Introduction

Treatment of pediatric acute lymphoblastic leukemia (ALL), has greatly improved over the last decades, leading to cure in approximately 80–85%. T-cell ALL (T-ALL) accounts for 15% of all newly diagnosed pediatric ALL cases, and is clinically regarded as a high-risk disease with a relapse rate of about 30% (reviewed in Pui et al.¹ and Thiel et al.²).

In about half of the pediatric T-ALL patients recurrent chromosomal translocations are being found (reviewed in De Keersmaecker et al.,³ Raimondi⁴ and Rubnitz and Look⁵). For most of these abnormalities, the T-cell receptor (TCR)-/ or TCR- loci are involved⁶ presumably as mistakes during attempted gene segment rearrangements. Other recurrent translocation partners include basic helix–loop–helix (bHLH) genes (MYC, TAL1, TAL2, LYL1 and bHLHB1), cysteine-rich (LIM-domain containing) genes (LMO1 and LMO2), homeodomain genes (HOX11/TLX1, HOX11L2/TLX3 or the HOXA gene cluster) or, as identified most recently, the MYB oncogene.⁷ Other translocations leading to the formation of specific fusion genes like CALM-AF10,⁸ or MLL rearrangements have also been described.^{4, 5} These rearrangements in general occur in a mutually exclusive manner and allows for the identification of specific T-ALL subgroups. However, other recurrent chromosomal abnormalities such as 6q deletions, deletion of 9p21 including the p15 and p16 loci, episomal amplification of a NUP214–ABL1 fusion or MYB duplications are shared by various of these T-ALL subgroups.^{3, 9, 10} Activating mutations in NOTCH1 have been identified in more than 50% of all T-ALL cases that are also shared by various T-ALL subgroups.¹¹

The presence of specific molecular-cytogenetic aberrations may predict for outcome in T-ALL. CALM-AF10 may define a poor prognostic subgroup,^{8, 12} but the prognostic relevance of HOX11L2 has proved diverse and may depend on the therapy protocol.^{12, 13, 14, 15, 16, 17} HOX11 abnormalities have been associated with a favorable outcome.^{14, 18, 19, 20} The prognostic significance of NOTCH1 mutations is currently unknown, but in one study mutations were associated with a favorable outcome.²¹

The prognostic significance of these molecular-cytogenetic subgroups may depend on differences in maturation arrest during T-cell development.²² CALM-AF10 and HOX11L2 rearrangements are predominantly found in cases with an early T-cell development along the -lineage, whereas HOX11 and TAL1 abnormalities have been associated with the cortical and mature stage along the -lineage of T-cell development.^{8, 23, 24, 25} The European Group for the Immunological Characterization of Leukemias (EGIL)²⁶ has previously defined four developmental stages in T-ALL for the classification into specific development subgroups depending on the expression of specific cluster of differentiation markers (CD-markers).^{27, 28} Macintyre and co-workers²³ have developed an alternative classification system that is based upon the TCR status that may better reflect T-cell development as it is currently understood. Alike recognition of specific molecular-cytogenetic subgroups, the recognition of specific T-cell developmental subgroups may also have prognostic relevance. For instance, adult T-ALL cases with a EGIL pro-/pre-T-cell immunophenotype have demonstrated reduced remission induction, early relapse and shortened overall survival time.^{2, 29, 30} Complete remission (CR) rate was also lower for adult T-ALL with an immature (IM) immunophenotype according to the TCR classification system on the Lala-94 protocol. However, the overall relapse incidence was comparable for all TCR classification subgroups.³¹ In children this is less well known, CD1-positive T-ALL have been associated with an excellent outcome,^{32, 33} and is expressed in most HOX11-positive T-ALL patients.^{14, 34}

In the present study, we have classified pediatric T-ALL patient samples into various immunophenotypic subgroups based upon EGIL²⁶ or TCR classification criteria.²³ For the various subgroups, the presence of cytogenetic abnormalities as well as the expression of early T-cell transcription factors was investigated. As cytogenetic abnormalities have been associated with outcome, it was investigated whether differentiation status may provide an explanation for the outcome differences between various cytogenetic subgroups.

