What is the relevance of Ikaros gene deletions as prognostic marker in pediatric Philadelphia negative B-cell precursor acutelymphoblastic leukemia?

by Chiara Palmi, Maria Grazia Valsecchi, Giulia Longinotti, Daniela Silvestri,Valentina Carrino, Valentino Conter, Giuseppe Basso, Andrea Biondi, Geertruy Te Kronnie, and Giovanni Cazzaniga

Haematologica 2013 [Epub ahead of print]

Citation: Palmi C, Valsecchi MG, Longinotti G, Silvestri D, Carrino V, Conter V, Basso G,Biondi A, Te Kronnie G, and Cazzaniga G. What is the relevance of Ikaros gene deletionsas prognostic marker in pediatric Philadelphia negative B-cell precursor acute lymphoblasticleukemia? Haematologica. 2013; 98:xxx doi:10.3324/haematol.2012.075432

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Copyright 2013 Ferrata Storti Foundation.Published Ahead of Print on April 12, 2013, as doi:10.3324/haematol.2012.075432.

 

What is the relevance of Ikaros gene deletions as prognostic marker in pediatric

Philadelphia negative B-cell precursor acute lymphoblastic leukemia?

Chiara Palmi,1 Maria Grazia Valsecchi,2 Giulia Longinotti,1 Daniela Silvestri,2 Valentina

Carrino,1 Valentino Conter,3 Giuseppe Basso,4 Andrea Biondi,3 Geertruy Te Kronnie,4 and

Giovanni Cazzaniga1

1Centro Ricerca Tettamanti, Clinica Pediatrica, Università di Milano Bicocca, Ospedale

San Gerardo, Monza, Italy.

2Centro di Biostatistica per l’Epidemiologia Clinica, Università di Milano Bicocca, Monza,

Italy.

3Clinica Pediatrica, Università di Milano Bicocca, Ospedale San Gerardo, Monza, Italy.

4Laboratorio di Oncoematologia, Dipartimento di Pediatria, Università di Padova, Italy.

Correspondence: Andrea Biondi, Clinica Pediatrica, Università di Milano Bicocca,

Ospedale San Gerardo, Via Pergolesi 33, 20900 Monza (MB), Italy.

E-mail: abiondi.unimib@gmail.com

Key words: IKZF1 deletions, pediatric Ph- BCP-ALL, prognosis.

Running head: IKZF1 deletion and prognosis in Ph- BCP-ALL

DOI: 10.3324/haematol.2012.075432

 

Acknowledgments

The authors would like to thank Simona Songia, Lilia Corral, Eugenia Mella, Tiziana Villa

(Monza), Elena Seganfreddo and Katia Polato (Padova) for AIEOP MRD monitoring; all

medical doctors of the AIEOP centers. This work was supported by grants from:

Fondazione Tettamanti (Monza), Fondazione Città della Speranza (Padova), Associazione

Italiana Ricerca sul Cancro (AIRC) (to GB, AB, MGV, GteK and GC), MIUR (to GB and

AB), Fondazione Cariplo (to AB, GC and GteK), CARIPARO project of excellence (to

GteK).  This work was (partly) funded by the European Commission (FP7) under the

contract ENCCA (NoE-2011-261474).

Authorship and Disclosures

CP, and GL performed the molecular analyses; VC represents the team who performed

MRD analyses; CP analyzed data and wrote the manuscript; DS and MGV collected the

Trial data and performed all the statistical analyses; GB, AB and GtK supervised the

research; VC is responsible of the AIEOP ALL2000 study and collaborated in writing the

manuscript; GC designed the study and supervised the research.

The authors reported no potential conflicts of interest.

DOI: 10.3324/haematol.2012.075432

  

Abstract

We herewith focused the analysis of Ikaros gene deletions in a homogeneous cohort of

410 pediatric non-Down syndrome and Philadelphia chromosome-negative, B-cell

precursor Acute Lymphoblastic Leukemia patients enrolled in Italy into the AIEOP-BFM

ALL2000 study. We confirm their reported poor prognostic value, although the associated

Event-free survival was relatively high (approximately 70%).

