Original Article | Published:

Acute Leukemias

Immunobiological diversity in infant acute lymphoblastic leukemia is related to the occurrence and type of MLL gene rearrangement

Leukemia volume 21, pages 633641 (2007) | Download Citation

Abstract

The aim of this study was to identify immunobiological subgroups in 133 infant acute lymphoblastic leukemia (ALL) cases as assessed by their immunophenotype, immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangement pattern, and the presence of mixed lineage leukemia (MLL) rearrangements. About 70% of cases showed the pro-B-ALL immunophenotype, whereas the remaining cases were common ALL and pre-B-ALL. MLL translocations were found in 79% of infants, involving MLL-AF4 (41%), MLL-ENL (18%), MLL-AF9 (11%) or another MLL partner gene (10%). Detailed analysis of Ig/TCR rearrangement patterns revealed IGH, IGK and IGL rearrangements in 91, 21 and 13% of infants, respectively. Cross-lineage TCRD, TCRG and TCRB rearrangements were found in 46, 17 and 10% of cases, respectively. As compared to childhood precursor-B-ALL, Ig/TCR rearrangements in infant ALL were less frequent and more oligoclonal. MLL-AF4 and MLL-ENL-positive infants demonstrated immature rearrangements, whereas in MLL-AF9-positive leukemias more mature rearrangements predominated. The immature Ig/TCR pattern in infant ALL correlated with young age at diagnosis, CD10 negativity and predominantly with the presence and the type of MLL translocation. The high frequency of immature and oligoclonal Ig/TCR rearrangements is probably caused by early (prenatal) oncogenic transformation in immature B-lineage progenitor cells with germline Ig/TCR genes combined with a short latency period.

Introduction

Despite considerable improvement in clinical outcome in childhood acute lymphoblastic leukemia (ALL), infant ALL (within the first year of life) is still associated with a poor survival. Virtually all infants with ALL achieve complete remission but many patients relapse, often within 1 year after diagnosis (reviewed in1, 2, 3, 4.

The immunophenotype of infant ALL mainly represents the CD10-negative pro-B-ALL, often with coexpression of myeloid-associated antigens. This is in contrast to the predominantly CD10-positive immunophenotype (common-ALL and pre-B-ALL) in childhood precursor-B-ALL, indicating that infant ALL reflects an earlier stage of B-cell development. The remaining group of B-lineage infant ALL mainly consists of common ALL and pre-B-ALL. Mature B-ALL (Burkitt leukemia) is reported exceptionally and T-lineage ALL is found in only 4% of infant cases.5, 6, 7

Molecular analysis of infant ALL reveals chromosome abnormalities in about 70–90% of patients, mainly representing translocations involving the mixed lineage leukemia (MLL) gene on chromosome 11q23.6, 8 In the total group of acute leukemias, a broad spectrum of 50 different MLL partner genes have been identified,9 with a predominance in infant ALL of the AF4 gene in t(4;11) (q21;q23), followed by the ENL gene in t(11;19)(q23;p13) and the AF9 gene in t(9;11) (p22;q23). Detection of a rearranged MLL gene is clinically important for identification of oncogenetically different subgroups, as its occurrence has frequently been associated with poor clinical outcome.10, 11, 12, 13, 14

Most infant ALL do not express the CD10 molecule and it is assumed that at least in MLL-AF4-positive infant ALL patients the leukemic cells arise prenatally.15 Therefore, it can be anticipated that they also show more immature patterns of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements. So far, analysis of Ig/TCR gene rearrangements was only performed in small series of infant ALL and mainly focused on IGH using limited polymerase chain reaction (PCR) primer sets and/or limited probes for Southern blot analysis.16, 17, 18, 19, 20, 21, 22 IGH rearrangements were found in the vast majority of patients (>90%), whereas IGK genes were in germline configuration in most patients (>85%). TCRB and TCRG rearrangements were less frequently observed (<20% of the patients), whereas TCRD rearrangements occurred in almost half of the patients. Thus, these data suggest an immature Ig/TCR pattern in infant ALL.

It has previously been shown in childhood precursor B-ALL that Ig/TCR gene rearrangement patterns are related with age23, 24, 25 as well as the presence of TEL-AML1 fusion gene transcripts.26

Therefore, we aimed to perform detailed analysis of Ig/TCR gene rearrangements in 133 infant ALL cases to investigate whether Ig/TCR gene rearrangement patterns were related to the presence and type of MLL rearrangement, immunophenotype (CD10 expression) and age at diagnosis.