Materials and methods

Patients

Viably frozen diagnostic bone marrow (n=41) or peripheral blood samples (n=31) from 72 pediatric T-ALL patients, diagnosed between 1991 and 2000, treated according to the Dutch Childhood Oncology Group (DCOG) protocols ALL-7 (n=4), ALL-8 (n=26) or ALL-9 (n=42), were studied.^{35, 36} Clinical and immunophenotypic data of these patient samples were kindly provided by the DCOG. The median follow-up time for this cohort was 63.5 months. All patient samples were processed as previously described and contained >90% of leukemic blasts.³⁷ Informed consent was obtained in accordance to the Declaration of Helsinki. Bone marrow biopsies from 28 non-leukemic children (10 without evidence of a hematological malignancy, 2 Burkitt non-Hodgkin's lymphomas, 5 Hodgkin's lymphomas, 5 neuroblastomas, 4 rhabdomyosarcomas and 2 Ewing's sarcomas) were used as a control group to determine TAL1, LYL1, LMO1 and LMO2 reference levels. For all patients, the clinical parameters, T-ALL subgroup, NOTCH1 status, karyotypic results and individual immunophenotypic parameters have been summarized in Supplementary Table 1.

Quantitative real-time RT-PCR

RNA extraction and reverse transcription were performed according to the procedure described previously.³⁷ Expression levels of HOX11, HOX11L2, TAL1, LYL1, LMO1, LMO2 transcripts and SIL-TAL and CALM-AF10 fusion products, were quantified relative to the expression level of the endogenous housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by real-time RT-PCR in an ABI 7700 sequence detection system (PE Applied Biosystems, Foster City, CA, USA) such as described previously.^{12, 37, 38} The SIL-TAL1 primers (ENF601, ENR664) and probe (ENP641) for the detection of a SIL-TAL1 deletion were used as recommended by the Europe Against Cancer Program.³⁹

FISH analysis

Experiments were performed on interphase preparations as described before.⁴⁰ Dual-colored FISH experiments to study the presence of the CALM-AF10 fusion have been described before.¹² For detection of LMO1 and LMO2 rearrangements, BAC probe (BacPac Resources, Oakland, CA, USA) combinations RP11-79E12 and RP11-21N2 or RP1189C11 and RP11580K7 were used, respectively. BAC DNA was nick translated using biotin-16-dUTP/digoxigenin-11-dUTP (Roche, Penzberg, Germany). Patient samples were scored positive when more than 10% of interphase cells demonstrated fusion or split apart signals for 100 counted cells by two independent observers. MLL-, HOX11-, HOX11L2- or TAL1- translocations, or the SIL-TAL1 deletion⁴¹ was determined by commercially available FISH kits provided by DakoCytomation (Glostrup, Denmark), hybridized and scored as described by the manufacturer.