The difference in the Cumulative incidence of relapse between patients positive or not for

IKZF1 deletions was not marked (24.2% (5.9) vs 13.1% (1.8) overall and 23.9% (6.6) vs

16.5% (2.5) in the Intermediate risk subgroup). In line with this, IKZF1 deletions were not

an independent prognostic factor of the hazard of relapse.

Moreover, most IKZF1 deleted cases stratified in the high risk group relapsed, thus

suggesting that their identification would then require an alternative treatment.

In conclusion, the need and benefit of introducing IKZF1 deletions as an additional

stratification marker for Ph negative BCP-ALL patients remains questionable.

DOI: 10.3324/haematol.2012.075432

  

INTRODUCTION

In the AIEOP-BFM ALL2000 study, the risk group stratification largely based on Minimal

Residual Disease (MRD) monitoring as a measure of early response to therapy allowed to

achieve more than 80% cure rate. However, relapse is still the most frequent adverse

event, occurring mainly in the largest and heterogeneous subgroup of non-high risk (non-

HR) patients. (1) This emphasizes the need for new prognostic markers for upfront

identification of patients with a high risk of relapse or of patients who are likely not to

respond to the most aggressive chemotherapy.

Recently, genomic abnormalities of Cytokine Receptor like Factor 2 (CRLF2) and Ikaros

(IKZF1) genes have been reported, not only in Down Syndrome (DS) and Philadelphia

chromosome positive (Ph+) patients, but also in patients without known chromosomal

aberrations, although with different incidence. (2-6) Indeed, IKZF1 deletions are rare in T-

ALL (about 5%) (7) , highly frequent in Ph+ ALL (about 80%) (8) and an incidence of 35%

was reported in Down Syndrome ALL patients (9). The most frequent IKZF1 alterations

identified in ALL patients were deletions encompassing the whole gene or involving only

some exons. (5, 7-15) All these deletions cause the loss of IKZF1 activity. (16) 

IKZF1 deletions were shown to be related to poor outcome in pediatric ALL patients, (5, 7,

11-15) but their prognostic impact could be different in specific subgroups.

The potential benefit of the early identification of a new prognostic marker should be

assessed within the subgroup of patients who are not at HR due to other features and

evaluated in a homogeneous cohort of cases. A recent paper published by Dorge et al (7)

showed that patients with IKZF1 deletion had an inferior outcome compared to non deleted

and accordingly it was concluded that IKZF1 deletions may be a strong candidate for

changing the stratification strategy. However, although inferior, their outcome was still

relatively favorable, since patients with deletions had a 5-year EFS of about 70%. Thus,

IKZF1 deletions, although potentially useful for stratification, is not associated to a really

DOI: 10.3324/haematol.2012.075432

 

poor prognosis. Our work aims to assess the prognostic value of IKZF1 deletions in a

cohort of patients who underwent a stratification and treatment very similar to that reported

in (7, 17) .

Our investigation was focused on the ALL subcohort where a change in risk stratification

could be more relevant in clinical practice. We thus screened a cohort of 410 non Down

patients, non T, Ph negative BCP-ALL enrolled into AIEOP-BFM ALL2000 study in Italy

and recently analyzed for CRLF2 alterations for evaluation of the prognostic role of IKZF1

(18).

METHODS

Patients

The study cohort was constituted by 410 non-DS, Ph-, BCP-ALL patients consecutively

enrolled in the AIEOP-BFM ALL2000 study in AIEOP Centers from February 2003 to July

2005, who were included in the previous study on CRLF2 alterations and for which DNA

was still available. (18) Data on recurrent genomic aberrations were available for most

patients. (19) P2RY8-CRLF2 rearrangement was tested by RT-PCR in 372 (90.7%)

patients. (18)

As shown in Supplementary Table 1, there is an unbalance toward more unfavorable

features in respect to treatment response (PPR and high MRD levels) in the non

investigated group. Despite this difference, however, the event free survival curve of the

analyzed patients is not different from that of not analyzed patients diagnosed in AIEOP

centers in the study period (2003-2005) (Supplementary Figure 1).

The project was approved by AIEOP ALL Scientific Committee.