Materials and methods

Patients and cell samples

This study is part of the INTERFANT-99 study, an international treatment protocol for infant ALL (n=482), supported by a large group of national study groups. A total of 133 infant precursor B-ALL samples (28%; 133/482) were included in the present laboratory study based on the availability of cytospin slides for split-signal fluorescence in situ hybridization (FISH) analysis and/or the availability of peripheral blood (PB) and/or bone marrow (BM) samples at initial diagnosis for the isolation of RNA and/or DNA. Our study group was representative for the total group of patients in the INTERFANT-99 study, according to gender, age, CD10 expression and MLL translocations. No additional information on the presence of other translocations frequently observed in childhood ALL (TEL-AML1, E2A-PBX1, BCR-ABL) was available. Mononuclear cells were isolated from PB and/or BM using Ficoll–Paque density gradient centrifugation.

The immunophenotype was assessed by flow cytometry in the different collaborating study groups and was known in 132 out of 133 infants (99%). Ninety-two infant ALL cases (69%) showed the CD10-negative pro-B-ALL immunophenotype, whereas the remaining cases were common ALL (n=17, 13%) or pre-B-ALL (n=22, 17%). In one patient with precursor-B-ALL the precise immunophenotype was not established.

Infant ALL cases were subdivided into six groups according to their age at diagnosis: 0–2 months of age (n=18), 2–4 months (n=27), 4–6 months (n=20), 6–8 months (n=22), 8–10 months (n=24) and 10–12 months of age (n=22).

Detection of a rearranged MLL gene

The vast majority of infant ALL cases (>90%) could be evaluated for the presence of MLL rearrangements by use of FISH, reverse transcriptase PCR (RT–PCR) and/or sequencing. In 89 infant ALL cases (67%), the split-signal FISH (DAKO, Glostrup, Denmark) was used as described previously.27 The presence of MLL-AF4, MLL-AF9 and MLL-ENL fusion gene transcripts was studied by RT–PCR in 128 (96%), 96 (72%) and 98 (74%) infant ALL cases, respectively. RNA was isolated using a GeneElute Mammalian Total RNA isolation kit (Sigma, St Louis, MO, USA) according to the manufacturer's instructions or phenol chloroform extraction according to Chomczynsky and Sacchi.28

RT–PCR was performed for the detection of the three main types of MLL fusion gene transcripts. Cell lines with the relevant fusion gene were included as positive controls: MV-11; RS4;11 for MLL-AF4 (DSMZ, Braunschweig, Germany) and ALL-PO;29 MonoMac6, THP-1 (DSMZ) for MLL-AF9 and KOPN-1, KOCL-33 (kindly provided by Dr M Seto, Nagoya, Japan) and patient control samples for MLL-ENL.

MLL-AF4 fusion gene transcripts were detected with the MLL-A and AF4-B primers as described previously.30 For the detection of MLL-AF9, a newly designed primer in exon 9 of the AF9 gene (AF9-B 5′-AGCGAGCAAAGATCAAAATC-3′) was used in combination with MLL-A. For the detection of MLL-ENL, two new primers were designed: one in exon 2 (ENL-Bs 5′-GGCTTGGGGAAGCTGTC-3′) used in combination with MLL-A and one primer in exon 9 of the ENL gene (ENL-Dl 5′-CCTCGCCTGACGAAGAGT-3′) that was combined with the previously published MLL-C primer.30 RT–PCR conditions for the detection of MLL-AF4, MLL-AF9 and MLL-ENL fusion gene transcripts were described previously.30, 31

In eight infant ALL cases (6%) with an unknown partner gene rearranged to the MLL gene, the MLL DNA breakpoint region was sequenced using the recently described long-distance inverse PCR technique.32 In addition, DNA of four out of the five MLL-negative CD10-negative infant ALL cases were sequenced to detect unusual MLL gene rearrangement including inversions or duplications that might be missed by split-signal FISH.

PCR analysis of Ig/TCR gene rearrangements

DNA was isolated from 133 infant ALL cases using QIAamp DNA mini kit (Qiagen, Hilden, Germany) or DNA Purification System Blood kit (Gentra systems, Minneapolis, MN, USA).