Immunophenotypic cytoplasmic TCR- (Cyt-) and TCR- or TCR- analyses

Indirect cytoplasmic TCR- (Cyt-) staining was performed on acetone-fixed cytospin preparations using the antibody F1 (BioAdvance, Emerainville, France). Following 15 min incubation with the F1 antibody, a secondary incubation with GM-FITC was performed for visualization. Cyt- positivity was scored using fluorescence microscopy by two independent observers. TCR expression on the T-ALL samples was analyzed by standard flowcytometry using antibodies against TCR- (BMA031; Beckman Coulter, Fullerton, CA, USA) and TCR- (11F2; Becton Dickinson, San Jose, CA, USA) in combination with staining for CD3 expression. Measurement and acquisition of data were done on FACScan and FACSCalibur flowcytometers (Becton Dickinson). The central reference laboratory of the DCOG determined other immunophenotypic parameters, following standard procedures and a predefined diagnostic panel of reagents.⁴² A sample was considered positive when 25% of the leukemic cells or higher expressed a specific immunophenotypic marker. T-ALL patients were assigned into specific subgroups comparable to the EGIL²⁶ or the TCR classification²³ systems. Briefly, patients were assigned to the pro-/pre-T-cell subgroup (CD7⁺, CD2⁺ and/or CD5⁺ and/or CD8⁺, but CD1^- and sCD3^-), the cortical T (CD1⁺) or the mature T (sCD3⁺/CD1^-) subgroup in accordance to EGIL criteria. In accordance to the TCR-based classification system, patients were assigned to the IM (Cyt-^-, sCD3^-, TCR-^- and TCR-^-), the pre- (Cyt-⁺, sCD3^-, TCR-^- and TCR-^-) or the TCR- (sCD3⁺, TCR-⁺) or TCR- subgroups (sCD3⁺, TCR-⁺).

NOTCH1 mutations

NOTCH1 mutation screening was performed according to the procedure as described previously.¹¹ All samples were directly sequenced.

Statistical analysis

All statistical analyses were performed using SPSS 12.0. software. The correlation between the EGIL and TCR classification to assign T-ALL patients to various subgroups was tested using the Spearman's correlation test. Kaplan–Meier curves were constructed, and the log-rank P-values were calculated. For the calculation of the disease-free survival (DFS), events were defined as relapse or non-response to induction therapy. The Mann–Whitney U-test (MWU) was used to analyze differences in age, WBC and gene expression levels between subgroups. Distribution of positive cases among EGIL or TCR classification subgroups was tested using the Fisher's exact test.

Results

To study the relation between recurrent molecular-cytogenetic abnormalities, T-cell development stage and outcome, patients were assigned into T-cell development subgroups as developed by EGIL²⁶ and by the Macintyre group³¹ (TCR classification). In accordance to EGIL criteria, T-ALL patients were assigned to the pro-/pre-T subgroup (n=24), the cortical subgroup (n=30) and the mature T-cell subgroup (n=18). For 67 patients, the Cyt- expression status was successfully determined allowing classification according to the TCR classification criteria: 26 patients were assigned to the IM subgroup, 19 patients to the pre- subgroup and 22 patients to the mature subgroup with 5 cases expressing TCR- and 17 cases expressing TCR- (Table 1). The IM T-ALL cases were not further differentiated into IM0, IM, IM and IM subgroups³¹ for this study.

Table 1 - Relation between clinical parameters and immunophenotype.

Full table

Clinical parameters of EGIL and TCR subgroups

Clinical characteristics in relation to EGIL or TCR classification subgroups are depicted in Table 1. For these, no significant relationships were observed with gender, white blood cell count (WBC) or mediastinal involvement, except for age. The median age for TCR--positive patients (10.4 years) was significantly higher compared to other patients (6.3 years; P=0.02).

Using both classification systems, more than half of the patient samples were assigned to comparable developmental stages, that is, pro-/pre-T vs IM, cortical-T vs pre- or mature vs TCR- or TCR- based upon the EGIL^{8, 31} or TCR classification²³ criteria (Table 2; Spearman's =0.474, P<0.001 (two-tailed)). Multiple samples, however, were classified to discordant stages. Twelve IM T-ALLs lacking Cyt- expression were assigned to the cortical T-cell subgroup based on their CD1 positivity. Eleven pre- T-ALLs were denoted as pro-/pre-T (seven cases) or mature T-ALLs (four cases) according to EGIL criteria. Seven mature TCR- or TCR--positive T-ALLs were denoted as cortical-T based upon their CD1 positivity despite the fact that these seven cases expressed mCD3. One TCR--positive T-ALL was found to be negative for both mCD3 and CD1 and was assigned to the pro-/pre-T subgroup.