Risk group definitions and treatment outlines were previously reported (17) and are briefly

summarized in the Supplementary materials.

DOI: 10.3324/haematol.2012.075432

 

DNA copy number variations

IKZF1 deletions, together with deletions in the additional genes CDKN2A/B, PAX5, ETV6,

BTG1, RB1 and EBF1 were investigated by Multiplex Ligation-dependent Probe

Amplification (MLPA) technique using the Salsa MLPA kit P335-A3 ALL-IKZF1 kit (MRC-

Holland, Amsterdam, the Netherlands), according to the manufacturer’s instructions. (7,

18, 20). Patients positive for IKZF1 deletions were further analyzed by the more specific

Salsa MLPA P202-A1 IKZF1 kit (MRC-Holland, Amsterdam, the Netherlands) to confirm

and better define the extension of the alteration.

Samples of pediatric ALL patients in complete remission were used as wild type controls.

Statistical analysis

Event-free survival (EFS) time was calculated from date of diagnosis to date of event,

which was resistance, relapse, death or second neoplasms, whichever occurred first (and

censored at last follow-up if no events occurred). EFS was estimated according to Kaplan-

Meier, and compared according to log-rank test. Cumulative incidence of relapse (CIR) at

5 years was estimated by adjusting for competing risks of other events and comparison

performed with the Gray test. (18) The multivariate Cox model on EFS and on the cause

specific hazard of relapse was applied to assess, with the Wald test, the impact of IKZF1

deletions, after accounting for the risk group, age and white blood cell count at diagnosis,

and the presence of P2RY8-CRLF2 aberration. The Cox model was also applied on each

variable separately (univariate analysis).

RESULTS AND DISCUSSION

IKZF1 deletions at diagnosis

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IKZF1 deletions were detected in 54/410 cases (13.2%), in keeping with incidence data

reported in the literature. (3, 13) In 25 cases (6.1%) the deletion was intra-genic, involving

only some exons of the IKZF1 gene, while in 29 cases (7.1%) the deletion was

encompassing the whole IKZF1 gene. In particular, we identified 9 cases with lack of

exons 4-7 ( Δ4-7), 3 cases with Δ2-8, 2 cases for each of the following deletions: Δ2-7, Δ4-

8, Δ1 -3, Δ2 -3 and single cases for: Δ1 -4, Δ4 -5, Δ4 -6, Δ6-8 and Δ2 (exon numbering is

according to ref.6) (Supplementary Table 2).

Clinical characteristics of patients are described in Table 1. The major difference regards

treatment response, with less patients with PPR in IKFZ1 deleted and less patients with

high MRD levels in non IKFZ1 deleted patients. The percentage of patients allocated to the

HR group however is the same (7%) in IKFZ1 deleted or not deleted patients. The relative

incidence of major deletions subgroups did not vary according to final risk group

assignment (Supplementary Table 2). Among the IKZF1 deletion positive patients one was

positive for the chromosomal translocation t(12;21), none was positive for t(4;11). Only 3

IKZF1 deleted cases carried also P2RY8-CRLF2 fusion (Table 1). The differences in the

incidence of double deleted cases as reported in this and other studies (2,3,5,7,11) is

probably due to the relatively low number of patients in all studies.

By MLPA technique we further analyzed the presence of copy number variations of other

genes frequently deleted in BCP-ALL patients, and known to be involved in lymphoid

development (PAX5, ETV6, EBF1) or in cell cycle regulation (CDKN2A/B, BTG1, RB1).

(10, 20-22) We did not find a statistically significant difference in the incidence of these

genetic alterations in children positive or negative for IKZF1 deletions (Supplementary

Table 3). Most of these genetic alterations occurred simultaneously in the same patients

and are described in detail in Supplementary Table 2. Twenty-five of the 54 IKZF1 deletion

positive patients, and in particular 7/9 patients carrying Δ4-7 deletion, resulted negative for

any additional tested alterations (Supplementary Table 2), although copy number

DOI: 10.3324/haematol.2012.075432

 

variations of exons not detected by MLPA assays cannot be excluded. Moreover,

aberrancies present in less than 20-50% of cells could not be detected due to the limited

sensitivity of the MLPA assay.