Subsequently, PCR heteroduplex analysis of IGH (VH-DH-JH, DH-JH), IGK (Vκ-Jκ, Vκ-Kde, intron-Kde), IGL (Vλ-Jλ), TCRG (Vγ-Jγ), TCRD (Dδ2-Dδ3, Vδ2-Dδ3, Vδ-Jδ, Vδ2-Jα) and TCRB (Vβ-Dβ-Jβ, Dβ-Jβ) gene rearrangements were performed as described previously.33 Clonal PCR products were sequenced and analyzed as described previously.33, 34, 35, 36 Ig and TCR gene rearrangements in infant ALL cases were compared with the same rearrangements recently reported in 274 childhood precursor-B-ALL cases (>1 year at diagnosis).23, 24, 25

Southern blot analysis of Ig/TCR gene rearrangements

Southern blot analysis was performed in 74 of 135 infant ALL cases (55%) for IGH and TCRD gene rearrangements and in 61 out of 135 infant cases (45%) for IGK-Kde gene rearrangements. Southern blot analysis was performed using BglII-digested DNA in combination with IGHJ6, TCRDJ1 and/or IGKDE probes (DAKO Corporation, Carpinteria, CA, USA).37, 38, 39 The lower number of patients analyzed for IGK-Kde was because of insufficient DNA quality after rehybridization of the blots in 13 patients.

Real-time quantitative PCR for RAG1 and RAG2 transcript levels

Levels of recombination activating gene (RAG)1 and RAG2 transcripts were analyzed in 42 infant ALL patients with enough RNA available as described previously.40 The results were compared with 32 previously reported childhood ALL cases.40

Statistical analysis

To compare the prevalence of Ig/TCR gene rearrangements between different groups (MLL groups, infant ALL vs childhood ALL patients, CD10-positive vs CD10-negative) the χ2-test (two-sided) was used (with calculation of Fisher's exact P-values in case of low prevalence). Relations of Ig/TCR gene rearrangements with different age groups were calculated using the χ2-test for trend. The Mann–Whitney U-test was used for comparison of RAG1 and RAG2 transcript levels between different groups. In addition, Spearman correlations (rs) were used to evaluate the relation between RAG1/RAG2 transcription levels and the number of gene rearrangements at different Ig/TCR loci.

P0.05 were considered statistically significant.

Results

MLL translocations and relation to age and immunophenotype

In 124 out of 133 infant ALL cases (93%) the presence or absence of a rearranged MLL gene could be analyzed by split-signal FISH, RT–PCR analysis and/or by sequencing the MLL DNA breakpoint region. For the nine infant ALL cases with an unknown MLL status, cytogenetic data were not available. In 26/124 infant ALL cases (21%), no MLL rearrangement was observed. In 98/124 cases (79%), an MLL rearrangement was detected, which concerned 51 patients (41%) with MLL-AF4 fusion gene transcripts, 22 infants (18%) with MLL-ENL fusion gene transcripts, 13 infants (11%) with MLL-AF9 fusion gene transcripts, and 12 cases (10%) with other MLL partner genes: AF10 (10p12; n=3), EPS15 (1p32; n=2), SELB (3q21; n=1), MSF (17q23; n=1), LAF4 (2q11; n=1), unidentified (n=4).

To investigate the relationship between MLL rearrangements and age, infant ALL cases were subdivided into six age groups. The majority of infant ALL cases without an MLL translocation were present in the oldest age groups (6–8, 8–10 and 10–12 months; mean age 9.0 months), whereas MLL-AF4 fusion gene transcripts were predominantly present in the younger infants (0–2, 2–4 and 4–6 months; mean age 4.5 months) (Figure 1a). Infant ALL cases with the MLL-ENL (mean age 5.8 months), MLL-AF9 (mean age 7.8 months) and other MLL (mean age 5.0 months) fusion gene transcripts were more equally distributed over the six age categories (Figure 1a).

Figure 1
Figure 1

Age distribution of MLL gene rearrangements and their relation with CD10 expression. (a) Distribution of five MLL groups in six different age groups. (b) Frequency of CD10-positive and negative patients in the different MLL groups.

