clinico-biologic features of 5202 acute lymphoblastic ... features of 5202 acute lymphoblastic...

Clinico-biologic features of 5202 acute lymphoblastic leukemiapatients enrolled in the Italian AIEOP and GIMEMA Protocols andstratified in age-cohorts

by Sabina Chiaretti, Antonella Vitale, Giovanni Cazzaniga, Sonia Maria Orlando,Daniela Silvestri, Paola Fazi, Maria Grazia Valsecchi, Loredana Elia, Anna Maria Testi, Francesca Mancini, Valentino Conter, Geertruy te Kronnie,Felicetto Ferrara, Francesco Di Raimondo, Alessandra Tedeschi, Giuseppe Fioritoni,Francesco Fabbiano, Giovanna Meloni, Giorgina Specchia, Giovanni Pizzolo, Franco Mandelli, Anna Guarini, Giuseppe Basso, Andrea Biondi, and Robin Foa'

Haematologica 2013 [Epub ahead of print]

Citation: Chiaretti S, Vitale A, Cazzaniga G, Orlando SM, Silvestri D, Fazi P, Valsecchi MG, Elia L, Testi AM, Mancini F, Conter V, Te Kronnie G, Ferrara F, Di Raimondo F, Tedeschi A, Fioritoni G, Fabbiano F, Meloni G, Specchia G, Pizzolo G,Mandelli F, Guarini A, Basso G, Biondi A, and Foa' R. Clinico-biologic features of 5202acute lymphoblastic leukemia patients enrolled in the Italian AIEOP and GIMEMAProtocols and stratified in age-cohorts. Haematologica. 2013; 98:xxx doi:10.3324/haematol.2012.080432

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1

Clinico-biologic features of 5202 acute lymphoblastic leukemia patients enrolled in the Italian

AIEOP and GIMEMA Protocols and stratified in age-cohorts

Short title for the running head: ALL features in different age cohorts

Sabina Chiaretti1, Antonella Vitale1, Gianni Cazzaniga2, Sonia Maria Orlando3, Daniela Silvestri4,

Paola Fazi3, Maria Grazia Valsecchi4, Loredana Elia1, Anna Maria Testi1, Francesca Mancini1,

Valentino Conter5, Geertruy te Kronnie6, Felicetto Ferrara7, Francesco Di Raimondo8,

Alessandra Tedeschi9, Giuseppe Fioritoni10, Francesco Fabbiano11, Giovanna Meloni1,

Giorgina Specchia12, Giovanni Pizzolo13, Franco Mandelli14, Anna Guarini1, Giuseppe Basso6,

Andrea Biondi2,4, and Robin Foà1

1Division of Hematology, Department of Cellular Biotechnologies and Hematology, “Sapienza”

University of Rome, Italy; 2Tettamanti Research Center, Pediatric Clinic, University of Milano-

Bicocca, Milan, Italy; 3GIMEMA Data center, GIMEMA Foundation, Rome, Italy; 4Medical

Statistics Unit, Department of Clinical Medicine and Prevention, University of Milano-Bicocca,

Milan, Italy; 5Department of Pediatrics, Ospedali Riuniti di Bergamo, Bergamo, Italy; 6Pediatric

Onco-hematology Clinic, University of Padua, Azienda Ospedaleria Universitaria, Padova, Italy; 7Division of Haematology and Stem Cell Transplantation Unit, Cardarelli Hospital, Naples, Italy; 8Department of Clinical and Molecular Bio-Medicine, Section of Hematology, Oncology and

Clinical Pathology, University of Catania, Italy; 9Department of Transfusion Medicine and

Division of Hematology, Niguarda Ca’ Granda Hospital, Milan, Italy; 10Department of

Hematology, Civic Hospital, Pescara, Italy; 11Institute of Hematology, Ospedali Riuniti Villa Sofia-

Cervello Hospital, Palermo, Italy; 12Hematology Section, Department of Pathological Anatomy,

University of Bari, Italy; 13Department of Medicine, Haematology Section, University Hospital of

Verona, Italy; 14GIMEMA, GIMEMA Foundation, Rome, Italy

Correspondence

Robin Foà, Division of Hematology, “Sapienza” University of Rome, Phone: +39-06-85795753;

Fax: +39-06-85795792. E-mail: [email protected]/[email protected]

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Funding

This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) Special

Program Molecular Clinical Oncology, 5 x 1000, Milan, Italy; Ministero dell’Università e Ricerca

(MIUR), Fondo per gli Investimenti della Ricerca di Base (FIRB), Rome, Italy; Associazione

Italiana per la Ricerca sulla leucemia (AIL), Italy; Compagnia di San Paolo, Turin, Italy; Progetto

“Oncologia”, Ministero della Salute, Rome, Italy; Fondazione Cariplo, Milano, Italy, ENCCA

(European network of Cancer in Children and Adolescents).

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ABSTRACT

Acute lymphoblastic leukemia shows marked differences in outcome between children and adults.

Since there is limited information on the distribution of clinico-biologic variables in different age

cohorts, we analyzed 5202 acute lymphoblastic leukemia patients enrolled in the Italian multicenter

AIEOP and GIMEMA protocols and stratified them in 9 age cohorts. The highest prevalence of

acute lymphoblastic leukemia was observed in children, although a second peak was recorded from

the 4th decade onwards. Interestingly, the lowest incidence was found in females between 14-40

years. Immunophenotypic characterization showed a B-lineage in 85.8% of patients: a pro-B stage,

associated to MLL/AF4 positivity, was more frequent in patients between 10-50 years. A T-lineage

(14.2%) was rare among small children and increased in 10-40 years patients. The BCR/ABL1

rearrangement increased progressively with age, starting from the 10-14 cohort and impacting for

52.7% of cases in the 6th decade. Similarly, the MLL/AF4 rearrangement constantly increased up to

the 5th decade, while the ETV6/RUNX1 rearrangement disappeared from the age of 30 onwards.

This study shows that adolescents and young adults are characterized by a male prevalence, greater

T-lineage acute lymphoblastic leuekmia percentage, an increase of poor prognostic molecular

markers with aging compared to children, and conclusively quantifies the progressive increase with

age of BCR/ABL+ patients, potentially manageable by targeted therapies.

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INTRODUCTION

Acute lymphoblastic leukemia (ALL) is the most frequent neoplasm in children, whereas is

relatively rare in adults. Over the last decades, there has been a considerable improvement in the

outcome of children, with complete remission (CR) and long-term survival rates reaching 95% and

80% (1, 2), respectively. Contrariwise, in adults the survival rates generally do not exceed 40% (1,

4-6).