Prognostic impact of IKZF1 deletions

Compared to negative patients, the deletion of IKZF1 was associated to an inferior EFS

(70.2% (6.2) vs 85.2% (1.9) at 5 years, p-value=0.007) and a significantly higher CIR

(24.2% (5.9) vs. 13.1% (1.8) at 5 years, p-value= 0.049) (Figure 1A-B). The corresponding

survival figures at 5 years were: 87.0%(4.6) vs 93.0%(1.4) (p-value=0.10).

These data are in accordance with other studies reported in the literature, in particular with

the recent paper by Dorge et al. analysing ALL patients enrolled in AIEOP-BFM ALL2000

study in Germany (EFS 69% (5) vs 85% (1), p-value=<0.001 and CIR 21% (4) vs 10% (1),

p=0.001). (7)

The negative prognostic impact of IKZF1 deletions was retained, although without

statistical significance, when the favorable factor t(12;21) was excluded from the analysis

(Supplementary Figure 2 A-B) and when also patients with hyperdiploidy were excluded

(Supplementary Figure 2 C-D). Patients positive for Δ4-7 deletion, predicted to encode a

dominant-negative IKZF1 isoform, did not show a worse outcome (3/9 relapsed)

(Supplementary Table 2). We also analyzed the impact of IKZF1 deletions alone or in

combination with additional copy number abnormalities. Interestingly, only 3 out of 25

patients positive for IKZF1 deletion only relapsed vs. 10/28 when additional alterations

were present, pointing to a poor outcome when a major genetic instability was observed.

Specifically, 3/3 cases positive for both IKZF1 deletions and the P2RY8-CRLF2 fusion

relapsed (Supplementary Table 2), but the limited numbers do not allow to draw any

conclusion on a possible synergic effect of IKZF1 alterations with other coexistent

abnormalities.

DOI: 10.3324/haematol.2012.075432

      

The Cox model analysis was performed on the 410 patients to assess whether, after

adjusting for other relevant risk factors, IKZF1 deletions retained a prognostic impact on

EFS (table 2A) or on the specific hazard of relapse (table 2B). IKZF1 alterations were

significantly related to a higher rate of events (Hazard ratio on EFS of 1.87; 95% CI 1.05-

3.32, p-value=0.03) and, although not significantly, to a higher rate of relapse (Hazard ratio

1.7; 95% CI 0.9-3.18, p-value=0.1). More precisely, two deaths in induction and 1 death in

CCR (total n=3) occurred in the 54 IKFZ1 deleted patients versus 3 deaths in induction

and 2 in CCR and 1 second malignant neoplasm (total n=6) in the 356 non IKFZ1 deleted

patients. These events contributed to the significance of the statistical difference in the

EFS.

In both COX model analyses, P2RY8-CRLF2 aberration and risk group were significantly

associated with outcome. Of note, when individually analyzed, IKFZ1 deletion had a

statistically significant effect on EFS and relapse, in keeping with results in Figure 1 (A and

B).

We further analyzed the prognostic value of IKZF1 deletions within the subgroups

according to protocol stratification. IKZF1 deletions was less frequent within the Standard

Risk (SR) group, being found in 8 out 117 SR patients (6.8%), 42 out of 264 Intermediate

Risk (IR) patients (15.9%) and 4 out of 29 High Risk (HR) patients (13.8%) (Table 1).

Interestingly, none of the 8 IKZF1 deletions positive SR patients relapsed, vs 10/42 cases

(23.8%) stratified in the Intermediate Risk (IR) group and 3/4 cases in the HR group. In

particular, in the largest IR subgroup, IKZF1 deletions positive patients showed an inferior

EFS and a higher CIR compared to the negative patients, but the differences did not reach

statistical significance (EFS: 69.0%(7.2) vs. 82.2%(2.6), p-value=0.052; CIR: 23.9%(6.6)

vs. 16.5%(2.5), p-value=0.30 Figure 1C-D).

