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[Cancer Research 61, 8547-8553, December 1, 2001]
© 2001 American Association for Cancer Research

Molecular Biology and Genetics

Molecular Analysis of Single Colonies Reveals a Diverse Origin of Initial Clonal Proliferation in B-Precursor Acute Lymphoblastic Leukemia that Can Precede the t(12;21) Translocation1

Victoria J. Weston, Carmel M. McConville, Jillian R. Mann, Philip J. Darbyshire, Sarah Lawson, John Gordon, Paul A. H. Moss, A. Malcolm R. Taylor and Tatjana Stankovic2

CRC Institute for Cancer Studies [V. J. W., P. A. H. M., A. M. R. T., T. S.], Division of Medical Genetics [C. M. M.], MRC Centre for Immune Regulation [J. G.], University of Birmingham, Birmingham B15 2TT, and Birmingham Children’s Hospital, Steelhouse Lane, Birmingham B4 6NH [V. J. W., J. R. M., P. J. D., S. L.], United Kingdom


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pathogenesis of pediatric B-precursor acute lymphoblastic leukemia is largely unknown, and even with nonrandom chromosomal translocations present, the precise order of clonal molecular events is undefined. We developed an in vitro system using cytokines interleukin (IL)-3, IL-7, IL-10, and FMS-like tyrosine kinase 3 ligand with CD40 ligand-expressing fibroblasts to obtain single blast colonies from which clonal immunoglobulin heavy chain (IgH), T-cell receptor {delta} gene rearrangements, and, in t(12;21)-positive cases, TEL-AML1 fusion transcripts could be simultaneously PCR amplified. The proliferation of early tumor progenitors increased subclone detection enabling us, in seven diagnostic samples, to determine the stage of differentiation at which each leukemia occurred. Four were derived from the stage before initiation of IgH rearrangement, one during recombination of variable, joining, and diversity segments of the heavy chain gene VDJH, and two after completion of IgH rearrangement. Furthermore, analysis of a t(12;21)-positive leukemia with unusually late onset, identified both TEL-AML1-positive and -negative colonies carrying a clonal T-cell receptor {delta} rearrangement, inferring the presence of clonal expansion before the occurrence of the t(12;21). In contrast, in a typical, early onset t(12;21)-positive leukemia, the t(12;21) appeared to be the first clonal event. In both leukemias, the t(12;21) occurred before recombination of variable, joining and diversity segments of the heavy chain gene VDJ.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Clonal immune system gene rearrangements are a distinctive feature of lymphoid malignancies and can serve as a useful disease "fingerprint" for individual tumors (1) . Pediatric B-precursor ALLs3 in 90% of cases carry clonal IgH gene rearrangements, and 55–90% of tumors show the presence of clonal cross-lineage rearrangements, either TCR{delta}, TCR{gamma}, or both (2, 3, 4, 5, 6) .

Tumor transformation in pediatric B-precursor ALL may precede the stage of differentiation indicated by the maturity of the dominant leukemic clone. In some patients, analysis of immune system gene rearrangements suggests the presence of multiple subclones (7, 8, 9, 10) , and by comparing the subclonal rearrangements within a leukemia and exploiting the sequential process of IgH VDJ recombination, the maturity of the proliferating tumor progenitor can be ascertained. The tumor may potentially arise at three distinguishable stages during B-cell differentiation: (a) it can arise from a progenitor with VDJH rearrangement completed, rarely producing any detectable subclonal variation; (b) the tumor progenitor can arise at the earlier stage of D-JH recombination, recognized by the presence of subclones using the same D-JH segment but a different variable segment of the heavy chain gene VH; and (c) the tumor can arise before the initiation of VDJH recombination with IgH genes still segment in germ-line configuration. This stage of tumor transformation is recognized by the presence of multiple leukemic subclones with independent immune system gene rearrangements. We have observed previously that relapses in ALL patients are often associated with the emergence of subclones carrying independent IgH rearrangements, not detectable at presentation, as a consequence of the proliferation of an early nonrearranged tumor progenitor (9) .

Access to the earliest population of tumor blasts harboring the molecular changes crucial for initiation of leukemogenesis is difficult, mostly because of its size and the absence of detectable clonal markers. Also, indirect evidence for the proliferation of an IgH nonrearranged progenitor, through the presence of multiple unrelated IgH-rearranged subclones at presentation of the disease, is hampered by a practical consequence of the competitive nature of PCR, which favors amplification of the rearrangement from the dominant leukemic clone.