Several factors can explain this marked difference, including the more intensive regimens used in

children, comprising high dose methotrexate, asparaginase and reinduction therapy, less toxic

effects, a greater compliance to high doses of chemotherapy, and an increased “physician and parent

compliance” of pediatric oncologist/hematologists wards (7,8). As a proof of principle, several

studies (9-15) showed a superior outcome for adolescents and young adults (AYA) treated with

pediatric-like regimens. In adults, the most important risk factors rely on age and white blood cell

count (WBC), a pro-B ALL and poor prognosis molecular markers (6).

Some biologic characteristics are partly responsible for the different clinical scenario, like the

increased incidence of the BCR/ABL1 (16, 17) and MLL/AF4 transcripts, negatively impacting on

prognosis, and a decreased incidence of ETV6/RUNX1 rearrangement, associated with a favorable

outcome, in older patients (18). Only few studies have focused on a detailed analysis of the clinico-

biologic features among various age cohorts and usually took into account only few age groups in

heterogeneous populations.

In this study, we retrospectively evaluated the clinical-biological features of 5202 ALL patients,

enrolled in the Italian multicenter protocols AIEOP (Associazione Italiana di Ematologia ed

Oncologia Pediatrica) and GIMEMA (Gruppo Italiano Malattie EMatologiche dell’Adulto)

protocols for the treatment of pediatric and adult ALL. All patients were uniformly characterized at

presentation. Although comparison of outcome in the various age cohorts considered was not a

purpose of this study, event-free survival (EFS) was not different from that previously reported (2-

5): it decreased proportionally with aging and was below 50% in patients > 18 years, who, at the

time of data collection, were treated with non-intensive regimens (Supplementary Figure 1).

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METHODS

Patients’ enrollment

Between April 1995 and June 2009, 5202 patients were included in the Italian pediatric (AIEOP)

and adult (GIMEMA) multicenter protocols, and evaluated for clinico-biologic features at

presentation. Of 3753 children, 1711 were enrolled in the ALL 95 and 2042 in the ALL 2000

AIEOP trials - designed for children with an age between 1-18 years; of 1449 adults, 457 were

enrolled in the GIMEMA LAL 0496 (age 14-60 years), 565 in the GIMEMA LAL 2000 (age 14-60

years), 385 in the GIMEMA LAL 0904 (age 15-60 years), and 42 in the GIMEMA LAL 1205

protocols; the latter protocol, enrolling patients >18 years harboring the BCR/ABL1 rearrangement,

was included because partly simultaneous to the LAL 0904; these trials were previously approved

by the local ethical committees.

All patients, parents or guardians gave their informed consent to blood/marrow collection and to

biologic analyses, in agreement with the Declaration of Helsinki.

For this study, patients were stratified into 9 age cohorts: 1-5, 5-10, 10-14, 14-18, 18-25, 25-30, 30-

40, 40-50, 50-60 years. Infants were excluded from the analysis.

Clinico-biologic features

Clinical parameters included gender, WBC count, platetet (Plt) count and hemoglobin (Hgb) levels,

mediastinal, spleen and liver enlargement, and central nervous system (CNS) involvement.

For WBC, Plt counts and Hgb levels, the following cut-points were considered: 50 x 109/L, 100 x

109/L and 10 g/dL, respectively.

Mediastinum, spleen and liver were considered enlarged if > 3 cm. CNS involvement was defined

as described (19).

ALL diagnosis was performed on May-Grumwald-Giemsa smears and immunophenotype: the latter

allowed defining the lineage derivation and degree of differentiation of the leukemic cells. The cut-

off for positivity was ≥ 20% for surface and 10% for intracytoplasmatic antigens.

B-lineage ALL (B-ALL) cases were subdivided into B1 (pro-B ALL, CD10-), B2 (common-ALL,

CD10+ and intracytoplasmic (cy) Igµ−) and B3 (pre-B ALL, CD10+ and cyIgµ+), and NC if not

further classified cases (cyIgµ not tested) (20).

T-lineage ALL (T-ALL) cases were subdivided into T1 (pro-T and pre-T ALL, cyCD3+ and

CD7+,CD2+ and/or CD5+ and/or CD8+, respectively); T2 (cortical T-ALL, CD1a+) and T3

(mature T-ALL, surface CD3+ and CD1a-) (20).

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Molecular analysis of adults (21) included BCR/ABL1, E2A/PBX1, ETV6/RUNX1 and MLL

rearrangements (i.e. MLL/AF4 and MLL/ENL) screening for B-ALL patients, and BCR/ABL1 and

MLL rearrangements for T-ALL cases; SIL/TAL1, NUP298/RAP and NUP214/ABL1 were

investigated only in more recent trials, therefore, since data were not consistently available, were

not considered.

Children were screened for BCR/ABL1, ETV6/RUNX1, MLL/AF4 and partly for E2A/PBX1 (22).

Since karyotyping data were not uniformly available, this parameter was not considered.

Flow-cytometry and molecular analyses were centrally performed in 2 laboratories, one for

pediatric and the other for adult cases.

Statistical analysis

Patients’ characteristics were summarized by cross-tabulations (categorical variables) and quantiles

(ordinal factors). Non-parametric tests, as appropriate, were applied for comparisons between

groups (Pearson's χ2, Mantel-Haenszel χ2or Fisher Exact test for categorical variables, Mann-

Whitney or Kruskal-Wallis test for continuous variables). Analyses were performed using the SAS

v9.2 software; all tests were two-sided, accepting p ≤0.05 as statistically significant.

RESULTS

Incidence of ALL

The ALL distribution among the different age cohorts was evaluated (Figure 1A). The majority of

ALL cases was included within the 1-5 year age cohort (37% of the whole cohort) and

progressively decreased up to the 3rd decade; however, a slight increase in ALL was again recorded

starting from the 4th decade onwards (> 5% in the 30-40, 40-40 and 50-60 age groups).

Immunophenotye

Flow cytometry analysis revealed an overall prevalence of B-ALL in the whole cohort (85.8%),

while T-ALL was much less frequent (14.2%). The distribution of B- and T-ALL was of interest

(Table 1A, Figure 1B). An increase in the incidence of T-ALL was observed from the 10-14 age

cohort up to the 4th decade of life, with a tendency to decline thereafter. Conversely, B-ALLs were

less frequent in the same age framework (p<.0001).