In summary, previous studies (12-14) reported that the presence of IKZF1 deletions is a

risk factor in childhood ALL and this finding was recently confirmed in Dorge et al (7) in the

DOI: 10.3324/haematol.2012.075432

 

framework of a BFM treatment strategy, also for patients so called at intermediate risk. Our

findings are substantially in keeping with those reported in (7), yet the value of including

IKZF1 deletions as a new marker for risk stratification is challenged by our results.

Indeed, overall EFS for patients with IKZF1 deletions, after excluding the confounding

effect of DS, T-immunophenotype and Ph+ patients, is around 70% at 5 years also in our

experience. The 3 patients with IKZF1 deletions who were at HR and relapsed had poor

response to treatment (high MRD levels) and accordingly were all eligible to transplant,

thus identification of IKZF1 deletions would not contribute to a better stratification. In the IR

group, with a 5-year EFS of 70%, treatment intensification could be justified to improve

results. In our context, the recent AIEOP-BFM ALL 2009 study, with a more intensive use

of L-asparaginase, might already provide a benefit that reduces the impact of IKZF1

deletion. This is especially true if we consider that in our data the difference in the

cumulative incidence of relapse is not so marked, being approximately 7% in IR and 11%

overall. This, as well as the lower number of events in the multivariate analysis, may

explain why the presence of IKZF1 deletions is an independent prognostic factor on EFS

but not on the hazard of relapse alone.

In conclusion, based on our data, the suitability of IKZF1 deletions as an additional

stratification marker for Ph- BCP-ALL patients remains questionable, at least until new

target therapy will be available.

DOI: 10.3324/haematol.2012.075432

  

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DOI: 10.3324/haematol.2012.075432

 

Table 1. Clinical features of the study cohort patients positive or negative for IKZF1 deletion.

IKZF1 deletions No Yes p-value N % N % All patients

356 54

GENDER Male Female

187 169

52.5 47.5

27 27

50.0 50.0

0.73

AGE 1-5 yrs 6-9 yrs 10-17 yrs

239

68 49

67.1 19.1 13.8

27 12 15

50.0 22.2 27.8

0.02

WBC(x1000/µl) <20 20-100 ≥100

254

79 23

71.4 22.2

6.5

39 12

3

72.2 22.2

5.6

0.97

Translocations t(4;11) Positive Negative Not known t(12;21) Positive Negative Not known

3 351

2

82 256

18

0.8 99.2

24.3 75.7

0 54

0

1 50

3

100.0

2.0 98.0

0.50

<0.001

Prednisone response Good Poor Not known

333

21 2

94.1

5.9

53

1 0

98.2

1.8

0.22

MRD SR IR HR Not known

116 162

2 76

41.4 57.9

0.7

8

27 3

16

21.1 71.0

7.9

<0.001

Final protocol strata SR IR HR

109 222

25

30.6 62.4

7.0

8

42 4

14.8 77.8

7.4

0.05

P2RY8-CRLF2 No Yes Not known

307

16 33

95.0

5.0

46

3 5

93.9

6.1

0.73

NCI criteria Standard High

267

89

75.0 25.0

34 20

63.0 37.0

0.06

DNA index ≥1.16 and <1.6 <1.16 or ≥1.6 Not known

76

255 25

23.0 77.0

5

44 5

10.2 89.8

0.04

 

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Pagina 16 di 17  

 Table 2. Results of the univariate and multivariate analyses. Cox model on EFS (hazard of

first event among resistance, relapse, death, second malignant neoplasm) and the hazard of

relapse in 410 patients.

2a: ANALYSIS ON EFS

UNIVARIATE ANALYSIS MULTIVARIATE ANALYSIS

Characteristics Hazard ratio 95% CI P-value Hazard

ratio 95% CI P-value

IKZF1 No Yes

2.1

1.21-3.68

0.009

1

1.87

1.05-3.32

0.03 P2RY8-CRLF2 No Yes Not Known

3.14 1.36

1.49-6.60 0.65-2.86

0.003 0.42

1

3.25 1.37

1.54-6.85 0.65-2.89

0.002 0.42

AGE 1-9 years 10-17 years

1.87

1.08-3.23

0.02

1

1.69

0.97-2.93

0.06 WBC (X1000/µl) <100 ≥100

2.31

1.10-4.82

0.03

1

2.19

0.97-4.92

0.06 FINAL RISK Standard risk Intermediate risk High risk

2.78 5.15

1.37-5.63 2.04-12.98

0.005 <0.001

1

2.60 4.30

1.27-5.33 1.64-11.23

0.009 0.003

WBC, White Blood Cell count; MRD, Minimal Residual Disease 2b: ANALYSIS ON RELAPSE