The t(12;21) is a single genetic event detectable in ~25% of pediatric B-precursor ALL cases (11, 12, 14, 14) , most frequently occurring between the ages of 1 and 5 years (15 , 16) . This translocation results in an "in-frame" fusion of two critical regulators of hematopoiesis, TEL (ETV6) at chromosome 12p13 and AML1 (CBFA2) at chromosome 21q22 (17, 18, 19, 20) . The gene fusion is consistently detected in neonatal blood spots of leukemia patients suggesting its origin in fetal development (21) . It is not clear, however, whether the occurrence and pathogenicity of this translocation is limited to a particular stage of early B-cell development. As tumor-specific clonal immune system gene rearrangements can also have an in utero origin (22 , 23) , the timing of the t(12;21) with respect to clonal immune system gene rearrangements remains to be established. Although initially considered as an event defining a good prognostic subgroup, it has become evident more recently that the biological behavior of leukemias carrying the t(12;21) is heterogeneous (11 , 15 , 24 , 25) .

The aim of this study was to establish the stage of B-cell differentiation at which clonal expansion is initiated in different B-precursor ALLs, as well as the relative timing of various clonal events. We optimized the in vitro culture of single blast colonies from leukemic bone marrow to improve the sensitivity of detection of multiple leukemic subclones. Twelve pediatric B-precursor ALL samples were subsequently studied. In 7 of those, we analyzed the DNA or cDNA from individual colonies for the presence of clonal IgH or TCR{delta} rearrangements. In two leukemias positive for the t(12;21), we searched for the simultaneous presence of TEL-AML1 transcripts and clonal immune system gene rearrangements. We established that in four of the seven analyzed cases, clonal proliferation initiated at the stage before IgH rearrangement. In one of the two t(12;21)-positive cases, we also demonstrated that the t(12;21) occurred after the initiation of clonal proliferation.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In Vitro Culture of Single Blast Colonies from Leukemic Bone Marrow.
Leukemic mononuclear cells were separated from total bone marrow samples using Lymphoprep (Nycomed). Cells were cultured in duplicate 96-well flat-bottomed plates in 100-µl volume of RPMI (with 10% FCS and L-Glutamine) at a concentration of 1 x 105/well in the following combinations: (a) on their own; (b) with cytokines added at concentrations known to be saturating, IL-3 (10 ng/ml), IL-7 (60 ng/ml), IL-10 (10 ng/ml), and Flt3L (10 ng/ml; R & D Systems); (c) with 1 x 104 cells/well CD40LMFs (kindly donated by Yong Jun Lui) irradiated with 50 Gy {gamma} rays; (d) with 1 x 104 cells/well control MFs irradiated with 50 Gy {gamma} rays; (e) with irradiated CD40LMFs and cytokines (at the same concentration as in c); and (f) with irradiated MFs and cytokines (at the same concentration as in d). Control wells were established without leukemic blasts but with either irradiated CD40LMFs or MFs alone. On days 4 and 6, proliferation in a single plate was assayed by pulsing for 18 h with 0.5 µCi/well [methyl-3H]Thymidine (Amersham Pharmacia Biotech). Between days 6–8, 30 single blast colonies (each containing 50–250 cells) were picked individually for each leukemia from different wells, and RNA or DNA was extracted as described previously (26 , 27) . cDNA was synthesized using the Qiagen Sensiscript cDNA synthesis kit with oligodeoxythymidylate primer (Life Technologies, Inc.).

PCR Analysis of Single Blast Colony DNA or cDNA.
DNA or cDNA from each colony was subjected to two rounds of PCR amplification of IgH or TCR V{delta}2-D{delta}3 immune system gene rearrangements and in t(12;21)-positive cases, TEL-AML1 transcripts, using either Taq Expand High Fidelity or Taq Expand Long Template DNA Polymerase (Roche). For the first round of IgH VDJ amplification, the Fr1 forward primer (5'-SAG GTR CAG CTG BWG SAG TCN G-3') was used together with the J1H reverse primer (5'-ACC TGA GGA GAC GGT GAC CAG GGT-3'). The second round was performed using either: (a) the Fr3A forward primer (5'-ACA CGG CYS TGT ATT ACT GT-3') with the J1H reverse primer; or (b) the Fr1 forward primer with the JPS (9) reverse primer (5'-ACC AGG GTC CCT TGG CCC CA-3'). The first round of amplification of the TCR V{delta}2-D{delta}3 region was performed with the VDOUT forward primer (5'-ACC AAA CAG TGC CTG TGT CAA TAG G-3') together with the DDIN reverse primer (5'-GTT TTT GTA CAG GTC TCT GTA GG-3'), whereas for the second round, the VDIN forward primer (5'-ACC TGG CTG TAC TTA AGA TAC TTG C-3') was used with the DDIN reverse primer. The TEL-AML1 fusion transcript was amplified as described before (28) . PCR products were separated by 10% polyacylamide gel electrophoresis (PAGE), and bands were cut out and crush eluted in water overnight for direct sequencing using a 3100 automated capillary sequencer (PE Applied Biosystems).

