘AIEOP-BFMALL2000 protocol’) and the potential impact of P2RY8-CRLF2fusion within MRD-based subgroups.

We also present data on CRLF2 over-expression and P2RY8-CRLF2 fusion at BCP-ALL relapse and speculate on P2RY8-CRLF2as a secondary event in disease progression.

PATIENTS AND METHODSPatientsBCP-ALL patients consecutively enrolled in the AIEOP-BFM ALL2000 studyin AIEOP Centers from February 2003 to July 2005, not affected by Downsyndrome (DS) nor Philadelphia chromosome positive (Phþ ), wereincluded in the study cohort. CRLF2 expression was analyzed in 464patients at diagnosis and in 33 paired diagnosis and relapse samples, while

P2RY8-CRLF2 rearrangement was tested in 365 patients at diagnosisand in 26 paired diagnosis and relapse samples for which leftover RNA wasavailable. IGH@-CRLF2 translocation was screened in 8 out of 12 patientswho were positive for CRLF2 over-expression (X20 times higher thanoverall median) but negative for P2RY8-CRLF2 fusion at diagnosis. DNA wasavailable from 34 patients positive for CRLF2 over-expression or P2RY8-CRLF2 fusion at diagnosis, and the following were analyzed: CRLF2mutations (in 25 out of 34 patients), JAK2 mutations (in 32 out of 34patients) and IKZF1 deletions (in 33 out of 34 patients). CRLF2 and JAK2mutations were also analyzed in 6 and 32 paired diagnosis and relapsesamples, respectively.

The clinical characteristics of the analyzed patients compared with thosenot analyzed (AIEOP-BFM ALL2000 study patients diagnosed betweenSeptember 2000 and July 2006) are shown in Supplementary Table 1, andtheir event-free survival (EFS) curve is shown in Supplementary Figure 1a.Details of the study cohort are shown in the Supplementary Materials.

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P2RY8-CRLF2 pos 22/365 (6.0%) - - 2/14 4/21 4/21

hiCRLF2 &P2RY8-CRLF2 pos 10/365 (2.7%) - - 2/10 3/10 3/10

hiCRLF2 &P2RY8-CRLF2 neg 10/365 (2.7%) - 1/8 0/10 1/10 1/10

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Figure 1. CRLF2 expression and genomic alterations. (a) CRLF2 expression in 464 BCP-ALL patients. For each case results are reported as foldchanges on the median expression value of all patients in the cohort. Expression ranged from 0.006- to 810-fold change. (b) Additionalgenomic aberrations in hiCRLF2 patients. In bright blue are cases not tested for P2RY8-CRLF2 fusion. (c) Additional genomic aberrations inloCRLF2 P2RY8-CRLF2-positive patients. (d) Details on the combination of CRLF2 expression, P2RY8-CRLF2 fusion and other genomic aberrations.

Poor outcome of P2RY8-CRLF2 in childhood ALLC Palmi et al

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In addition, 194 SR and 286 IR consecutive patients (non-DS and Ph� )enrolled in the AIEOP-BFM ALL2000 study and treated in German Centers(BFM-G)12 were analyzed by reverse transcriptase PCR (RT-PCR) for P2RY8-CRLF2 rearrangement, as a validation cohort.

Informed consent to participate in the study was obtained for allpatients by parents or legal guardians. Risk group definitions and finalstratification, treatment outlines, details and differences between AIEOPand BFM were previously reported15 and briefly summarized below.

Protocol stratificationPatient risk groups were defined as follows. The HR group includedpatients with any of the following criteria: t(4;11) or MLL/AF4, prednisonepoor response (X1000 blasts/ml on day 8 peripheral blood after 7 days ofprednisone and one dose of intrathecal methotrexate on day 1), inability toachieve clinical remission after induction phase IA and high burden(X10� 3) of PCR-MRD at day 78. The SR group included patients wholacked high-risk criteria and tested negative to PCR-MRD performed by

Table 1. Clinical features of AIEOP study cohort patients positive or negative for CRLF2 over-expression and P2RY8-CRLF2 fusion