UNIVARIATE ANALYSIS MULTIVARIATE ANALYSIS

Characteristics Hazard ratio 95% CI P-value Hazard

ratio 95% CI P-value

IKZF1 No Yes

1.94

1.05-3.58

0.03

1

1.70

0.90-3.18

0.10 P2RY8-CRLF2 No Yes Not Known

3.76 1.40

1.78-7.98 0.63-3.10

<0.001 0.41

1

3.73 1.47

1.75-7.93 0.69-3.26

<0.001 0.35

AGE 1-9 years 10-17 years

1.59

0.86-2.94

0.14

1

1.45

0.78-2.70

0.24 WBC (X1000/µl) <100 ≥100

1.28

0.46-3.52

0.64

1

1.27

0.43-3.71

0.67 FINAL RISK Standard risk Intermediate risk High risk

2.48 3.54

1.21-5.06 1.26-9.94

0.01 0.02

1

2.33 3.46

1.13-4.81 1.19-10.01

0.02 0.02

WBC, White Blood Cell count; MRD, Minimal Residual Disease

DOI: 10.3324/haematol.2012.075432

 

LEGEND TO FIGURES

Figure 1. Association of IKZF1 deletions to treatment outcome.

(A) EFS and (B) CIR of study cohort patients according to the presence or absence of IKZF1 deletions. (C) EFS and (D) CIR of IR patients according to the presence or absence of IKZF1 deletions.

DOI: 10.3324/haematol.2012.075432

DOI: 10.3324/haematol.2012.075432

Supplementary Design and Methods

Protocol stratification

Patient risk groups were defined as follows. The HR group included patients with any of

the following criteria: t(4;11) or MLL/AF4; prednisone poor response (≥ 1,000 blasts/µL on

day 8 peripheral blood after 7 days of prednisone and one dose of intrathecal

methotrexate on day 1); inability to achieve clinical remission after Induction Phase IA;

high burden (≥ 10-3) of PCR-Minimal Residual Disease (MRD) at day 78. The SR group

included patients who lacked high-risk criteria and tested negative to PCR-MRD performed

by using two sensitive markers (≥ 1×10-4) at both day 33 and day 78. The IR group

included the remaining patients, and those not evaluated by PCR-MRD.

PCR-MRD

PCR-MRD was detected by RQ-PCR of Immunoglobulin and/or T-cell receptor gene

rearrangements in bone marrow samples collected at the end of the TP1 (day 33), and

TP2 (day 78) induction phases; (17) data were interpreted according to EuroMRD

published guidelines (van der Velden VHJ, Cazzaniga G, Schrauder A, Hancock J, Bader

P, Panzer-Grumayer ER et al. On behalf of the European Study Group on MRD detection

in ALL (ESG-MRD-ALL). Analysis of minimal residual disease by Ig/TCR gene

rearrangements: Guidelines for interpretation of real-time quantitative PCR data. Leukemia

2007; 21: 604-611).

DOI: 10.3324/haematol.2012.075432

Supplementary Tables

Supplementary Table 1. Clinical features of the study cohort patients versus not investigated patients.

Analyzed for IKZF1

Not analyzed for IKZF1 p-value

N % N % All patients

410 472

GENDER Male Female

214 196

52.2 47.8

252 220

53.4 46.6

0.72

AGE 1-5 yrs 6-9 yrs 10-17 yrs

266 80 64

64.9 19.5 15.6

292 96 84

61.9 20.3 17.8

0.60

WBC(x1000/µl) <20 20-100 ≥100

293 91 26

71.5 22.2 6.3

338 107 27

71.6 22.7 5.7

0.92

Translocations t(4;11) Positive Negative Not known t(12;21) Positive Negative Not known