When looking at the differentiation stage of B-ALL, the most significant (p<0.0001) finding was a

constant and significant increase in the percentage of pro-B up to the 5th decade of life: in small

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children (age cohorts 1-5 and 5-10 years) the incidence of pro-B ALL was 3.27% and 3.88%, in

older children (10-14 years) and adolescents (14-18 years) it was 8.9% and 10.5%, while in adults it

was 17.58%, 13.92%, 17.89% and 18.78% for the 18-25, 25-30, 30-40, 40-50 age cohorts,

respectively (Supplementary Figure 2).

In T-ALL, a peak of pro-T/ pre-T ALL cases was recorded in the 5th and 6th decade, with an

incidence of 68.18% and 58.33% of cases, respectively; consistently, the percentage of cortical T-

ALL was very low in adults and young elderly (Supplementary Figure 3).

Gender distribution

The cohort evaluated included 2889 males and 2313 females, with a M/F ratio of 1.25. Gender

distribution was of interest: in fact, while there was an overall prevalence of the male gender in

almost all age cohorts, this phenomenon was particularly evident between the age of 14 and 40,

started to revert in the 5th decade of life and the females were prevalent over the age of 50 (p<.0001;

Figure 2A). Furthermore, a striking association was found between lineage derivation and gender,

T-ALL being more frequently diagnosed in male subjects: this association was statistically

significant up to the 4th decade of life (Figures 1B, 2A and 2B).

Hematologic parameters and correlation with immunophenotype

A WBC count >50 x 109/L was significantly less frequent in children, being recorded in 18.87%

and 16.34% of patients aged 1-5 and 5-10, while it progressively increased from pre-adolescence

onwards (p<.0001); a Plt count <100 x 109/L was more frequently detected in patients older than the

age of 30 years (p<.0001); finally, Hgb levels <10g/dL were more frequently detected in younger

patients and even more in small children (1-5 years, p<.0001) (Supplementary Figure 4A, Table

1B).

A WBC count >50 x 109/L was more frequently recorded in T-ALL than in B-ALL patients (53.41

% vs 16.56%, p<.0001), regardless of the age cohorts considered. However, in B-ALL a WBC

count >50 x 109/L was relatively rare up to 25 years, while it was more recurrent form 25 years

onwards. At variance, in T-ALL the scenario was almost opposite, with a WBC count >50 x 109/L

detected in > 45% of patients aged <30 years, and tended to decrease thereafter (p<.0001,

Supplementary Figure 4B).

Finally, a significantly higher percentage of cases with Plt count <100 x 109/L and Hgb levels

<10g/dL was observed in B-ALL compared to T-ALL (Plt count 70.12% vs 64.94% and Hgb levels

77.14% vs 42.94%; p=0.0053 and p<.0001, respectively, Supplementary Figure 4C and 4D).

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Organ involvement

Organ involvement was considered; since reactive adenopathies are a frequent event in healthy

children, this parameter was not taken into account.

Mediastinal, spleen and liver enlargements were prevalently recorded in T-ALL (p<.0001). The first

two were more frequently detected between 10 and 25 years (p<.0001 and p=0.028, respectively;

Table 1B, Supplementary Table 1, Figure 3A and 3B), while liver enlargement decreased with age

(p<.0001; Table 1B, Supplementary Table 1 and Figure 3C).

Finally, CNS involvement was a rare event (2%) and was more frequently detected in T-ALL rather

than in B-ALL (5.95% vs 1.47%, p<0.0001); furthermore, in T-ALL, , it was associated with

hyperleukocytosis (p<0.0001). When patients were stratified according to age, a peak of CNS

positivity incidence was recorded in the groups aged 10-14, 25-30 and 50-60 (3.91%, 5% and

4.91%, respectively). Interestingly, if patients were stratified for both lineage derivation and age, in

T-ALL CNS positivity was a more frequent event in children, particularly in the age groups 5-10,

10-14 and 14-18 (8.67, 10.34 and 5.41%, respectively), and then progressively decreased, whereas

in B-ALL meningosis was more frequent in older patients, particularly in the 25-30 (5.48%) and 50-

60 age groups (5.56%) (Table 1B, Supplementary Table 1 and Figure 3D): notably, in the latter age

group, a significant association was found with the BCR/ABL1 rearrangement (10% of BCR/ ABL1

had a CNS involvement, p=0.028).

Molecular analysis in B-ALL

The overall frequency of molecular aberrations is reported in Table 1C and Figure 4.

ETV6/RUNX1 represented the most frequent alteration in small children (age cohorts 1-5: 26.14%;

5-10: 23.96%), being detected in more than 20% of cases; its incidence progressively decreased in

pre-adolescents, adolescents and young adults (age cohorts 10-14: 7.04%; 14-18: 4.71%; 18-25:

1.49%; 25-30: 1.41%), and disappeared in adults from 30 years onwards (p<.0001).

The E2A/PBX1 aberration was relatively infrequent, being detected in ≤7% of cases in all age-

cohorts. Its detection did not show a specific trend:, it was slightly higher in the 10-14 and 14-18

groups, reaching 7% and 5.7% respectively, but it showed no significant variation (p=0.06).

The MLL/AF4 rearrangement was virtually absent in the 1-5 and 5-10 age groups (0.48% and 0.7%,

respectively), while it progressively increased up to the 5th decade of life, being detected in 2.78%;

2.15%; 3.86%, 6.45%, 7.94% and 11.74% in the 10-14, 14-18, 18-25, 25-30, 30-40, 40-50 age

cohorts, respectively, and decreased again to 5.24% in the 50-60 age cohort (p<.0001).Finally, the

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BCR/ABL1 rearrangement showed a striking behavior: in fact, it was very rare in small children

(1.68% and 2.79% in the 1-5 and 5-10 age groups, respectively), while it progressively increased

with age (age cohorts 10-14: 5.52%; 14-18: 5.81%; 18-25: 14.41%, 25-30: 26%; 30-40: 37.33%;

40-50: 42.86%) reaching a frequency of 52.74% in patients of the 50-60 age-group (p<.0001).

Correlation between molecular aberrations, WBC count and flow cytometry in B-ALL

To investigate if the presence of specific aberrations might influence the WBC count at

presentation, this parameter was correlated with the presence of the ETV6/RUNX1, E2A/PBX1,

MLL/AF4 and BCR/ABL1 rearrangements in the different age cohorts.

ETV6/RUNX1 was significantly associated to a lower WBC count when the whole cohort was

considered (p=0.0001); the same trend was also observed in the 1-5, 5-10, 10-14 and 14-18 age

groups, although it did not reach statistical significance. For the remaining groups, a statistical

analysis was not feasible due the small/absent number of positive cases (Supplementary Table 2).