Characteristics P-value hiCRLF2 P-value P2RY8-CRLF2

No Yes No Yes

N % N % N % N %

All patients 442 95.30 22 4.70 343 93.97 22 6.03

Gender 0.47 0.19Female 216 48.87 9 40.91 167 48.69 14 63.64Male 226 51.13 13 59.09 176 51.31 8 36.36

Age 0.18 0.521–5 Yrs 288 65.16 12 54.55 224 65.31 12 54.556–9 Yrs 87 19.68 8 36.36 69 20.12 6 27.27X10 Yrs 67 15.16 2 9.09 50 14.58 4 18.18

WBC(� 1000/ml) 0.45 0.66o20 309 69.91 18 81.82 235 68.51 14 63.6420–100 101 22.85 4 18.18 82 23.91 7 31.82X100 32 7.24 0 0.00 26 7.58 1 4.55

Translocationst(4;11) — —Pos 5 1.13 0 0.00 5 1.46 0 0.00Neg 435 98.42 22 100.00 336 97.96 22 100.00Unknown 2 0.45 0 0.00 2 0.58 0 0.00

t(12;21) 0.01 0.59Pos 92 20.81 0 0.00 71 20.70 3 13.64Neg 328 74.21 22 100.00 254 74.05 19 86.36Unknown 22 4.98 0 0.00 18 5.25 0 0.00

Prednisone response 1.00 1.00Good 413 93.44 21 95.45 317 92.42 21 95.45Poor 27 6.11 1 4.55 25 7.29 1 4.55Unknown 2 0.45 0 0.00 1 0.29 0 0.00

MRD 0.09 0.69SR 134 30.32 3 13.64 108 31.49 6 27.27IR 205 46.38 13 59.09 154 44.90 12 54.55HR 6 1.36 1 4.55 3 0.87 0 0.00Unknown 97 21.95 5 22.73 78 22.74 4 18.18

Final protocol strata 0.28 0.94SR 126 28.51 3 13.64 100 29.15 6 27.27IR 283 64.03 17 77.27 214 62.39 15 68.18HR 33 7.47 2 9.09 29 8.45 1 4.55DNA index 1.00 0.0061.16–1.6 92 20.81 5 22.73 80 23.32 0 0.00Other 319 72.17 17 77.27 240 69.97 21 95.45Unknown 31 7.01 0 0.00 23 6.71 1 4.55

hiCRLF2 — o0.001No — — — — 333 97.08 12 54.55Yes — — — — 10 2.92 10 45.45Unknown — — — — 0 0.00 0 0.00

P2RY8-CRLF2 o0.001 — — — — —No 333 75.34 10 45.45 — — — —Yes 12 2.71 10 45.45 — — — —Unknown 97 21.95 2 9.09 — — — —

Abbreviations: HR, high risk; IR, intermediate risk; MRD, minimal residual disease; SR, standard risk; WBC, white blood cell count.


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using two sensitive markers (X1� 10� 4) at both day 33 and day 78. The IRgroup included the remaining patients, and those not evaluated by PCR-MRD. Details of PCR-MRD are reported in the Supplementary Materials.

Quantitative expression of CRLF2CRLF2 transcript levels on diagnostic and relapse samples were analyzedusing TaqMan Gene Expression Assay Hs00913509_s1 (Applied Biosystems,Foster City, CA, USA); the housekeeping GUS gene transcript was tested asan internal control by using Universal Probe Library System (RocheDiagnostics, Basel, Switzerland), following the manufacturer’s instruc-tions.16 Optimal primers and probe for GUS amplification were selectedusing the Roche ProbeFinder software (https://www.roche-appliedscience.com/sis/rtpcr/upl). Each cDNA sample (20 ng RNA equivalent) was tested induplicate (Ct range between replicates o1.5). The amplification reactionwas performed on the 7900HT FAST Real Time PCR System instrument(Applied Biosystems) with the following protocol: initial step at 95 1C for10 min, then 50 cycles at 95 1C for 15 s and at 60 1C for 1 min.