Analysis of immunophenotype in the different MLL groups revealed that 50 out of 51 (98%) infant ALL cases with MLL-AF4 were pro-B-ALL (CD10-negative). Interestingly, the only pre-B-ALL (CD10-positive) MLL-AF4-positive case had a DNA breakpoint in intron 10 of the AF4 gene, that is, outside the classical AF4 breakpoint cluster region (data not shown). MLL-ENL and MLL-AF9 infants were predominantly pro-B-ALL (CD10-negative), whereas MLL-negative cases were mainly CD10-positive (Figure 1b). All patients with a fusion partner other than AF4, AF9 or ENL were CD10-negative. Four out of 22 pre-B-ALL cases (18%) were CD10-negative of which two cases were MLL-AF9, one case was MLL-ENL and one patient was MLL-AF10-positive (Figure 1b).

In a total group of 132 patients, CD10-positive patients had a significant older age at diagnosis as compared to CD10-negative patients (Mann–Whitney U-test, P<0.05).

Ig and TCR gene rearrangements in infant ALL versus childhood precursor-B-ALL (1 year)

Frequencies of Ig and TCR gene rearrangements and clonality status for IGH, IGK-Kde and TCRD are shown in Table 1 for 133 infant ALL cases. IGH gene rearrangements were found in almost all patients (91%), whereas light-chain gene rearrangements (IGK, IGL) as well as cross-lineage TCR gene rearrangements were rare in infant ALL patients, except for TCRD gene rearrangements (46%). Southern blot analysis of the IGH locus (Figure 2), as well as the IGK-Kde and TCRD loci revealed an unusually high frequency of oligoclonality at all three loci.

Table 1: Frequencies of Ig/TCR gene rearrangements and oligoclonality in infant and childhood precursor-B-ALL
Figure 2
Figure 2

Oligoclonality of the IGH locus in infant ALL. Southern blot analysis of the IGH locus using BglII-digested DNA in combination with IGHJ6 probe in six infant ALL cases.

Ig and TCR gene rearrangements in infant ALL cases were compared with the same rearrangements in 274 recently reported childhood precursor-B-ALL cases (1 year at diagnosis)23, 25 (Table 1). These data demonstrate that the frequency of IGK, TCRB (Vβ-Dβ-Jβ), TCRG and TCRD gene rearrangement in infant ALL is significantly lower compared to childhood precursor-B-ALL (P<0.05). No significant difference was observed for Vλ-Jλ rearrangements between infant and childhood ALL cases. Although no differences were observed for overall frequency of IGH gene rearrangements, the IGH pattern of infant ALL is less mature with frequent involvement of incomplete (DH-JH) rearrangement, whereas in childhood precursor-B-ALL mainly complete (VH-DH-JH) rearrangements were observed (P<0.05). We also observed a statistically significant (P<0.05) higher frequency of patients with oligoclonality defined by combined Southern blot and PCR data in infant ALL (IGH 76%; TCRD 54%) as compared to childhood ALL (IGH 38%; TCRD 25%) (Table 1). In addition, a higher level of oligoclonality (higher number of rearranged bands on Southern blot) was observed in infant ALL (Figure 2).

Spectrum of gene segment usage in infant ALL versus childhood precursor-B-ALL (1 year)

To determine the involved gene segments of the detected Ig/TCR gene rearrangement, sequence analysis was performed. The majority of DH-JH sequences (n=87) contained a DH2 (24%), DH3 gene (37%), JH4 (30%) and/or JH6 gene segments (41%). No statistically significant differences for gene segment usage of DH-JH rearrangements were observed between infant and childhood ALL cases. Analysis of 86 VH-DH-JH sequences showed that VH3 (33%) and VH6 (29%) were most frequently used in infant ALL, whereas VH3 was predominantly used in childhood ALL (52%, P<0.0001).

No differences could be observed for gene segment usage in IGK rearrangements (Kde, Vκ-Jκ) between infant and childhood ALL patients.

TCRG rearrangements in both infant ALL and childhood ALL involved mainly Vγ9 (48% and 29%, respectively). Jγ1.1 was most frequently used in infants (57%) compared to Jγ2.3 in childhood ALL cases (64%, P<0.05). Sequence analysis of Vδ2-Jα rearrangements in infant ALL cases revealed a broad spectrum of Jα gene segment usage, whereas in childhood ALL Jα29 is predominantly used (51%, P<0.0001). The Dβ-Jβ and Vβ-Dβ-Jβ gene rearrangements were not analyzed because of their low frequency.