The E2A/PBX1 rearrangement did not show any significant association with the WBC count,

neither in the global series nor in the various age cohorts (Supplementary Table 3).

At variance, the MLL/AF4 rearrangement was associated to a higher WBC count in almost all age

groups and in the whole cohort (p<0.0001, Supplementary Table 4).

Finally, the BCR/ABL1 rearrangement displayed a peculiar behavior (Supplementary Table 5): this

aberration was associated with a higher percentage of patients with a WBC >50 x 109/L in the

whole cohort (p<0.0001) and in younger children (1-5 years), adolescents and young adults (10-30)

and older patients (>50). There was no statistical association between BCR/ABL positivity and

WBC >50 x 109/L in the remaining age groups. The overall results are shown in Figure 5A.

Next, a correlation between molecular aberrations and flow cytometry was carried out

(Supplementary Tables 6-9). The ETV6/RUNX1 rearrangement was more frequently identified in

common ALL (Figure 5B): this association reached statistical significance when the whole cohort

was considered (p<.0001) and in the 1-5 age cohort (p=0.025; Supplementary Table 6). Similarly,

the E2A/PBX1 aberration was never detected in pro-B ALL cases, while it was more frequent in

pre-B ALL cases (Figure 5C); nevertheless, this finding was significant only when the whole cohort

was considered (p<0.0001), and in the 1-5, 5-10 and 18-25 age cohorts (p=0.013, 0.018 and 0.012,

respectively), probably because of the small number of positive cases in the other subgroups

(Supplementary Table 7).

Contrariwise, the MLL/AF4 rearrangement was detected almost exclusively in pro-B ALL cases

(Figure 5D).with a highly significant in every age cohort (Supplementary Table 8).

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Finally, the BCR/ABL1 fusion product was rarely detected in pro-B ALL cases (Figure 5E): the

association between a non-pro-B stage and the presence of BCR/ABL transcript was statistically

significant when the whole cohort was considered (p=0.0017) and starting from the 4th decade of

life onwards, (p<0.0001, Supplementary Table 9).

DISCUSSION

ALL is a clinically and biologically heterogeneous disease affecting both children and adults with a

distinct incidence. According to the US-SEER (National Cancer Institue, Surveillance,

Epidemiology and End Results Program) (23), approximately 60.3% of ALL cases are diagnosed

under the age of 20; 10.3% between 20 and 34, 5.9% between 35 and 44, 6.7% between 45 and 54,

6.1% between 55 and 64, 5.0% between 65 and 74, 4.0% between 75 and 84 and 1.7% 85+ years of

age.

Since the clinical scenario differs profoundly among age groups, mostly in terms of outcome (1-6),

in this study we considered a data set that comprised a series of children and adults ALL to evaluate

the clinical, hematologic and biologic features at diagnosis among different age groups, subdivided

in 9 age cohorts, i.e. 1-5, 5-10, 10-14, 14-18, 18-25, 25-30, 30-40, 40-50,50-60 years.

Few previous studies have looked at these features in a prospective and uniform manner. In

pediatric cohorts it was shown that a hyperdyploid karyotype and the ETV6/RUNX1 aberration

decrease with age, whereas the presence of BCR/ABL1, as well as the increased incidence of T-

lineage ALL are more frequent in older children and young adults (7, 24, 25). A study on a

relatively small cohort of adult patients (36) reported that older patients (>60 years) have a

decreased male/female ratio, a decreased incidence of mediastinal involvement, adenopathy,

splenomegaly, a reduced median WBC count, as well as a lower percentage of T-lineage ALL in the

elderly.

The current study included 5202 patients (3753 children and 1449 adults) enrolled between 1995

and 2009 respectively in two and four consecutive multicenter Italian protocols for children

(AIEOP) and adults (GIMEMA). All cases were prospectively and uniformly characterized at the

time of diagnosis.

The highest peak of incidence was detected, as expected, in small children between 1 and 5 years;

the occurrence of the disease increased again from the 4th decade, reaching a steady percentage of

about 5.5%, as reported (26) and similar to the US-SEER data; in fact, in our cohort, the overall

percentage of ALL cases is 74% in patients aged < 18 years, 8.5% in cases between 18 and 30, and

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6.2%, 5.5% and 5.3% in patients aged 30-40, 40-50 and 50-60 years, respectively. Patients ≥ 60

years were, at that time, eligible for only one protocol enrolling exclusively BCR/ABL1+ patients;

we thus cannot determine ALL incidence in these individuals, although it is likely that the

frequency of ALL further increases in the over sixties.

Gender distribution was remarkable: starting from the 10-14 age cohort there was a striking

decrease of the ALL rate in females; this tendency is evident up to the 4th decade of life, and

disappeared in the 50-60 years age cohort. This phenomenon seems to be associated with lineage

derivation: in fact, the incidence of ALL is higher in male adolescents and young adults with a T-

ALL and only to a lesser extent with a B-ALL. Although these data were partly known (27, 28), the

analysis on this broad cohort strongly suggests that the age related sex hormones levels may play a

“protective factor” in ALL initiation in females and leads to further investigate in a prospectively

the contribution of hormonal changes occurring during menarche and menopause.

Similarly, organ involvement was widely distributed: beyond mediastinal enlargement, frequently

detected in older children and young adults (age 10-25 years) and significantly associated with a T-

lineage phenotype, spleen and liver enlargement, also associated with a T-lineage phenotype, were

less frequent in older patients, in line with earlier reports (27, 29),thus suggesting a physiological

atrophy of lymphoid organ with aging. Also, consistent with published work (19, 30), CNS was a

rare event, more frequently found in adults than children. In children, it was more frequently

detected in T-ALL patients aged 5-18. The reason for the preferential contamination of CNS by

leukemic T cells in children is not clear, but it might be postulated that adhesion and/or metastatic

molecules are more expressed in pediatric leukemic T cells as opposed to adult leukemic T cells.

This hypothesis is corroborated, by the high levels of IL-15, a proinflammatory cytokine that

promotes T-cell proliferation, detected in childhood ALL with CNS involvement (31) Otherwise,

this peculiar behavior might be sustained by more invasive properties of pediatric T-ALL, as also

suggested by the association with a higher WBC count, liver and spleen enlargement observed in

pediatric, but not in adult T-ALL.