Relative gene expression (indicated as fold change) was quantified bythe 2�DDCt method.17 The DDCts were calculated by subtracting themedian of the DCt of all the (n¼ 464) tested patients at diagnosis to theDCt of each sample.

CRLF2 aberrationsThe presence of the fusion transcript P2RY8-CRLF2 was investigated byRT-PCR. The patients analyzed at diagnosis were representative of theentire study cohort (Supplementary Table 2 and Supplementary Figure 1b).In order to verify that the RT reaction worked properly, the house-keepinggene Abelson tyrosine kinase (ABL) expression was tested in parallel for eachcDNA sample. RT-PCR for P2RY8-CRLF2 was performed using primersdesigned in the first exon of P2RY8 (50-GGACAGATGGAACTGGAAGG-30)and the third exon of CRLF2 (50-GTCCCATTCCTGATGGAGAA-30), giving riseto a PCR fragment of B511 bp. Amplification conditions were: initialdenaturation at 94 1C for 2 min; 40 PCR cycles: denaturation at 94 1C for

30 s, annealing at 63 1C for 30 s, extension at 72 1C for 1 min and finalextension at 72 1C for 10 min (PCR reagents from Roche).

RT-PCR to detect the P2RY8-CRLF2 fusion in the BFM-G cohort wasperformed as previously described.12

IGH@-CRLF2 translocation was inquired by fluorescence in situ hybridiza-tion (FISH) on interphase nuclei using the Vysis LSI IGH Dual Color Break-Apart Rearrangement Probe (Abbott Molecular, Abbott Park, IL, USA) andthe CEP (chromosomes enumeration probes) DNA FISH Probe to detectchromosome X (Abbott Molecular). The nuclei were counterstained withDAPI (40 ,6-diamino-2-phenylindol). Results were recorded using a fluores-cence Leica DMRB microscope fitted with a � 100/1.30 oil objective, CCDcamera and digital imaging software from Metasystem (ISIS, FISH imagingsystem).

To identify CRLF2 mutations, intronic primers of human CRLF2 sequence(accession number NM_022148.2) were used to amplify exons 1–6 ofthe gene with PCR.11 Fragments were analyzed by denaturinghigh-performance liquid chromatography (WAVE, Transgenomic, Omaha,NE, USA). Fragments with abnormal chromatography patterns weresequenced.

Other genetic aberrationsHigh resolution melting (HRM) analysis was performed to identify JAK2mutations in exon 16 using High Resolution Melting Master (RocheDiagnostics), following the manufacturer’s instructions. In detail 20 ng ofDNA was amplified in a final volume of 20ml with 10ml of ResolutionMelting master mix (Roche Diagnostics), 2.8ml of magnesium chloride,3.5 mM (Roche Diagnostics) and 4 pmol of each primer (forward: 50-TTTGGGGGCTTGAACATACT-30 and reverse: 50-TTCAAGGAAAATTAACAACATGC-30).The reaction was performed on the LightCycler 480 instrument (RocheDiagnostics) with the following protocol: initial denaturation at 95 1C for10 min, 45 cycles of amplification at 95 1C for 10 s, 53 1C for 10 s and 72 1Cfor 15 s, then for HRM analysis samples were denatured at 95 1C for 1 min,cooled at 40 1C for 1 min and heated from 60 to 95 1C with a ramping rateof 0.02 1C/s. DNA from a pool of peripheral blood buffy coat of healthy

P2RY8-CRLF2 -P2RY8-CRLF2 +


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Figure 2. Association of CRLF2 over-expression and P2RY8-CRLF2 fusion to treatment outcome. (a) EFS and (b) CIR of study cohort patientsaccording to CRLF2 expression: hiCRLF2 and loCRLF2. (c) EFS and (d) CIR of investigated patients according to the presence or absence ofP2RY8-CRLF2 fusion gene.