No differences in gene segment usage at Ig and TCR loci were observed between the different MLL groups.

Relation between Ig/TCR gene rearrangement and MLL translocations in infant ALL

To analyze the relation between Ig/TCR gene rearrangements and MLL aberrations, we first compared Ig/TCR gene rearrangement patterns of infants with (n=98) and without (n=26) a rearranged MLL gene (Table 1). Patients with an MLL rearrangement showed a significantly higher frequency of incomplete IGH (DH-JH) and a lower frequency of complete IGH (VH-DH-JH), IGK-Kde and cross lineage TCRB, TCRG and TCRD rearrangements compared to MLL-negative patients. Southern blot analysis of the IGH, IGK-Kde and TCRD loci revealed that, although not statistically significant, IGK-Kde as well as TCRD were more frequently oligoclonal in MLL-negative patients.

Further analysis of 98 infants with a rearranged MLL gene (MLL-AF4, MLL-ENL, MLL-AF9 or MLL rearrangements with other partner genes) revealed that incomplete IGH rearrangements (DH-JH) were present at a higher frequency in infants with MLL-AF4 compared to patients with MLL-ENL, MLL-AF9 and other MLL rearrangements, whereas complete IGH rearrangements (VH-DH-JH) were predominantly found in patients with MLL-AF9 (Table 1). IGK (Vκ-Jκ, Kde), IGL, TCRB (Dβ-Jβ) and Vδ2-Jα rearrangements were detected at significantly higher frequencies in patients with the MLL-AF9 fusion gene transcript as compared to MLL-AF4 and MLL-ENL-positive patients.

Although not statistically significant, analysis of oligoclonality at the IGH locus demonstrated a higher frequency and higher level of oligoclonality in patients with MLL-AF4, MLL-ENL and other MLL rearrangements (Table 1; Figure 2), whereas oligoclonality of the TCRD locus was lower in cases with MLL-AF9 (Table 1). Overall, these data indicate that both MLL-AF4 and MLL-ENL-positive patients have more immature Ig gene rearrangements and that TCR gene rearrangements are less frequent. In contrast, Ig gene rearrangements of MLL-AF9-positive infant ALL cases are more mature and TCR gene rearrangements occur more frequently, both comparable with infant ALL without an MLL rearrangement. The Ig and TCR gene rearrangement patterns in the latter two groups are comparable with the Ig/TCR pattern of childhood ALL cases between 1 and 3 years old at diagnosis, except for TCRG that is much less frequent in all infant ALL subgroups.

Ig/TCR gene rearrangements in relation to immunophenotype in infant ALL

To investigate the relation between Ig/TCR gene rearrangements and immunophenotype, we analyzed Ig/TCR gene rearrangements in CD10-positive (n=36) and CD10-negative (n=96) infant ALL cases, irrespective of MLL status. A significantly higher frequency of incomplete IGH (DH-JH) and lower frequencies of IGH (VH-DH-JH), IGK (Kde, Vκ-Jκ) and TCRB (Dβ-Jβ) rearrangements were observed in CD10-negative infant ALL cases as compared to CD10-positive cases (P<0.05) (Figure 3a). Ig light chain rearrangements were particularly present in CD10-positive pre-B-ALL patients (60 vs 25% in other patients).

Figure 3
Figure 3

Frequency of Ig and TCR gene rearrangements. (a) Frequency of Ig and TCR gene rearrangements in six different age groups. (b) Frequency of Ig and TCR gene rearrangements in CD10-positive and negative patients. (* statistically significant differences (P<0.05))

Reliable multivariate analysis could not be performed because of the low numbers in the different subgroups. Therefore, we analyzed the frequency of Ig/TCR gene rearrangements in both CD10-positive as well as CD10-negative infants of the different MLL groups. No statistically significant differences in Ig/TCR patterns were observed, implying that the immunophenotype in infant ALL patients with a particular MLL rearrangement does not affect the Ig/TCR pattern. In contrast, within CD10-negative infant ALL cases statistically significant differences in Ig/TCR patterns were observed between patients with different MLL partner genes, and these differences resembled those observed in the total group of patients (both CD10-negative and -positive). Likewise, in the CD10-positive group, significantly more IGK and IGL gene rearrangements were observed in MLL-AF9-positive patients as compared to MLL-ENL-positive patients. Overall, these results strongly suggest that the Ig/TCR gene rearrangement patterns are related to the presence and type of MLL rearrangement but not to the immunophenotype of the ALL.