Immunophenotypic analysis confirmed the overall higher incidence of B-ALL vs T-ALL, but also

showed that T-ALL is significantly more frequent in patients whose age is comprised between 14

and 40 years, thus confirmingthat this immunophenotypic subset is more represented in adolescents

and young adults. Moreover, hyperleukocytosis (WBC >50 x 109/L) was infrequent in pediatric B-

ALL and tended to progressively increase with age in this group, whereas in T-ALL

hyperleukocytosis was less frequent in patients > 30. Furthermore, it also showed that in B-ALL

there is a progressive increase of pro-B cases up to the 5th decade of life, a finding significantly

12

associated to the presence of the MLL/AF4 rearrangement. Regarding to T-ALL, T1 cases were

instead more frequent detected in the 5th and 6th decades; this finding is of interest, since early T-

ALL cases tend to have a more unfavorable prognosis (32).

Molecular screening showed the complete disappearance of the ETV6/RUNX1 rearrangement and

the progressive increase of the BCR/ABL1 and MLL/AF4 fusion transcripts with age progression.

The ETV6/RUNX1 rearrangement disappearance with aging further supports the notion that this

ALL subset has indeed a prenatal origin, as indicated by neonatal blood spots or Guthrie cards

screening (33). Moreover, it is in agreement with the finding that a “second leukemogenic hit” must

occur during childhood in order to be leukemogenic (34, 35). It could be speculated that, upon

puberty, the pre-leukemic clone harboring the ETV6/RUNX1 rearrangement is physiologically

cleared out by other factors, such as hormonal changes or a normal degeneration of lymphoid

development.

In our analysis the MLL/AF4 rearrangement is virtually absent in the 1-5 age cohort and its

detection becomes more frequent with age, suggesting that the rearrangement detected in these

patients is probably different from that of infant ALL. To validate this hypothesis, the 1-5 age

cohort was further subdivided into single years and we did not detect an increased incidence of the

transcript in the 1-2 years age group (data not shown).

Given this distribution pattern, it is unlikely that the leukemogenic clone originates from in utero

conditions, as opposed to infant MLL-rearranged leukemias (36): it is intriguing to speculate that

infant MLL-rearranged leukemias require the concomitant presence of residual cord blood and/or

maternal cells to occur, whereas in the pediatric and adult MLL-rearranged leukemias these

“nurturing” cells are no more required. In line with this, previous gene expression profiling studies

have shown that infant MLL-rearranged leukemias share a common expression pattern with MLL-

germline infant leukemias (37).

Finally, the constant and progressively increase of the BCR/ABL1 rearrangements in older patients

indicates that the genomic instability associated with aging leads to the appearance of the leukemic

clone and the accumulation of additional oncogenic hits.

Overall, the variable incidence of the aforementioned lesions leads to hypothesize that in children

harboring ETV6/RUNX1 and, to a lesser extent, MLL/AF4 the driver event is prenatal, whereas in

BCR/ABL1+ ALL, more frequently detected in adults, the driver event occurs during life-time and

induces an overt leukemia only when several additional hits occur.

While ETV6/RUNX1+cases were generally characterized by a low WBC count in the whole cohort,

a striking association between WBC >50 x 109/L was found in all MLL/AF4 positive patients, but

13

not in all patients harboring the BCR/ABL1 rearrangement. This latter result, particularly evident in

patients aged 30 and 50 years, did not change if a different cut-off point was used (i.e. 30 x 109/L,

not shown). The fact that BCR/ABL1+ patients have a different clinical presentation is in line with

the well-documented biological heterogeneity of this subset at the transcriptional level (38, 39) and

with the presence of additional molecular lesions, such as IKZF1 deletions (40, 41).

Overall, this study - that represents the largest cohort of ALL cases broadly and uniformly

characterized throughout ages – confirms and extends previous results on the incidence and

distribution of clinico-biological characteristics (7, 16-18, 23-29). Furthermore, it provides at least

three important information. First, it conclusively shows that the AYA subset (14-30 year-old

patients) shows a higher occurrence of hyperleukocytosis, higher frequency of low platelet count

and a prevalence of male sex; biologically, this group is characterized by a greater percentage of T-

ALL, an increase of unfavorable prognosis aberrations, such as BCR/ABL1 and MLL/AF4, and the

virtual absence of ETV6/RUNX. Altogether, these data help to understand the worse outcome of this

age-group and support the use of more intensive regimens (9-15).

Second, it shows that the incidence of molecular aberrations varies with age progression.

Nevertheless, the clinical characteristics associated, i.e. lineage derivation, differentiation stage and

WBC counts do not change among the different age-cohorts, indicating that the most important

factor for leukemia initiation is represented by the biological mechanism/s responsible for the

emergence of the molecular lesion/s. Finally, it shows that MLL/AF4 and BCR/ABL1

rearrangements constantly increase with age. This is particularly relevant for the BCR/ABL1 fusion,

detected in 52.7% of the 50-60 year-old subgroup. Given the profound changes in the management

of Ph+ ALL following the advent of tyrosine kinase inhibitors (TKI) (42-45), also less fit patients

can nowadays be successfully treated. Indeed, the possibility of effectively treating patients with

TKI plus steroids without systemic chemotherapy (42, 45) makes this a most viable option for

elderly ALL patients, in whom the BCR/ABL1 rearrangement is frequently detected.

In conclusion, this large retrospective study on adult and pediatric ALL uniformly characterized at

presentation has allowed to better define the clinico-biologic features that characterize the various

age subgroups and helps to unravel the differences in clinical behavior and outcome, and provides

therapeutic indications for specific subgroups of patients.

14

Authorship and Disclosures

SC analyzed the data and wrote the manuscript, AV analyzed the data, GC performed molecular

screening and revised the manuscript; SMO, PF, DS, MGV generated the database and performed

statistical analysis, LE performed molecular screening, AMT provided clinical data, FM performed

cytomorphological analysis, VC provided clinical data, GTK performed molecular screening, FF

FDR, AT, GF, FF, GM, GS, GP, FM provided clinical data, AG, GB, AB and RF designed research

and critically revised the manuscript.

The authors have no conflicts to disclose.