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donors was used as a wild-type reference, whereas DNA from the MUTZ5human cell line (DSMZ), carrying R683G Jak2 mutation, was used as apositive control. All the fragments with abnormal melting profile weresequenced. In particular, 200 ng of DNA samples positive for HRM analysiswere amplified using 2.5 U High Fidelity Polymerase Optimase (Transge-nomic, Glasgow, UK) and 20 pmol JAK2 oligos described above at thefollowing PCR conditions: 95 1C for 5 min, 35 cycles at 95 1C for 30 s, 54 1Cfor 40 s, 72 1C for 45 s and a final extension at 72 1C for 7 min.

The PCR products were cloned by the Zero Blunt PCR Cloning Kit(Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions,and then sequenced using the ABI-3130 Genetic Analyzer instrument(Applied Biosystems).

IKZF1 deletions were investigated by Multiplex Ligation-dependentProbe Amplification (MLPA) technique using the Salsa MLPA kit P335-A3ALL-IKZF1 kit (MRC-Holland, Amsterdam, the Netherlands), accordingto the manufacturer’s instructions. Samples of pediatric ALL patients incomplete remission were used as wild-type controls. The fragmentswere separated with the ABI-3130 Genetic Analyzer instrument (AppliedBiosystems) and the data analyzed using Coffalyser software (http://old.mlpa.com/coffalyser).

Statistical analysisEFS and survival were calculated from the date of diagnosis to the date ofevent, which for EFS was resistance, relapse, death or second malignantneoplasm, whichever occurred first, and for survival was death from anycause. EFS and survival curves at 5 years were estimated according toKaplan–Meier, and compared according to log-rank test. Cumulativeincidence of relapse (CIR) at 5 years was estimated by adjusting for

competing risks of other events and comparison performed with the Graytest.

The Cox regression model was applied to evaluate the prognostic valueof P2RY8-CRLF2 fusion and hiCRLF2 expression on the cause-specific hazardof relapse after adjusting by main prognostic features: sex, age, whitecell count and MRD.

Follow-up was updated in February 2010.

RESULTSCRLF2 aberrations at diagnosisCRLF2 expression, evaluated by real-time quantitative (RQ)-PCR on464 pediatric BCP-ALL patients at diagnosis, ranged from 0.006-to 810-fold change compared to the overall median value(Figure 1a). Outcome was evaluated within subgroups definedby different categories of CRLF2 expression. In particular, weevaluated potential cut-points starting from five times the overallmedian value and moving by steps of 5. The lowest threshold forCRLF2 expression showing a significant difference in CIR betweentwo groups was 20 times the median that was then adopted ascut-point (Supplementary Figure 2). Higher values of the thresholddid not show an increase in discriminating power of theCIR for the two groups separated by the threshold.

Twenty-two patients (4.7%) presented an expression higherthan 20 times (hiCRLF2).

The P2RY8-CRLF2 fusion was detected in 22 patients of the 365cases analyzed (6.0%). In all, 10 of them were also hiCRLF2 (10/20

p-value=0.005

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Figure 3. Association of CRLF2 over-expression and P2RY8-CRLF2 fusion to overall survival (OS) in the whole cohort and in MRD IR subgroup.(a) OS of study cohort patients according to CRLF2 expression: loCRLF2 and hiCRLF2. (b) OS of investigated patients according to the absenceor presence of P2RY8-CRLF2 fusion gene. (c) OS of IR patients according to the absence or presence of P2RY8-CRLF2 fusion gene.


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tested for the deletion, 50.0%), whereas the remaining 12 casespresented low levels of CRLF2 expression (less than 20 times themedian, range: 0.006–18.5, ‘loCRLF2’; Figure 1a). Within the hiCRLF2group, only one out of eight (negative for P2RY8-CRLF2) waspositive for the IGH@ translocation (Figure 1b).

We observed JAK2 mutations in 3/10 hiCRLF2 P2RY8-CRLF2positive patients, in 1/12 hiCRLF2 P2RY8-CRLF2 negative patientsand 1/11 loCRLF2 P2RY8-CRLF2 positive patients (two R683G,one R683S and two insertion mutations: L681-I682insGEEL andT705delinsRWE). IKZF1 deletions were detected in 5/22 hiCRLF2patients, 3 associated with P2RY8-CRLF2 fusion, and in 2/11 casespositive for P2RY8-CRLF2 but loCRLF2. The CRLF2 F232C mutationwas detected in two hiCRLF2 patients, positive for both P2RY8-CRLF2 and IKZF1 deletions (Figures 1b, c and d).

