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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: [email protected]
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
DOI: 10.3324/haematol.2012.075432
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
REFERENCES
1. Pui CH, Carroll WL, Meshinchi S, Arceci RJ. Biology, risk stratification, and therapy
of pediatric acute leukemias: an update. J Clin Oncol. 2011;29(5):551-65.
2. Russell LJ, Capasso M, Vater I, Akasaka T, Bernard OA, Calasanz MJ, et al.
Deregulated expression of cytokine receptor gene, CRLF2, is involved in lymphoid
transformation in B-cell precursor acute lymphoblastic leukemia. Blood.
2009;114(13):2688-98.
3. Mullighan CG, Collins-Underwood JR, Phillips LA, Loudin MG, Liu W, Zhang J, et
al. Rearrangement of CRLF2 in B-progenitor- and Down syndrome-associated acute
lymphoblastic leukemia. Nat Genet. 2009;41(11):1243-6.
4. Den Boer ML, van Slegtenhorst M, De Menezes RX, Cheok MH, Buijs-Gladdines
JG, Peters ST, et al. A subtype of childhood acute lymphoblastic leukaemia with poor
treatment outcome: a genome-wide classification study. Lancet Oncol. 2009;10(2):125-34.
5. Mullighan CG, Su X, Zhang J, Radtke I, Phillips LA, Miller CB, et al. Deletion of
IKZF1 and prognosis in acute lymphoblastic leukemia. N Engl J Med. 2009;360(5):470-80.
6. Iacobucci I, Storlazzi CT, Cilloni D, Lonetti A, Ottaviani E, Soverini S, et al.
Identification and molecular characterization of recurrent genomic deletions on 7p12 in the
IKZF1 gene in a large cohort of BCR-ABL1-positive acute lymphoblastic leukemia patients:
on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working
Party (GIMEMA AL WP). Blood. 2009;114(10):2159-67.
7. Dorge P, Meissner B, Zimmermann M, Moericke A, Schrauder A, Bourquin JP, et al.
IKZF1 deletion is an independent predictor of outcome in pediatric acute lymphoblastic
leukemia treated according to the ALL-BFM 2000 protocol. Haematologica. 2012 Aug 8.
[Epub ahead of print].
DOI: 10.3324/haematol.2012.075432
8. Mullighan CG, Miller CB, Radtke I, Phillips LA, Dalton J, Ma J, et al. BCR-ABL1
lymphoblastic leukaemia is characterized by the deletion of Ikaros. Nature.
2008;453(7191):110-4.
9. Buitenkamp TD, Pieters R, Gallimore NE, van der Veer A, Meijerink JP, Beverloo
HB, et al. Outcome in children with Down's syndrome and acute lymphoblastic leukemia:
role of IKZF1 deletions and CRLF2 aberrations. Leukemia. 2012;26:2204-11.
10. Mullighan CG, Goorha S, Radtke I, Miller CB, Coustan-Smith E, Dalton JD, et al.
Genome-wide analysis of genetic alterations in acute lymphoblastic leukaemia. Nature.
2007;446(7137):758-64.
11. Harvey RC, Mullighan CG, Chen IM, Wharton W, Mikhail FM, Carroll AJ, et al.
Rearrangement of CRLF2 is associated with mutation of JAK kinases, alteration of IKZF1,
Hispanic/Latino ethnicity, and a poor outcome in pediatric B-progenitor acute
lymphoblastic leukemia. Blood. 2010;115(26):5312-21.
12. Waanders E, van der Velden VH, van der Schoot CE, van Leeuwen FN, van
Reijmersdal SV, de Haas V, et al. Integrated use of minimal residual disease classification
and IKZF1 alteration status accurately predicts 79% of relapses in pediatric acute
lymphoblastic leukemia. Leukemia. 2011;25(2):254-8.
13. Kuiper RP, Waanders E, van der Velden VH, van Reijmersdal SV, Venkatachalam
R, Scheijen B, et al. IKZF1 deletions predict relapse in uniformly treated pediatric
precursor B-ALL. Leukemia. Jul;24(7):1258-64.
14. Yang YL, Hung CC, Chen JS, Lin KH, Jou ST, Hsiao CC, et al. IKZF1 deletions
predict a poor prognosis in children with B-cell progenitor acute lymphoblastic leukemia: a
multicenter analysis in Taiwan. Cancer Sci. 2011;102(10):1874-81.
15. Chen IM, Harvey RC, Mullighan CG, Gastier-Foster J, Wharton W, Kang H, et al.
Outcome modeling with CRLF2, IKZF1, JAK, and minimal residual disease in pediatric
DOI: 10.3324/haematol.2012.075432
acute lymphoblastic leukemia: a Children's Oncology Group study. Blood.
2012;119(15):3512-22.
16. Sun L, Liu A, Georgopoulos K. Zinc finger-mediated protein interactions modulate
Ikaros activity, a molecular control of lymphocyte development. Embo J.
1996;15(19):5358-69.
17. Conter V, Bartram CR, Valsecchi MG, Schrauder A, Panzer-Grumayer R, Moricke
A, et al. Molecular response to treatment redefines all prognostic factors in children and
adolescents with B-cell precursor acute lymphoblastic leukemia: results in 3184 patients of
the AIEOP-BFM ALL 2000 study. Blood. 2010;115(16):3206-14.
18. Palmi C, Vendramini E, Silvestri D, Longinotti G, Frison D, Cario G, et al. Poor
prognosis for P2RY8-CRLF2 fusion but not for CRLF2 over-expression in children with
intermediate risk B-cell precursor acute lymphoblastic leukemia. Leukemia.
2012;26(10):2245-53.
19. van Dongen JJ, Macintyre EA, Gabert JA, Delabesse E, Rossi V, Saglio G, et al.
Standardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in
acute leukemia for detection of minimal residual disease. Report of the BIOMED-1
Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia.
1999;13(12):1901-28.
20. Krentz S, Hof J, Mendioroz A, Vaggopoulou R, Dorge P, Lottaz C, et al. Prognostic
value of genetic alterations in children with first bone marrow relapse of childhood B-cell
precursor acute lymphoblastic leukemia. Leukemia. 2012 Jun 13. [Epub ahead of print].
21. Strefford JC, Worley H, Barber K, Wright S, Stewart AR, Robinson HM, et al.
Genome complexity in acute lymphoblastic leukemia is revealed by array-based
comparative genomic hybridization. Oncogene. 2007;26(29):4306-18.
22. Kuiper RP, Schoenmakers EF, van Reijmersdal SV, Hehir-Kwa JY, van Kessel AG,
van Leeuwen FN, et al. High-resolution genomic profiling of childhood ALL reveals novel
DOI: 10.3324/haematol.2012.075432
recurrent genetic lesions affecting pathways involved in lymphocyte differentiation and cell
cycle progression. Leukemia. 2007;21(6):1258-66.
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
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