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19

Table 1A, 1B and 1C. Immunophenotypic features (A), clinical parameters (B) and molecular

features (C) among different age cohorts. Median counts and p-values are also indicated. Age cohorts (yrs) p

1-5 5-10 10-14 14-18 18-25 25-30 30-40 40-50 50-60

A. Immunophenotypic features B-lineage ALL

(%)

1793

(94.97)

932

(84.27)

397

(77.24)

259

(76.4)

224

(73.93)

103

(75.18)

235

(72.98)

254

(88.19)

245

(87.81) <.0001 T-lineage ALL

(%)

95

(5.03)

174

(15.73)

117

(22.76)

80

(23.6)

79

(26.07)

34

(24.82)

87

(27.02)

34

(11.81)

34

(12.19) B. Clinical parameters

WBC>50x109/L (%)

Median WBC count (Range)

359 (18.87)

12.59 (0.35-1000)

182 (16.34)

8.99

(0.01-761)

138 (26.74)

10.45 (0.18-940)

79 (23.24)

9.62

(0.2-708)

77 (25.50)

13.20

(0.5-848)

43 (31.16)

20.05

(0.8-321)

93 (28.97)

17.30

(0.4-700)

85 (29.62)

17.90

(0.4-872)

73 (26.16)

15.00

(0.5-597)

<.0001

Plts<100x109/L (%)

Median Plts count

(Range)

1352 (71.61)

47

(1-982)

685 (61.88)

72

(2-875)

320 (62.99)

73

(2.8-582)

236 (71.08)

54.5

(1-730)

201 (69.79)

56

(4-461)

92 (69.17)

48

(1.4-290)

232 (75.08)

48

(4-517)

217 (76.41)

46

(1-376)

216 (80)

37

(1-298)

<.0001

Hgb<10g/dL (%)

Median Hgb count

(Range)

1600 (84.75)

7.40

(2.3-15)

812 (73.35)

8.30

(3-15)

314 (61.57)

9.10

(1-17.1)

188 (57.32)

9.5

(2.9-16.8)

163 (62.45)

9

(3.4-16.7)

68 (55.28)

9.5

(3.1-17.5)

169 (59.93)

9.4

(3-17)

142 (57.26)

9.45

(4.3-16)

147 (62.82)

9.2

(4-16.6)

<.0001

Mediastinum enlargement

(%)

79

(4.16)

90

(8.11)

58

(11.26)

35

(10.74)

28

(11.07)

10

(8.93)

26

(9.59)

10

(4.12)

7

(3.14)

<.0001

Spleen enlargement

(%)

516

(27.26)

331

(29.79)

174

(33.92)

104

(32)

75

(30.99)

31

(27.43)

73

(28.85)

61

(26.18)

46

(21.9)

0.028

Liver enlargement

(%)

421

(22.25)

233

(20.92)

107

(20.86)

54

(16.93)

33

(14.29)

10

(8.85)

39

15.48

34

(14.19)

22

(10.33)

<.0001

CNS involvement

(%)

20

(1.05)

26

(2.34)

20

(3.91)

4

(1.29)

5

(2.36)

5

(5.0)

6

(2.58)

5

(2.5)

8

(4.91)

n.a.

C. Molecular features ETV6/RUNX1

(%)

337

(26.14)

162

(23.96)

20

(7.04)

8

(4.71)

2

(1.79)

1

(1.71)

0

(0)

0

(0)

0

(0)

<.0001

E2A/PBX1

(%)

14

(2.12)

13

(3.99)

8

(7.08)

6

(5.71)

6

(3.28)

1

(1.22)

10

(5.08)

5

(2.22%)

5

(2.26)

0.06

MLL/AF4

(%)

8

(0.48)

6

(0.70)

10

(2.78)

5

(2.15)

8

(3.86)

6

(6.45)

17

(7.94)

27

(11.74)

12

(5.24)

<.0001

BCR/ABL

(%)

28

(1.68)

24

(2.79)

20

(5.52)

14

(5.81)

32

(14.41)

26

(26)

84

(37.33)

102

(42.86)

125

(52.74)

<.0001

20

FIGURE LEGENDS

Figure 1. A. Percentage of the distribution of ALL among various age groups; B. Lineage

derivation in the various age cohorts. B-ALL: grey line; T-ALL: black line.

Figure 2. Gender distribution. A. Gender distribution analysis among age cohorts. The analysis

reveals a decreased incidence of females between 14 and 50 years. The grey line represents females,

the black line males. B. Gender distribution and lineage derivation among different age groups. The

grey continuous line represents B-ALL females, the grey dotted line B-ALL males, the black

continuous line T-ALL males and the black dotted line T-ALL females.

Figure 3. Organ involvement among age cohorts in the whole cohort (continuous line) and

stratified according to the lineage derivation (B-ALL: dashed line, T-ALL: dotted line). A:

mediastinal involvement; B: spleen involvement; C: liver involvement; D: CNS involvement.

Figure 4. Incidence of the molecular aberrations in different age cohorts of B-ALL patients. A

significant decrease of ETV6/RUNX1 is observed with age progression, while BCR/ABL and

MLL1/AF4 rearrangements are more frequent in adults.

Figure 5. Hyperleukocytosis and stage of differentiation of B-ALL analyzed on the basis of the

molecular aberrations. A. Percentage of cases with hyperleukocytosis (WBC >50 x 109/L) in

ETV6/RUNX1+ (black bar), E2A/PBX1+ (dark grey bar), MLL1/AF4+ (light grey bar) and

BCR/ABL+ (white bar) cases. B-E. Stage of differentiation within the different molecular

aberrations (B: ETV6/RUNX1; C: E2A/PBX1+; D: MLL1/AF4; E: BCR/ABL+); the black bars

indicate a pro-B ALL, the light grey bars a common ALL and the dark grey bar a pre-B ALL.

Detailed p-values are provided in Supplementary Tables 2-9.

Supplementary Table 1. Organ involvement among various age cohorts; percentages are also provided within B- and T-ALL cohorts; p-value is also indicated.

Age cohorts (yrs) p 1-5 5-10 10-14 14-18 18-25 25-30 30-40 40-50 50-60

Mediastinum enlargement (%)

4.16 8.11 11.26 10.74 11.07 8.93 9.59 4.12 3.14 <.0001

B-ALL (%) 1.73 1.29 1.26 1.61 0.53 1.16 0 1.40 0 n.a. T-ALL (%) 49.27 43.10 45.3 40.79 41.54 34.62 33.77 25 26.92 0.19

Spleen enlargement (%) 27.26 29.79 33.92 32 30.99 27.43 28.85 26.18 21.9 0.028

B-ALL (%) 25.56 26.42 29.29 26.91 26.55 25.58 27.22 24.51 23.78 0.90 T-ALL (%) 57.89 48.84 50.43 49.33 43.08 33.33 32.88 37.93 8.00 0.0003

Liver enlargement (%) 22.25 20.92 20.86 16.93 14.29 8.85 15.48 14.19 10.33 <.0001

B-ALL (%) 21.26 19.64 17.93 15.66 10.59 5.88 13.48 15.00 10.58 <.0001 T-ALL (%) 41.05 28.74 31.3 21.74 24.59 17.86 20.27 14.29 8.33 0.006

CNS involvement 1.05 2.34 3.91 1.29 2.36 5.0 2.58 2.5 4.91 n.a.