Clinical characteristics of patients positive for CRLF2 aberrations atdiagnosis are described in Table 1 and in Supplementary Results.

All patients with P2RY8-CRLF2 fusion had DNA index o1.16,while 5/22 hiCRLF2 patients were classified as high hyperdiploid(Table 1).

All hiCRLF2 patients were negative for the recurring chromosomal translocations t(4;11) and t(12;21). In particular, byregression analysis t(12;21) and CRLF2 expression show asignificant negative correlation.

Prognostic impact of CRLF2 over-expression and P2RY8-CRLF2fusion at diagnosisHiCRLF2 patients have a significantly inferior EFS (62.9%±10.5 vs82.6%±1.8, P¼ 0.05) and increased CIR (37.1%±10.5 vs 15.2%±1.7, P¼ 0.02) compared with loCRLF2 patients. All the eight eventsin hiCRLF2 patients were relapses (Figures 2a and b). The 5-yearsurvival estimates were 85.9%±7.6 and 92.1%±1.3, respectively(P¼ 0.35; Figure 3a).

In all, 9 out of the 22 patients carrying P2RY8-CRLF2 fusionexperienced relapse. Kaplan–Meier analysis revealed an inferiorEFS (5-year EFS of 57.2%±10.9 vs 83.5%±2.0, P¼ 0.004) andincreased CIR (42.8%±10.9 vs 14.5% ±1.9, P¼ 0.001) for patientscarrying the fusion gene (Figures 2c and d). The 5-year survivalestimates were significantly different, 80.9%±8.6 and 93.4%±1.4,respectively (P¼ 0.04; Figure 3b).

The worst outcome (EFS 37.5%±16.1 and CIR 62.5%±16.1)was observed for patients with the combination of hiCRLF2 andP2RY8-CRLF2 fusion, while hiCRLF2 patients who are negative forP2RY8-CRLF2 fusion seem to have an outcome comparable withpatients lacking CRLF2 over-expression and fusion. Twelve patientswere loCRLF2 and positive for P2RY8-CRLF2 fusion, three of whomrelapsed (Figures 4a and b).

Cox model analysis was performed to assess the relativeprognostic value of P2RY8-CRLF2 fusion and hiCRLF2 expressionafter adjusting for main prognostic features (Table 2). Results showthat P2RY8-CRLF2 fusion retains prognostic significance with athreefold increase in the risk of relapse for positive versus negativepatients (hazard ratio 3.30; 95% CI 1.29–8.48; P¼ 0.01), whilehiCRLF2 is not an independent prognostic factor (hazard ratio 1.05;95% CI 0.37–2.97; P¼ 0.93).

Outcome and risk groupWe further analyzed the prognostic value of P2RY8-CRLF2 fusionseparately within SR and IR patient groups. P2RY8-CRLF2 fusionwas similarly distributed within the two non-HR subgroups, beingfound in 6 out 106 SR patients (5.7%) and 15 out of 229 IR patients(6.7%; Table 1). However, none of the 6 P2RY8-CRLF2-positiveSR patients relapsed (Figure 5a), while 9 out of 15 cases inthe IR group relapsed with a significant difference in the CIR(61.1%±12.9 vs 17.6%±2.6, Po0.0001; Figure 5b), and in survival(72.7%±11.7 vs 93.1%±1.8, P¼ 0.005; Figure 3c). EFS curves areprovided as Supplementary Figure 3. Four of the nine relapses hadBM localization, three were extramedullary and two combined

10N. pts

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Figure 4. Association of combined CRLF2 over-expressionand P2RY8-CRLF2 fusion to treatment outcome. (a) EFS and (b) CIRaccording to hiCRLF2 and loCRLF2 expression and to the presence(POS) or absence (NEG) of P2RY8-CRLF2 fusion gene.