Ig/TCR gene rearrangements in relation to age at diagnosis in infant ALL

To investigate the relation between Ig/TCR gene rearrangements and age at diagnosis, we analyzed Ig/TCR rearrangements in the six different age groups (Figure 3b). We observed significantly more immature incomplete DH-JH rearrangements in the younger age groups and more mature VH-DH-JH rearrangements in older patients (P<0.05). Also IGK-Kde and cross-lineage Vδ2-Jα rearrangements occurred more frequently in older infants (P<0.05).

Analysis of relation between Ig/TCR and age at diagnosis could also be performed in the two largest MLL groups, that is, MLL-AF4 and MLL-ENL. These analyses showed that within the MLL-AF4-positive group significantly less VH-DH-JH rearrangements were detected in younger (0–2 months) infant ALL patients compared to older patients (2–4, 4–6 and 8–10 months); in MLL-ENL-positive patients significantly less VH-DH-JH rearrangements were observed in young patients (0–2, 2–4 and 4–6 months) compared to older patients (6–8 and 8–10 months) (P<0.05). These data indicate that within these two large MLL groups the Ig/TCR gene rearrangement pattern is also related to age.

Analysis of Ig/TCR gene rearrangements in individual age groups showed significant differences between the different MLL groups, indicating that the presence and type of MLL rearrangement is more dominantly affecting the Ig/TCR pattern than age.

RAG1 and RAG2 gene expression

No significant difference in the levels of RAG1/RAG2 in 42 infant ALL cases was observed between the different MLL groups. The mean transcription levels of RAG1 and RAG2 in infant ALL cases (n=42) were 3.2 and 14.0, respectively, which is much higher than previously determined RAG1/RAG2 levels of 0.1 and 0.2, respectively, in childhood ALL cases (Mann–Whitney U-test, P<0.001).40 The level of RAG1 expression correlated with the total number of rearrangements at TCRD locus (rs=0.35, P=0.024) as well as the total number of TCR gene rearrangements (rs=0.33, P=0.032), but not with the total number of rearrangements at IGH, IGK, IGL, TCRB, TCRG loci or the total number of Ig gene rearrangements. In addition, the level of RAG2 expression was significantly higher in patients with oligoclonality at the IGH locus as compared to monoclonal patients (Mann–Whitney U-test, P=0.010). There was no correlation between levels of RAG1 and RAG2 expression, and age at diagnosis or CD10 expression.

Discussion

Our study shows that in infant ALL Ig and TCR gene rearrangements were significantly less frequent, more often oligoclonal and more immature compared to the same rearrangements described in childhood precursor-B-ALL.23, 24, 25 Within infant ALL, different Ig/TCR patterns were observed, depending on age of the infant at diagnosis, immunophenotype (CD10 expression) and particularly depending on the presence and type of MLL rearrangement. Immature Ig/TCR gene rearrangements were found in MLL-AF4 and MLL-ENL-positive infant ALL patients, which generally presented at young age (0–6 months) with a CD10-negative ALL. In contrast, a more mature Ig/TCR pattern was observed in MLL-AF9-positive patients and in MLL leukemias without a MLL rearrangement, which generally presented at older age and more often presented with a CD10-positive ALL.

It has previously been described in infant, childhood and adult leukemia patients that Ig/TCR gene rearrangements are related to age.18, 20, 23, 24, 26, 41, 42 We demonstrated that infant ALL patients of 0–6 months of age at diagnosis as compared to older infants (6–12 months) showed a more immature Ig/TCR pattern, particularly characterized by a high prevalence of incomplete IGH gene rearrangements and low frequencies of complete IGH, IGK and TCRD gene rearrangements (Figure 4). Infants with MLL-AF4 were more prevalent in the youngest age groups, whereas older infant leukemias more frequently lacked a rearranged MLL gene.

Figure 4
Figure 4

Frequencies of CD10 negativity, MLL groups and Ig/TCR gene rearrangements according to age at diagnosis. y-axes are depicted as a 0–100% scale. Each bar represents the frequency relative to 100%.