ASO Hybridization.
To prove that additional rearrangements were not induced in vitro, but present in the total tumor population before culture, we performed ASO hybridization. Oligonucleotides were designed to the most variable part of the IgH VDJ join of each rearrangement. The oligonucleotide sequences were as follows: ALL 9 (IgH clone 2), 5'-CTG TAC CAC AGA TCG GCG ATT T-3'; ALL 10 (IgH clone 2), 5'-GCC TGG TAT AGC AGC TCG T-3'; and ALL 2 (IgH clone 2), 5'-GCA GCA GCT GGA CAG GGG GGA TTT-3'. For each case, DNA from the total tumor population before culture, at dilutions of 1:0, 1:10, 1:100, and 1:1000 with normal DNA, together with the appropriate controls (DNA from the total tumor population of another leukemia, positive colony DNA and no DNA), were subjected to a single PCR amplification using the Fr3A and J1H primers. PCR products were separated by 8% PAGE, and blotting and hybridization were performed as described previously (29) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
In Vitro Culture System Allows Proliferation of Leukemic Cells and Formation of Single Blast Colonies.
We selected and optimized a combination of culture conditions to provide a universal method to enable the proliferation of leukemic blasts from the separated bone marrow of different B-precursor ALL cases. B-cell progenitors were shown previously to proliferate in the presence of the CD40L and cytokines IL-3, IL-7, and IL-10 (30, 31, 32, 33) . Flt3L was also shown to induce the proliferation of very early CD34+ cells (34 , 35) . Therefore, this combination of culture conditions was expected to sustain the proliferation of the earliest B-cell progenitors in vitro. Subsequently, 12 cases of B-precursor ALL bone marrow samples were subjected to the optimized procedure.

All 12 B-precursor ALLs (ALL 1–12) started forming visible blast colonies by day 4 of culture. On days 4 and 6, 18-h [methyl-3H]Thymidine incorporation was performed on pooled colonies in all cases to show that the apparent colonies were the result of the active proliferation of cells rather than physical clustering (data not shown). Although the initial proliferative responses to the different culture conditions were heterogeneous between tumors, by day 6, the response became uniform. In all leukemias, optimal colony formation occurred in the presence of both cytokines and CD40LMFs (Fig. 1A)Citation . Individual colonies, by this time, consisted of between 50–250 densely packed cells (Fig. 1B)Citation , the equivalent of up to about eight mitoses. The combination of cytokines and control nontransfected MFs did not support the proliferation of blasts and, hence, colony formation. We concluded that the positive effect of the CD40LMFs was the result of stimulatory signaling through CD40 and that the in vitro culture system is feasible to obtain single B-precursor ALL blast colonies.



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  		Fig. 1. Proliferation of B-precursor ALL blast cells in vitro. A, 18-h [methyl-3H]Thymidine incorporation showing proliferation of primary B-precursor ALL blasts from a representative case after 4 and 6 days in four distinct culture conditions. Day 4, ALL 5 showed maximum proliferation in the presence of cytokines. Day 6, ALL 5 required both CD40LMFs and cytokines for optimal colony formation. B, a single blast colony consisting of ~250 cells (up to eight mitoses) after 6 days in culture in the presence of irradiated CD40LMFs and cytokines (IL3, IL7, IL10, and Flt3L). Original magnification: x125.

 
B-Precursor ALL Is Derived from Different Stages of B-cell Differentiation.
Of the 12 presentation B-Precursor ALL bone marrow samples used to optimize the in vitro culture system, 7 were analyzed for the presence of multiple leukemic subclones by the detection of IgH gene rearrangements, both from the total tumor population before culture and from individual colonies after culture (Table 1)Citation .