Table 2. Results of the multivariate analyses. Cox model on hazardof relapse in 365 patients

Characteristics P-value Hazard ratio 95% CI

hiCRLF2No 1.00Yes 0.93 1.05 0.37–2.97

P2RY8-CRLF2No 1Yes 0.01 3.30 1.29–8.48

GenderFemale 1Male 0.80 0.94 0.56–1.58

Age1–9 Yrs 110–17 Yrs 0.35 1.37 0.71–2.65

WBC (� 1000/ml)o100 1X100 0.006 2.8 1.35–5.8

MRDSR 1IR o0.001 4.74 2.12–10.62HR o0.001 38.54 7.67–193.58Unknown 0.44 1.51 0.53–4.34

Abbreviations: HR, high risk; IR, intermediate risk; MRD, minimal residualdisease; SR, standard risk; WBC, white blood cell count.


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(compared with only 6/39 extramedullary relapses in the P2RY8-CRLF2-negative IR patients).

In order to validate those findings, we analyzed the prognosticvalue of P2RY8-CRLF2 fusion for SR and IR patients enrolled inthe AIEOP-BFM ALL2000 study and treated in German Centers(BFM-G).12

In the BFM-G cohort, 2 out of 8 SR patients carrying the fusionexperienced relapse, whereas 6 relapses out of 11 IR patients wereobserved (Figures 5c and d). In the same BFM-G cohort, IR patientspositive for P2RY8-CRLF2 fusion showed a significant higher CIRthan patients without the fusion (54.5%±15 vs 10.7%±1.9,Po0.0001). Two of the six relapses had BM localization, three wereextramedullary and one combined (compared with 10/31 extra-medullary relapses in the P2RY8-CRLF2-negative IR patients).

BFM-G SR patients with or without the fusion have a CIR of25.0%±15.3 vs 5.6%±1.7, P¼ 0.05. Thus, apparently even in theBFM-G cohort, the SR patients who are positive for the fusion havea lower incidence of relapse than IR patients.

CRLF2 expression and genomic aberrations at relapseThirty-three paired diagnostic and relapse specimens wereanalyzed for CRLF2 expression level. Samples at relapse showeda median value of CRLF2 expression two times higher than therespective samples at diagnosis (2.60 vs 1.25; Figure 6a).The relapse sample with the highest CRLF2 expression level wasthe only sample positive for both P2RY8-CRLF2 rearrangement andJAK2 mutation (L681-I682insLR). Two P2RY8-CRLF2-positivepatients at diagnosis lost the rearrangement at relapse and no

de novo P2RY8-CRLF2 fusion was detected. CRLF2 mutationsremained stable at relapse, while JAK2 mutations were lost in 1/34patients at relapse (2.9%), acquired in 2/34 cases (5.9%) and in1/34 case the mutation was different between diagnosis andrelapse (respectively, R683G and L681-I682insRD; Figure 6b).Detailed description of CRLF2-expression level and genomicaberrations of diagnosis/relapse pairs is shown in SupplementaryTable 3.

DISCUSSIONIn childhood ALL, risk-adjusted therapy has brought aboutimpressive improvements of survival rates during the last decades.In this context, clinicians and researchers have two majorobjectives: to find new prognostic markers to identify upfrontpatients with high probability to relapse,1,5 including relapsesduring long-term follow-up, and to recognize good prognosticfeatures that can be used to guide treatment-intensity reductions,aiming to spare children from acute and long-term side effects.15

Although the advent of MRD monitoring has revolutionizedthe stratification approach of the risk-based treatments,15,18 newgenomic discoveries are expected to be very useful, forrecognizing previously undetectable subgroups of childhoodALL patients with specific risk levels, thus complementing MRD.