We believe that the maturity status of the Ig/TCR gene rearrangements in infant ALL is mainly determined by the MLL partner gene based on clear differences in Ig/TCR gene rearrangements which are independent of age at diagnosis and immunophenotype. However, there was an influence of age in the MLL-AF4 and MLL-ENL leukemia patients, with a higher frequency of VH-DH-JH rearrangements at higher age (>6 months).

Other studies also demonstrated that Ig/TCR gene rearrangement patterns might be influenced by the presence of a specific chromosomal aberration, that is, TCRG appeared to be less frequent in infant ALL and in MLL-AF4-positive precursor B-ALL and were completely absent in E2A-PBX1 positive precursor B-ALL cases.20 Furthermore, the presence of TEL-AML1 fusion gene in childhood precursor-B-ALL patients is associated with a more mature Ig/TCR gene rearrangement pattern.26

The high prevalence of oligoclonality at the IGH locus in our infant ALL patients, particularly in MLL-AF4-positive patients, supports the immaturity of the malignantly transformed cell with a DH-JH gene rearrangement or germline IGH genes. Interestingly, oligoclonality and ongoing rearrangements of IGH and TCRD genes both correlated with higher transcriptional RAG1/2 levels, which were significantly higher in the entire infant ALL group as compared to childhood ALL.40 This would fit with the immaturity of infant ALL as RAG1/2 levels during normal B-cell development are highest during DH-JH and VH-DH-JH gene rearrangements.43 It has previously been shown that CD34 positivity was more common in MLL-AF4-positive ALL cases compared to E2A-PBX1-positive ALL cases, also supporting the immaturity of the malignant MLL-AF4-positive cell.20 Furthermore, there is convincing evidence for prenatal development of the MLL-AF4 fusion gene as the main initiating event in t(4;11) positive infant leukemias.15, 19, 44, 45, 46, 47, 48 This transformation is assumed to occur in early B-cell precursors with germline or incomplete IGH rearrangements, followed by second mutation(s) that lead to leukemia.49

Lack of CD10 expression on the cell surface might be related to the presence of a rearranged MLL gene. Almost all MLL-AF4-positive leukemias had a pro-B-ALL phenotype. Furthermore, all pre-B-ALL lacking CD10 had a rearranged MLL gene. This finding is supported by recent studies suggesting that CD10-negative pre-B-ALL cases may represent pro-B-ALL cases that maintain their tendency to rearrange and express their Ig heavy chain.50, 51

A recent study suggested that MLL-AF4-positive infant ALL is initiated later in fetal development than most MLL-AF4-negative childhood B-cell precursor ALL (<3 years), based on the presence and absence of nucleotide insertions at the DH-JH junction, respectively.22 In line with this observation, nucleotide insertion at the DH-JH junction was present in 73% of MLL-AF4 infant ALL cases in this study. The number of inserted nucleotides at the DH-JH junctions was not significantly different between the different MLL groups (data not shown).

An intriguing question is how the MLL gene or its fusion partner gene may be related to the Ig/TCR gene rearrangement pattern, either directly or indirectly. The oncogenic mechanism behind MLL fusion proteins has been obscured by the extraordinary diversity of fusion partners. Despite this diversity, all cases of MLL cluster together according to their gene expression pattern.52, 53, 54 Nevertheless, it has recently been demonstrated in Drosophila that MLL-AF9 confers a higher rate of cell proliferation, whereas MLL-AF4 appeared to delay cell-cycle progression.55, 56 Recently, Dupret et al.57 demonstrated a significant role for MLL partner genes in determining the stage of maturation arrest in acute leukemia in favor of a maturation arrest at the early stages of myeloid/lymphoid specification. The mechanism by which the MLL gene or fusion partner genes influence Ig/TCR gene rearrangements remains to be elucidated.

In summary, our results in infant precursor B-ALL show an immature Ig/TCR gene rearrangement pattern with a higher frequency of oligoclonality as compared to childhood precursor B-ALL. Within infant ALL the maturity status of Ig/TCR gene rearrangements is related to the type of MLL rearrangement and to some extent to the age at diagnosis. Our data suggest that the high frequency of immature and oligoclonal Ig/TCR gene rearrangements in infant ALL is caused by an early (prenatal) oncogenic transformation in early B-lineage progenitor cells, most of which probably had germline Ig/TCR genes at the time of transformation.