B-ALL (%) 0.95 1.07 2.03 0. 1.81 5.48 2.98 2.86 5.56 n.a. T-ALL (%) 3.19 8.67 10.34 5.41 4.35 3.70 1.54 0.00 0.00 n.a.

n.a. = not applicable since Chi-Square might not represent a valid test since some subgroups have expected counts less than five and Fisher test is not doable because the software cannot computed exact values.

Supplementary Table 2. Association between ETV6/RUNX1 rearrangement and WBC

Whole cohort

Age cohort: 1-5 yrs ETV6/RUNX1

ETV6/RUNX1

Negative Positive p

Negative Positive p WBC

<50x109/L 2136 462 Chi-Square

0.0001

WBC <50x109/L 775 284

Chi-Square 0.2377 WBC

>50x109/L 533 68

WBC >50x109/L 177 53

Age cohort: 5-10 yrs


ETV6/RUNX1

ETV6/RUNX1 Negative Positive p


<50x109/L 452 150 Chi-Square

0.098

WBC <50x109/L 214 17

Fisher test 1 WBC >50x109/L 62 12

WBC >50x109/L 50 3



ETV6/RUNX1

ETV6/RUNX1 Negative Positive p


<50x109/L 131 8 Fisher test

0.3534

WBC <50x109/L 104 2


WBC >50x109/L 28 0



ETV6/RUNX1

ETV6/RUNX1

Negative Positive p


<50x109/L 50 1

Fisher test 1

WBC <50x109/L 121 0

Not applicable WBC

>50x109/L 20 0

WBC >50x109/L 50 0



ETV6/RUNX1

ETV6/RUNX1

Negative Positive p


<50x109/L 140 0 Not

applicable

WBC <50x109/L 149 0

Not applicable WBC

>50x109/L 64 0

WBC >50x109/L 51 0

Supplementary Table 3. Association between E2A/PBX1 rearrangement and WBC count in B-ALL.

Whole cohort

Age cohort: 1-5 yrs

E2A/PBX1

E2A/PBX1

Negative Positive p

Negative Positive p

WBC <50x109/L 1597 54

Chi-square 0.83

WBC <50x109/L 515 9

Fisher test 0.179 WBC

>50x109/L 442 14

WBC >50x109/L 131 5



E2A/PBX1

E2A/PBX1

Negative Positive p

Negative Positive p

WBC <50x109/L 277 11

Fisher test 0.654

WBC <50x109/L 74 6


WBC >50x109/L 31 2

Age cohort:14-18 yrs


E2A/PBX1

E2A/PBX1

Negative Positive p

Negative Positive p

WBC <50x109/L 80 5

Fisher test 1

WBC <50x109/L 143 5


WBC >50x109/L 33 1



E2A/PBX1

E2A/PBX1

Negative Positive p

Negative Positive p

WBC <50x109/L 60 1

Fisher test 1

WBC <50x109/L 136 9


>50x109/L 21 0

WBC >50x109/L 51 1



E2A/PBX1

E2A/PBX1

Negative Positive p

Negative Positive p

WBC <50x109/L 152 3

Fisher test 0.6363

WBC <50x109/L 160 5


>50x109/L 64 2

WBC >50x109/L 56 0

Supplementary Table 4. Association between MLL/AF4 rearrangement and WBC count in B-ALL.

Whole cohort

Age cohort: 1-5 yrs

MLL/AF4

MLL/AF4

Negative Positive p Negative Positive p

WBC <50x109/L 3360 28

Chi-square <0.0001

WBC <50x109/L 1394 2

Fisher Test 4.085E-04 WBC

>50x109/L 635 71 WBC

>50x109/L 271 6

Age cohort: 5-10 yrs Age cohort: 10-14 yrs

MLL/AF4

MLL/AF4

Negative Positive p

Negative Positive p

WBC <50x109/L 770 0

Fisher Test 9.34E-07

WBC <50x109/L 296 1


>50x109/L 81 6

WBC >50x109/L 54 9



MLL/AF4

MLL/AF4

Negative Positive p

Negative Positive p

WBC <50x109/L 190 2

Fisher Test 0.0393

WBC <50x109/L 167 1


>50x109/L 38 3

WBC >50x109/L 31 7



MLL/AF4

MLL/AF4

Negative Positive p

Negative Positive p

WBC <50x109/L 69 2

Fisher Test 0.0264

WBC <50x109/L 158 2 Fisher Test

1.87E-08 WBC >50x109/L 18 4

WBC >50x109/L 39 15



MLL/AF4

MLL/AF4

Negative Positive p

Negative Positive p

WBC <50x109/L 151 11

Chi-Square 0.0003

WBC <50x109/L 165 7

Fisher Test 0.1787 WBC

>50x109/L 51 16

WBC >50x109/L 52 5

Supplementary Table 5. Association between BCR/ABL rearrangement and WBC count in B-ALL.

Whole cohort

Age cohort: 1-5 yrs

BCR/ABL

BCR/ABL

Negative Positive p

Negative Positive p

WBC <50x109/L 3140 296 Chi-square

<0.0001

WBC <50x109/L 1381 12


>50x109/L 557 158

WBC >50x109/L 260 16


Age cohort: 10-14 yrs BCR/ABL

BCR/ABL

Negative Positive p

Negative Positive p

WBC <50x109/L 753 19

Fisher Test 0.0859

WBC <50x109/L 289 9


>50x109/L 82 5

WBC >50x109/L 53 11



BCR/ABL

BCR/ABL

Negative Positive p

Negative Positive p

WBC <50x109/L 193 7

Fisher Test 0.0035

WBC <50x109/L 159 21


>50x109/L 34 7

WBC >50x109/L 30 11

Age cohort: 25-30yrs


BCR/ABL

BCR/ABL

Negative Positive p

Negative Positive p

WBC <50x109/L 60 16

Chi-Square 0.0447

WBC <50x109/L 109 60


>50x109/L 14 10

WBC >50x109/L 32 24



BCR/ABL

BCR/ABL

Negative Positive p

Negative Positive p

WBC <50x109/L 100 68

Chi-Square 0.2986

WBC <50x109/L 96 84


>50x109/L 36 33

WBC >50x109/L 16 41

Supplementary Table 6. Association between ETV6/RUNX1 rearrangement and the degree of B-cell differentiation.