Here, for the first time, we report the incidence and prognosticrelevance of CRLF2 over-expression and P2RY8-CRLF2 rearrange-ment in a study cohort of MRD-stratified childhood BCP-ALL,treated in Italy with the AIEOP-BFM ALL2000 protocol, excludingthe confounding effect of DS and Phþ subgroup of patients,

SR AIEOP patients

SR BFM-G patients

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P2RY8-CRLF2 -

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186

N. pts N. rel.

12 5.6%(1.7)

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11 6 54.5%(15)

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15 9 61.1%(12.9)

p-value<0.0001

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Figure 5. Association of P2RY8-CRLF2 fusion to treatment outcome in MRD subgroups. CIR from diagnosis of (a) SR and (b) IR AIEOP patientsaccording to the presence or absence of P2RY8-CRLF2 fusion gene. CIR from diagnosis of (c) SR and (d) IR BFM-G patients according to thepresence or absence of the P2RY8-CRLF2 fusion gene.


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which had been included in other studies.8,10–13 A high CRLF2expression (defined as an expression level 20 times higher thanthe median expression of all patients analyzed) was detected inabout 5% of patients, while the incidence of the P2RY8-CRLF2fusion was estimated as 6%, which is in agreement with theincidence found in other cohorts with similar Caucasiancomposition.12,13 Importantly, for the first time, we observedthat the P2RY8-CRLF2 fusion was not only found among patientswith hiCRLF2 expression but also among patients with loCRLF2expression. In the case of patients who are positive for the

P2RY8-CRLF2 fusion but with low expression of CRLF2, we mightspeculate that the deletion is present in subclones of the leukemicblast cell population. The latter would be consistent with themarked subclonal heterogeneity in ALL,19,20 but furtherexperiments will be necessary to show this.

The lack of absolute correlation between CRLF2 fusion andover-expression prompted us to explore the prognostic impact ofboth P2RY8-CRLF2 fusion and CRLF2 over-expression, alone and incombination.

Both hiCRLF2 and P2RY8-CRLF2 fusion were associated with ahigher relapse rate. As already reported12,13 late relapses wereprevalent, occurring mostly after the end of the 2-year treatment.Compared to the distribution of relapses in the AIEOP-BFMALL2000 protocol,19,20 the tendency toward an increasedpercentage of extramedullary relapse localization (either isolatedor combined) was observed.

Interestingly, even if the worst outcome was observed forpatients with the combination of hiCRLF2 and P2RY8-CRLF2 fusion,the latter turned out to be a poor prognostic factor regardless ofCRLF2 over-expression by the Cox model, with a threefold increasein the risk of relapse for positive versus negative patients evenafter adjusting for main prognostic features. Also the data of theOS analysis after 5 years observation time corroborated thefinding that P2RY8-CRLF2 is a stronger prognostic marker thanCRLF2 over-expression.

Moreover, P2RY8-CRLF2 fusion also proved to be a significantfactor of poor outcome when patients with the favorable factorst(12;21) and DNA index 1.16–1.6 were excluded from the analysis(Supplementary Figure 4).

In order to understand how to transfer the prognostic impact ofthis CRLF2 alteration into clinical practice, P2RY8-CRLF2 fusion wasanalyzed within the non-HR subgroups according to protocolstratification. In particular, it is relevant that the EFS and CIR of theP2RY8-CRLF2 fusion-positive patients in the IR group was similar tothat of HR patients in the AIEOP-BFM ALL2000 study and that theirOS was significantly reduced.15 Importantly, this was not the casefor SR patients.

The impact of P2RY8-CRLF2 fusion in the Italian cohort wasvalidated in a similar number of patients enrolled in the AIEOP-BFM ALL2000 protocol in Germany, further supporting theevidence that the detection of P2RY8-CRLF2 in MRD-based IRpatients would potentially qualify them for a high risk-basedtreatment. Overall, among IR patients in AIEOP and BFM cohorts,21 out of 26 P2RY8-CRLF2 fusion-positive patients were treated inthe IR prednisone arm, which is available to all patients in the newcurrent AIEOP-BFM ALL2009 protocol. Three out of the twenty sixpatients were highly MRD-positive at TP1 (d33) and still positive atTP2 (d78(ref.15); a new criterion for HR in the current protocol) and2/3 relapsed. Indeed, 13/23 relapsing patients would still not havebeen stratified by these means as HR patients in the currentAIEOP-BFM ALL2009 protocol.