Table 2 - Overlap between immunophenotypic classification systems.

Full table

Molecular-cytogenetic abnormality distribution over EGIL and TCR subgroups

For all 72 T-ALL patients, the rearrangements status of TLX3/HOX11L2, TLX1/HOX11, TAL1, CALM-AF10 or LMO2 genes was determined using FISH and quantitative real-time RT-PCR (RQ-PCR).^{12, 43} In Table 3, the distribution of these molecular-cytogenetic subgroups over various T-cell developmental stages according to EGIL or TCR classification criteria is shown.

Table 3 - Molecular-cytogenetic profiles in EGIL- and TCR-classification subgroups.

Full table

On the basis of EGIL criteria, two out of three CALM-AF10 rearranged cases were assigned to the pro-/pre-T subgroup. Both patients were also classified as IM T-ALL in accordance to the TCR classification system. One CALM-AF10 case expressed CD1, and was denoted as cortical-T.

HOX11L2-positive patients predominantly had an IM or cortical-T differentiation stage, and only 4 out of 17 patients had a mature T-cell stage according to EGIL criteria. Using TCR classification criteria, HOX11L2 rearranged T-ALL cases were classified as IM (7 cases) or pre- (7 cases) and only 3 cases were TCR- positive. None of the HOX11L2 patients were TCR- positive (P=0.015). All HOX11-positive samples expressed CD1 and were therefore assigned to the cortical T-cell subgroup (P=0.008). However, as most HOX11 patients lacked cytoplasmatic- (Cyt-) expression they were assigned to the IM subgroup based upon TCR classification criteria.

The 14 TAL1-rearranged cases were equally distributed among EGIL subgroups. However, only four TAL1-rearranged cases were classified as pre- based upon Cyt- expression, but nine TAL1-positive cases were TCR positive and exclusively associated with the -lineage (P<0.001). LMO2 cases were found in all developmental stages of EGIL and TCR classification systems. Both TCR positive, LMO2-rearranged cases were TCR- positive comparable to the TAL1 rearranged cases.

The NOTCH1 mutation status was successfully determined for 70 out of 72 patients. Mutations in the heterodimerization domain (HD; exon 26 or exon 27) of NOTCH1 was identified in 28 patients, 6 patients had a PEST (Proline (P), glutamate (E), serine (S) and threonine (T)) domain (exon 34) mutation and another 6 patients had mutations in both the HD and PEST domains (Figures 1a and b). The incidence of NOTCH1 mutations was less frequent in immunophenotypic mature cases according to both classification systems.

Figure 1.

Activating NOTCH1 mutations in pediatric T-ALL patients. (a) Position of point mutations or in-frame deletion/insertion mutations in the heterodimerization domain (exons 26 and 27) of NOTCH1. Amino-acid numbering is in accordance to Weng et al.¹¹ Amino-acid residues indicated in bold are conserved amino-acid residues between human, mouse and Xenopus NOTCH1. (b) The positions of missense and point mutations are given in the PEST domain of NOTCH1. Asterisks refer to stop codons. T-ALL, T-cell acute lymphoblastic leukemia.

Full figure and legend (359K)

Expression of TAL1, LYL1, LMO1 and LMO2 in EGIL or TCR- subgroups

In addition to recurrent cytogenetic abnormalities, the expression of several transcription factors including TAL1, LYL1, LMO1 and LMO2 was investigated in relation to the T-cell developmental subgroups. Rearrangements of the TAL1, LMO1 or LMO2 loci result in activation of these genes, but various patients also highly express TAL1, LYL1, LMO1 or LMO2 levels in the absence of chromosomal rearrangements. The expression levels for these transcription factors did not differ between the three EGIL subgroups (data not shown). For the TCR classification subgroups, the levels for TAL1, LYL1 and LMO2 were significantly different (Figure 2). The levels of LYL1 and LMO2 expression were significantly higher in the IM subgroup than in the pre- (P<0.001 and P=0.005, respectively) and TCR- subgroups (P=0.002 and P=0.003, respectively). TAL1 expression levels followed an opposite profile with the lowest expression in IM T-ALL cases and the highest levels for the pre- (P=0.002) and TCR- (P=0.003) subgroups.