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Acknowledgements

We are grateful to participants of the INTERFANT-99 study group for kindly providing infant ALL samples and to Paola de Lorenzo (Interfant-99 data center, Monza, Italy) for support in handling of the clinical data. We gratefully acknowledge DAKO (Glostrup, Denmark) for providing MLL probes for split-signal FISH. We are grateful to Danielle Jacobs and Ingrid Wolvers-Tetero for split-signal FISH analysis to AM Wijkhuijs for Southern blot analysis, to M de Bie and PG Hoogeveen for technical assistance, (Erasmus MC, Department of Immunology, Rotterdam, The Netherlands), to Jessica Buijs-Gladdines (Erasmus MC, Sophia Children's Hospital, Rotterdam, The Netherlands) and Elisabeth van Wering (Dutch Child Oncology Group, The Hague, Rotterdam) for sample collection and processing, to Wolf-Dieter Ludwig for immunophenotyping (Department of Hematology, Oncology and Tumor Immunology, Robert-Rössle-Clinic, HELIOS Clinic Berlin-Buch, Charité, Germany), to Oskar A. Haas for cytogenetics and FISH analysis (CCRI, Vienna, Austria), to T. Lion for RT–PCR analysis (CCRI, Vienna, Austria), and to Susi Fischer and Uli Monschein for technical assistance (CCRI, Vienna, Austria). We thank Marieke Comans-Bitter (Erasmus MC, Department of Immunology, Rotterdam, The Netherlands) for preparation of the figures. This study was performed on behalf of the INTERFANT-99 study group (coordinator: R. Pieters), which is composed of AIEOP (G. de Rossi, A. Biondi; Italy), ANZCHOG (R. Suppiah; Australia, New Zealand), Argentina (M. Felice), BFM-A (G. Mann; Austria), BFM-G (M. Schrappe; Germany), COALL (G Janka-Schaub; Germany), CWPGH (J. Stary; Czech Republic), DCOG (R. Pieters; the Netherlands), DFCI consortium (L Silverman; USA), EORTC-CLCG (A. Fester; France, Belgium, Portugal), FRALLE (F. Mechinaud; France), Hong Kong (CK Li), NOPHO (L Hovi; Scandinavian countries), PINDA (M Campbell; Chile), PPLLSG (T Szczepañski; Poland), SJCRH (JE Rubnitz; USA), UKCCSG (I Hann, A Vora; UK).

Financial support: Dutch Cancer Foundation (KWF grant EUR 2001–2441); Associazione Italiana Ricerca sul Cancro (AIRC): Regional Grant Cod.1105 to A.B; AIRC 2000 to G.B.; PIN 2004 to G.B.; Fonds zur Förderung der wissenschaftlichen Forschung (FWF P-13575-MED) to R.P-G; Deutsche Krebshilfe: 50-2698-Schr1; German BMBF Competence Network Pediatric Oncology/Hematology (Grant 01 Gi 9963/2).

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Affiliations

  1. Department of Immunology, Erasmus MC, Rotterdam, The Netherlands

    • M W J C Jansen
    • , V H J van der Velden
    •  & J J M van Dongen
  2. Centro Ricerca Tettamanti, Pediatric Clinic, University of Milano Bicocca, Monza, Italy

    • L Corral
    •  & A Biondi
  3. Children's Cancer Research Institute and, St Anna Kinderspital, Vienna, Austria

    • R Panzer-Grümayer
  4. Department of Pediatrics, University Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany

    • M Schrappe
    •  & A Schrauder
  5. Institute of Pharmaceutical Biology/ZAFES/DCAL, University of Frankfurt, Frankfurt/Main, Germany

    • R Marschalek
    •  & C Meyer
  6. Department of Pediatric Oncology/Hematology, Erasmus MC-Sophia Children's Hospital, Rotterdam, The Netherlands

    • M L den Boer
    •  & R Pieters
  7. Department of Epidemiology and Biostatistics, Erasmus MC, Rotterdam, The Netherlands

    • W J C Hop
  8. Interfant Trial Data Center, University of Milano-Bicocca, Department of Clinical Medicine and Prevention, Italy

    • M G Valsecchi
  9. Department of Pediatrics, University of Padova, Padova, Italy

    • G Basso

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https://doi.org/10.1038/sj.leu.2404578

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