In agreement with emerging evidence of mutual collaborationamong several genomic lesions leading to constitutive activationof the JAK2/STAT5 pathway,10–13 we found additional genomicabnormalities (CRLF2, JAK2 mutations and IKZF1 deletions) inhiCRLF2- and/or P2RY8-CRLF2-positive patients. However, thelimited numbers for each of the combinations of theseaberrations prevent any prognostic indication from being drawn.

Here, for the first time, we report the analysis on CRLF2alterations at relapse of childhood BCP-ALL. A total of 33 of the 79relapses occurring during the observation time of the study wereevaluated for CRLF2-expression levels. Samples at relapse showeda median value of CRLF2 expression that was slightly higher thanthe respective samples at diagnosis (2.60 vs 1.25). Interestingly, node novo P2RY8-CRLF2 fusion genes were detected in the 26 relapsecases for which the diagnostic correlate had been tested. On thecontrary, two positive patients at diagnosis (with low CRLF2expression levels) lost the P2RY8-CRLF2 fusion at relapse. This is an

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DIAGNOSES vs RELAPSES

P2RY8-CRLF2 (N=26)

DXREL

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DXREL

*different

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Figure 6. CRLF2 expression and genomic alterations at relapse. (a)Log–log plot of the CRLF2 expression value for 33 paired diagnosisand relapsed specimens. CRLF2 expression at relapse was, onmedian, double the paired diagnostic samples (median foldchange¼ 2.60 vs 1.25). (b) Table of genomic aberrations investi-gated in paired diagnosis and relapsed specimens.


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indirect clue that the deletion, even though it is a strong marker ofpoor prognosis as described above, is not a primary lesion and canbe acquired during the progression of the pre-leukemic clone andthus can contribute to the overt disease. Moreover, like P2RY8-CRLF2, JAK2 mutations appear to be secondary lesions; both donot remain stable at relapse in all cases.

In conclusion, we have shown that childhood BCP-ALL patientswith the P2RY8-CRLF2 fusion have a very poor outcome whentreated by the AIEOP-BFM approach that could justify a shift fromthe MRD-based intermediate to the high-risk group. It remains tobe demonstrated that a high risk-based therapy could be effectivein preventing relapse for these patients. In the long run,alternative therapies that interfere with the activation of theJAK2/STAT5 signaling pathway may hold a promise for treatmentof these patients.

CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGEMENTSThis study was supported by grants from: Fondazione Citta della Speranza (Padova),Fondazione Tettamanti (Monza), Associazione Italiana Ricerca sul Cancro (AIRC; toGteK, GB, GiC, AB and MGV), CARIPARO Project of Excellence (to GteK, EV and DF),MIUR (to AB and GB), Fondazione Cariplo (to AB and GiC), Deutsche Krebshilfe(to GuC, MSt and MSc), Madeleine-Schickedanz-Kinderkrebsstiftung (to GuC, MSt andMSc), Israel Science Foundation Legacy Program and Children with Leukaemia UK(to SI). We thank Simona Songia, Lilia Corral, Valentina Carrino, Eugenia Mella(Monza), Elena Seganfreddo and Katia Polato (Padova) for AIEOP MRD monitoring;Silvia Bungaro and Marta Galbiati (Monza) for cytogenetics and FISH; all medicaldoctors of the AIEOP and BFM centers.

AUTHOR CONTRIBUTIONSCP, GL, EV, DF and CS performed the molecular analyses on CRLF2 aberrationsand additional genetic mutations; CP, EV and SI collaborated in writing themanuscript; DS and MGV collected the Trial data and performed all thestatistical analyses; AMDM and AL performed cytogenetic analyses; VR, GF andTV represent the team that performed Trial molecular and MRD analyses;GB and AB supervised the research; EG performed DNA/RNA extraction and cellbanking; VC is responsible of the AIEOP ALL2000 study; MSc, MSt and GuCprovided the data of the BFM-G patients; GiC and GtK designed the study,supervised the research, analyzed data and wrote the manuscript.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)


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poor prognosis for p2ry8-crlf2 fusion but not for crlf2 over

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