Figure 2.

Expression levels of TAL1, LMO2 and LYL1 in TCR classification subgroups. Relative expression levels of (a) TAL1, (b) LMO2 and (c) LYL1 as percentage of housekeeping gene GAPDH expression levels for TCR classification subgroups and bone marrow control samples. All P-values below P=0.008 as indicated between various TCR classification subgroups are significant following the Bonferroni correction. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; TCR, T-cell receptor.

Full figure and legend (99K)

Outcome of T-ALL

Twenty out of 72 patients relapsed (28%). No relation was found between clinical parameters including age, WBC, mediastinal involvement, gender or treatment protocol and DFS (Table 4). None of the EGIL or TCR classification subgroups were associated with outcome (Table 4).

Table 4 - Outcome of clinical, immunophenotypic and molecular-cytogenetic subgroups.

Full table

The presence of HOX11, LMO2 abnormalities or NOTCH1 mutations was not related to DFS (Table 4). HOX11L2 and TAL1 abnormalities were associated with a nonsignificant trend toward poor and good DFS, respectively. The three CALM-AF10 rearranged patients had a poor DFS (P=0.0053).

Expression levels of LYL1, LMO2 and LMO1 did not predict for outcome. For T-ALL samples expressing comparable TAL1 levels as TAL1-rearranged cases (relative TAL1 expression levels >0.05%), no difference in outcome was observed compared to cases expressing lower TAL1 levels. In a trend analysis comparing the outcome of patients with the lowest TAL1 expression (below the 25th percentile) with patients between the 25th and 50th percentile, between the 50th and 75th percentile and above the 75th percentile of TAL1 expression, a trend toward better outcome was found for patients with higher TAL1 expression (P=0.09; Table 4).

Discussion

Molecular-cytogenetic abnormalities as identified in T-ALL may have prognostic significance. CALM-AF10-positive T-ALL has been associated with early relapse,^{8, 12} whereas HOX11L2 abnormalities have been associated with diverse treatment outcomes depending on the treatment given.^{22, 23, 24, 25, 26} HOX11L2 demonstrated a trend to poor outcome in our DCOG pediatric cohort, and was significantly associated with poor outcome for children treated according to the COALL-97 protocol.¹² As molecular-cytogenetic abnormalities are associated with the expression of specific immunophenotypic markers, we have studied whether arrest at specific developmental stages may provide a potential explanation for the observed differences in outcome between various cytogenetic subgroups. To this end, we have classified our T-ALL cases according to the criteria of two currently available T-cell classification systems, that is, the EGIL²⁶ and the TCR classification system.²³

Using these classification systems, about half of the T-ALL cases were assigned to more or less comparable developmental stages. However, various samples were assigned to different stages using both classification systems including five TCR- and 2 TCR--positive cases that were assigned to the cortical T-cell stage using EGIL based on their CD1 expression. All these cases expressed mCD3, and denoting cases as cortical T-cells on the basis of CD1 may be too stringent. The TCR classification system may therefore better relate pediatric T-ALL to normal T-cell development than EGIL. This is also supported by LYL1, LMO1 and LMO2 expression levels that were highest for IM T-ALL cases and significant lower levels for pre- and TCR- subgroups alike normal T-cell development.^{44, 45}