Whole cohort

Age cohort: 1-5 yrs

B1 B2 B3 p

B1 B2 B3 p ETV6/RUNX1

negative 170 1386 624 Chi-square

<.0001

ETV6/RUNX1 negative 30 611 278

Chi-square 0.0254 ETV6/RUNX1

positive 6 354 124

ETV6/RUNX1 positive 5 231 75



B1 B2 B3 p


negative 15 340 126 Chi-square

0.2508


Fisher test 0.4 ETV6/RUNX1

positive 1 106 41




B1 B2 B3 p


negative 17 97 21 Fisher test

0.1985


Fisher test 0.2328 ETV6/RUNX1

positive 0 2 2




B1 B2 B3 p


negative 8 17 7 Fisher test 1


not applicable ETV6/RUNX1

positive 0 1 0




B1 B2 B3 p


negative 34 39 38 not applicable


not applicable ETV6/RUNX1

positive 0 0 0


Supplementary Table 7. Association between E2A/PBX1 rearrangement and the degree

of B-cell differentiation.

Whole cohort

Age cohort: 1-5 yrs

B1 B2 B3 p

B1 B2 B3 p

E2A/PBX1 negative 161 784 552

Chi-square <.0001


Chi-square 0.0125 E2A/PBX1

positive 0 12 39 E2A/PBX1 positive 0 3 11



B1 B2 B3 p B1 B2 B3 p


Chi-square 0.0184


Fisher test 0.476

E2A/PBX1 positive 0 3 10

E2A/PBX1 positive 0 2 6


B1 B2 B3 p

B1 B2 B3 p

E2A/PBX1 negative 11 32 20 Fisher test

0.2896

E2A/PBX1 negative

23 41 26 Fisher test

0.0119 E2APBX1 positive 0 1 3

E2A/PBX1 positive

0 0 4


B1 B2 B3 p

B1 B2 B3 p


not applicable

E2A/PBX1 negative 25 47 28 Fisher test

0.4456

E2APBX1 positive 0 0 0 E2A/PBX1

positive 0 1 2


B1 B2 B3 p B1 B2 B3 p E2A/PBX1 negative 34 41 37 Fisher test

0.6384 E2A/PBX1 negative 18 47 38 Fisher test

1

E2APBX1 positive 0 1 2

E2APBX1 positive 0 1 1

Supplementary Table 8. Association between MLL/AF4 rearrangement and the degree

of B-cell differentiation.

Whole cohort

Age cohort: 1-5 yrs

B1 B2 B3 p

B1 B2 B3 p

MLL/AF4 negative 188 2242 910

Chi-square <.0001


Fisher test 3.21E-07 MLL/AF4

positive 68 3 5

MLL/AF4 positive 5 1 2


B1 B2 B3 p

B1 B2 B3 p


Fisher test 3.05E-07



positive 5 1 0



B1 B2 B3 p B1 B2 B3 p


Fisher test 1.42E-05


Fisher Test 0.0387 MLL/AF4

positive 5 0 0



B1 B2 B3 p B1 B2 B3 p


Fisher test 0.0006269



positive 5 0 1 MLL/AF4 positive 11 0 1


B1 B2 B3 p B1 B2 B3 p

MLL/AF4 negative 14 44 39 Chi-square



positive 20 0 0 <.0001 MLL/AF4 positive 7 0 0

Supplementary Table 9. Association between BCR/ABL rearrangement and the

degree of B-cell differentiation.

Whole cohort Age cohort: 1-5 yrs

B1 B2 B3 p B1 B2 B3 p

BCR/ABL negative 259 2105 832

Chi-square 0.0017


Chi-square 0.9805 BCR/ABL

positive 8 148 82

BCR/ABL positive 1 19 8


B1 B2 B3 p B1 B2 B3 p


Chi-square 0.5384


Fisher test 0.5746 BCR/ABL

positive 0 16 4


Age cohort:14-18 yrs Age cohort: 18-25 yrs

B1 B2 B3 p B1 B2 B3 p


Fisher test 0.653


Fisher test 0.3316 BCR/ABL

positive 0 8 1



B1 B2 B3 p B1 B2 B3 p


Fisher test 0.2599


Chi-square <.0001 BCR/ABL

positive 0 5 2 BCR/ABL positive 0 22 14


B1 B2 B3 p B1 B2 B3 p


Chi-square <.0001


Chi-square 0.0001 BCR/ABL

positive 2 23 17

BCR/ABL positive

3 34 27

Legends to Supplementary Figures

Supplementary Figure 1. EFS in the whole population analyzed, stratified according to the age

groups.

Supplementary Figure 2. Differentiation stages of B-ALL in the different age cohorts analyzed

(pro-B ALL: black bars; common ALL: light grey bar; pre-B ALL: dark grey bar). A significant

increase in the percentage of pro-B up to the 5th decade of life is observed (p<0.0001).

Supplementary Figure 3. Differentiation stages of T-ALL in the different age cohorts analyzed,

(pro-T & pre-T ALL: black bars; cortical T-ALL: light grey bar; mature T-ALL: dark grey bar). A

significant increase of T1 ALL is recorded in the 5th and 6th decade (p=0.0018).

Supplementary Figure 4. Hematologic parameters in the different age cohorts. A. White blood

count (black line), platelet count (light grey) and hemoglobin levels (dark grey) among different age

cohorts. B. Percentage of hyperleukocytosis (cut-point >50 x 109/L) in the different age cohorts

analyzed, stratified for lineage derivation (B-ALL: grey bars; T-ALL: black bars). A higher

percentage of hyperleukocytosis is evident in T-lineage ALL. C. Distribution of Plt count <100 x

109/L in the different age cohorts analyzed, stratified for lineage derivation (B-ALL: grey bars; T-

ALL: black bars). A significantly higher percentage of cases with Plt count <100 x 109/L is

observed in B-ALL. D. Distribution of percentage of Hgb levels <10g/dL in the different age

cohorts analyzed, stratified for lineage derivation (B-ALL: grey bars; T-ALL: black bars). A

significantly higher percentage of cases with Hgb levels <10g/dL is observed in B-ALL.

Supplementary Figure 1

Supplementary Figure 2.

clinico-biologic features of 5202 acute lymphoblastic ... features of 5202 acute lymphoblastic...

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