3 405

2

83 306 21

0.7 99.3

21.3 78.7

3 413 56

68

338 66

0.7 99.3

16.7 83.3

0.98

0.10

Prednisone response Good Poor Not known

386 22

2

94.6 5.4

425 47

0

90.0 10.0

0.01

MRD SR IR HR Not known

124 189

5 92

39.0 59.4 1.6

134 214 37 87

34.8 55.6 9.6

<0.001

Final protocol strata SR IR HR

117 264 29

28.5 64.4 7.1

129 266 77

27.3 56.4 16.3

<0.001

NCI criteria Standard High

301 109

73.4 26.6

338 134

71.6 28.4

0.55

DNA index ≥1.16 and <1.6 <1.16 or ≥1.6 Not known

81

299 30

21.3 78.7

110 330 32

25.0 75.0

0.21

DOI: 10.3324/haematol.2012.075432

Supplementary Table 2. Additional genetic alterations in patients positive for IKZF1 deletions.

IKZF1 CDKN2A/B PAX5 ETV6 BTG1 RB1 EBF1 P-CRLF2Pt. #1 ∆ 1-8 pos pos neg pos neg neg neg IR yesPt. #2 ∆ 1-8 neg neg pos neg neg neg neg IR noPt. #3 ∆ 1-8 pos neg neg neg neg neg nd IR noPt. #4 ∆ 1-8 neg neg pos neg neg neg neg IR noPt. #5 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #6 ∆ 1-8 neg neg pos neg neg pos neg HR noPt. #7 ∆ 1-8 pos pos pos neg neg neg neg SR noPt. #8 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #9 ∆ 1-8 pos pos neg pos neg neg neg IR no

Pt. #10 ∆ 1-8 neg neg pos neg neg neg neg HR yesPt. #11 ∆ 1-8 neg neg pos neg neg neg nd IR noPt. #12 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #13 ∆ 1-8 neg neg pos neg neg neg nd IR noPt. #14 ∆ 1-8 neg neg pos neg neg neg neg SR noPt. #15 ∆ 1-8 neg neg pos neg neg neg nd IR noPt. #16 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #17 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #18 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #19 ∆ 1-8 pos pos pos neg neg neg neg IR noPt. #20 ∆ 1-8 neg neg neg neg neg neg neg SR noPt. #21 ∆ 1-8 pos pos neg neg neg neg neg IR noPt. #22 ∆ 1-8 neg neg neg neg neg neg neg IR yesPt. #23 ∆ 1-8 neg neg neg neg neg neg neg IR yesPt. #24 ∆ 1-8 pos neg neg neg neg neg neg IR noPt. #25 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #26 ∆ 1-8 pos pos neg neg neg neg neg IR noPt. #27 ∆ 1-8 neg neg pos neg neg neg neg IR noPt. #28 ∆ 1-8 neg neg neg neg neg neg neg IR noPt. #29 ∆ 1-8 neg neg pos neg neg neg neg IR yesPt. #30 ∆ 1-3 neg pos pos neg neg neg neg SR noPt. #31 ∆ 1-3 neg neg pos neg neg neg neg IR noPt. #32 ∆ 1-4 neg neg neg neg neg neg neg IR noPt. #33 ∆ 2 neg neg neg neg neg neg neg IR noPt. #34 ∆ 2-3 neg neg pos neg neg neg neg IR noPt. #35 ∆ 2-3 pos pos neg pos neg neg neg IR yesPt. #36 ∆ 2-7 neg neg neg pos neg neg pos IR yesPt. #37 ∆ 2-7 neg neg neg neg neg neg neg IR noPt. #38 ∆ 2-8 pos neg neg neg neg neg neg SR noPt. #39 ∆ 2-8 neg neg neg neg neg neg nd HR yesPt. #40 ∆ 2-8 neg neg neg neg neg neg pos IR yesPt. #41 ∆ 4-5 neg neg neg neg neg neg neg IR noPt. #42 ∆ 4-6 neg neg neg neg neg neg neg SR noPt. #43 ∆ 4-7 neg neg neg neg neg neg neg IR yesPt. #44 ∆ 4-7 neg neg neg neg neg neg neg IR noPt. #45 ∆ 4-7 neg neg neg neg neg neg neg SR noPt. #46 ∆ 4-7 neg neg neg neg neg neg neg IR noPt. #47 ∆ 4-7 neg neg neg neg neg neg neg IR noPt. #48 ∆ 4-7 pos pos neg neg neg neg neg IR yesPt. #49 ∆ 4-7 neg neg neg neg neg neg neg IR noPt. #50 ∆ 4-7 pos pos neg neg neg neg pos IR yesPt. #51 ∆ 4-7 neg neg neg neg neg neg neg IR noPt. #52 ∆ 4-8 neg neg neg neg neg neg neg IR noPt. #53 ∆ 4-8 neg neg neg pos neg neg neg HR yesPt. #54 ∆ 6-8 neg neg neg neg neg neg neg SR no