All HOX11-rearranged cases in our study expressed CD1 and were denoted as cortical thymocytes using EGIL (P=0.008). As four out of five cases lacked Cyt- expression, developmental arrest for HOX11 T-ALL patients precedes -selection in most patients. HOX11L2-positive cases were predominantly associated with the pre-T/IM and cortical-T/pre- subgroups. A few HOX11L2 cases had a mature phenotype but exclusively associated with the TCR- lineage. On the other hand, expression of Cyt- in seven cases may indicate partial commitment to the -lineage in support of the hypothesis by Asnafi et al.²³ that HOX11L2-rearranged T-ALL cases resemble -lineage cortical thymocytes that are differentiated toward unusual TCR- expressing cells as intermediates between the -lineage and the -lineage. TAL1-rearranged cases were associated with all maturation stages according to EGIL, but were not associated with IM subtype nor with -lineage commitment according to the TCR classification system in accordance with previous studies.^{8, 24, 25} LMO2-rearranged cases including the three cases with the newly identified del(11)(p12p13) leading to elevated LMO2 levels⁴³ seem to arrest at all stages of T-cell maturation stages based on EGIL and the TCR classification criteria except for the -lineage. As TAL1 and LMO2 participate in the same transcriptional complex,^{46, 47, 48} we would have expected a similar distribution for TAL1 and LMO2 cases. On the other hand, observed differences may be due to the low numbers of LMO2-rearranged patient in our cohort.

Activating NOTCH1 mutations were present in 57% of T-ALL patients in line with previous reports.^{11, 21} Eleven out of 12 PEST domain mutations were missense mutations leading to truncation of NOTCH1 and deletion or disruption of the negative regulatory PEST domain.¹¹ One mutation was a point mutation of a conserved amino-acid residue in NOTCH1, and we speculate that this point mutation will interfere with the function of the PEST domain. The 34 mutations in the heterodimerization domain were all point mutations or in-frame deletion/insertion mutations, possibly promoting ligand-independent protease cleavage and release of intracellular NOTCH1 (ICN).¹¹ Nine out of 12 PEST domain mutations and 14 out of 34 HD mutations were new and have not been observed before in other T-ALL patient cohorts.^{11, 21} NOTCH1 mutations were predominantly associated with the cortical stage in another study.²¹ We did not observe significant differences in the distribution of NOTCH1 mutations between EGIL or TCR classification subgroups, in line with the presence of NOTCH1 mutations in all molecular-cytogenetic T-ALL subgroups. We also did not observe any differences regarding outcome in contrast to that same study in which activating NOTCH1 mutations were associated with favorable treatment response and long-term outcome in pediatric T-ALL.²¹

None of the EGIL or TCR classification subgroups predicted for outcome. Pro-/pre-T or IM immunophenotypes have been previously associated with reduced-remission induction^{2, 27, 30, 31} and poor outcome^{2, 29} in adult T-ALL. These observations could not be confirmed in our pediatric T-ALL cohort. Our HOX11-rearranged patients that were all CD1 positive did not have an improved outcome as published,^{14, 18, 19, 20, 28, 32} but the number of HOX11-rearranged patients in our study is low. As described earlier¹² and now analyzed over a prolonged follow-up, HOX11L2 demonstrated a trend towards poor outcome in our pediatric cohort.¹² Within this subgroup, CD1-positive cases relapsed as frequently as CD1-negative cases.¹²

TAL1 rearranged T-ALL demonstrated a trend for good outcome, in line with one study¹⁴ but in contrast to another study.³⁴ This was supported by analyzing the outcome of patients based on TAL1 expression levels in which cases with higher TAL1 levels demonstrated a trend for good outcome. Alternatively, the 18 cases with the lowest TAL1 levels included 10 HOX11L2-rearranged and 2 CALM-AF10-positive T-ALL cases, two cytogenetic entities that both have been associated with poor outcome in other studies.^{8, 13, 34}

In conclusion, the present study shows that differences in outcome for various molecular-cytogenetic subgroups cannot be attributed to differences in T-cell maturation stage.

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Acknowledgements

This study was sponsored by the Quality of Life and KOCR foundations.

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