Relapse

P-CRLF2 , P2RY8-CRLF2 .

Final RiskPt.Deletions

DOI: 10.3324/haematol.2012.075432

Supplementary Table 3.

Characteristics P-value

IKZF1 deletions

No Yes

N % N %

All patients 356 100,00 54 100,00 CDKN2A/B deletion 0,96

No 278 78,1 42 77,8 Yes 78 21,9 12 22,2

PAX5 deletion 0,24 No 311 87,4 44 81,5 Yes 45 12,6 10 18,5

ETV6 deletion 0,08 No 293 82,3 39 72,2 Yes 63 17,7 15 27,8

BTG1 deletion 0,37 No 335 94,1 49 90,7 Yes 21 5,9 5 9,3

RB1 deletion 0,06 No 331 93,0 54 100,0 Yes 25 7,0 0 0,0

EBF1 deletion 0,99 No 350 98,3 53 98,2 Yes 6 1,7 1 1,8

DOI: 10.3324/haematol.2012.075432

Supplementary Figures

Supplementary Figure 1 . Treatment outcome of study cohort. EFS between patients

included and non-included in the study cohort.

EF

S

0.0

0.2

0.4

0.6

0.8

1.0

YEARS FROM DIAGNOSIS

0 1 2 3 4 5

472N. pts

94N. events

80.2%(1.9)5 yrs EFS

not analysed

p-value=0.23

410

N. pts

70

N. events

83.2%(1.9)

5 yrs EFS

analysed

p-value=0.23

DOI: 10.3324/haematol.2012.075432

Supplementary Figure 2. Association of IKZF1 deletions to treatment outcome in the

absence of the favorable prognostic factors t(12;21) or hyperdiploidy. Event Free

survival, EFS (A, C) and Cumulative Incidence of Relapse, CIR (B, D) of the study cohort

for the presence or absence of IKZF1 deletions, excluding t(12;21) positive (A, B) or

hyperdiploid patients, respectively.

0.0

0.2

0.4

0.6

0.8

1.0

YEARS FROM DIAGNOSIS

0 1 2 3 4 5

256 N. pts

47N. events

81.8%(2.4)5 yrs EFS

NEG

p-value=0.06

50

N. pts

15

N. events

69.8%(6.5)

5 yrs EFS

POS

p-value=0.06

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

YEARS FROM DIAGNOSIS

0 1 2 3 4 5

256N. pts

42N. rel.

16.3%(2.3) 5 yrs Cum. Incidence

NEG

p-value=0.23

50

N. pts

12

N. rel.

24.2%(6.1)

5 yrs Cum. Incidence

POS

p-value=0.23

0.0

0.2

0.4

0.6

0.8

1.0

YEARS FROM DIAGNOSIS

0 1 2 3 4 5

165 N. pts

33N. events

80.4%(3.1)5 yrs EFS

NEG

p-value=0.20

40

N. pts

12

N. events

69.9%(7.3)

5 yrs EFS

POS

p-value=0.20

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

YEARS FROM DIAGNOSIS

0 1 2 3 4 5

165N. pts

28N. rel.

16.6%(2.9) 5 yrs Cum. Incidence

NEG

p-value=0.29

40

N. pts

10

N. rel.

25.1%(6.9)

5 yrs Cum. Incidence

POS

p-value=0.29

A B

C D

EF

SE

FS

CIR

CIR

DOI: 10.3324/haematol.2012.075432