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  		Table 1 Clonal VDJH and TCR{delta} rearrangements in seven paediatric B-precursor ALLs before and after culture in vitroa

 
PCR analysis of the total tumor population before culture revealed five cases (ALL 2, ALL 6, ALL 8, ALL 9, and ALL 11) with a single clonal IgH rearrangement and one (ALL 10) with two clonal IgH rearrangements of equal intensity. ALL 3 had no detectable clonal IgH rearrangement.

After 6–8 days in culture, the IgH status of the seven leukemias was reassessed by PCR analysis of individual colonies. Two of the seven leukemias (ALL 6 and ALL 8) both had the same IgH rearrangement detected in all of the colonies as detected from the total tumor population before culture (Table 1)Citation , inferring that ALL 6 and ALL 8 were each derived from a fully VDJH rearranged tumor progenitor.

The culture system increased the sensitivity of detection of minor subclones, and in ALL 9, ALL 10, ALL 2, and ALL 11, additional IgH rearrangements were detected after 6–8 days in culture (Table 1)Citation . To confirm that these additional rearrangements were not induced in vitro but present in the total tumor population before culture, we either simultaneously detected a second clonal marker, i.e., the TEL-AML1 transcript in ALL 11 and ALL 3, or performed ASO hybridization in ALL 9, ALL 10, and ALL 2 (Fig. 2)Citation . For each additional rearrangement, we designed an oligonucleotide corresponding to the most variable part of the VDJH sequence and performed hybridizations back to serial dilutions of the total tumor population before culture.



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  		Fig. 2. Three representative examples of ASO hybridization showing the pre-culture origin of three additional IgH rearrangements [see Table 1Citation (b)] from ALL 9 (IgH clone 2), ALL 10 (IgH clone 2), and ALL 2 (IgH clone 2) using the oligonucleotides described in "Materials and Methods" for hybridization. Lanes 1–4, serial dilutions of DNA from the total tumor population before culture with normal DNA [1:0 (1), 1:10 (2), 1:100 (3), and 1:1000 (4)]; 5, negative control 1 (DNA from the total tumor population of another leukemia); 6, positive control (positive colony DNA); and 7, negative control 2 (no DNA).

 
Compared with the total tumor population before culture, ALL 9 had an additional IgH rearrangement (ALL 9 IgH clone 2) detected in one of the colonies (Table 1Citation and Fig. 2Citation ). As this used the same partial D-JH sequence as the major clone (ALL 9 IgH clone 1), we speculated that transformation in ALL 9 had occurred after initiation of VDJH recombination (Fig. 3)Citation .



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  		Fig. 3. In vitro analysis of seven pediatric B-precursor ALLs identified three different stages of B-cell differentiation at which transformation occurred. ALL 10, ALL 2, ALL 11 and ALL 3 were each derived from an early tumor progenitor before the initiation of IgH VDJ recombination; ALL 9 was initiated during the process of VDJH rearrangement; and ALL 6 and ALL 8 were transformed after completion of IgH VDJ recombination.

 
In ALL 10, two clonal IgH rearrangements related in sequence and of equal intensity were detected from the total tumor population before culture. After culture, the same two related IgH rearrangements were simultaneously detected from different individual colonies indicating that they represented rearrangement of two IgH alleles. In an additional colony, another IgH rearrangement was identified with an independent sequence (Table 1)Citation . ASO confirmed that this additional rearrangement (ALL 10 IgH clone 2) was, indeed, present at a low level before culture (Fig. 2)Citation . Therefore, the two proliferating clones in ALL 10, one with two rearranged IgH alleles (ALL 10 IgH clones 1a and 1b) and the other with an independent IgH rearrangement (ALL 10 IgH clone 2), were derived from an immature progenitor at a stage before the initiation of VDJH recombination (Fig. 3)Citation .

In ALL 2, a single IgH rearrangement was identified from the total tumor population before culture, but after 6–8 days in culture, two additional IgH rearrangements were detected from separate colonies (Table 1)Citation . ASO, again, confirmed the presence of these additional rearrangements at a low level before culture (ALL 2 IgH clone 2 shown in Fig. 2Citation ; ALL 2 IgH clone 3 not shown). This suggested that ALL 2 had also arisen from an immature progenitor at the stage before the initiation of VDJH recombination (Fig. 3)Citation .

ALL 11 and ALL 3 were both t(12;21)-positive and were subsequently analyzed in greater detail (see "Discussion").

In Vitro Culture System Reveals the Timing of the t(12;21) with Respect to Clonal IgH and TCR{delta} Rearrangements.
The t(12;21) is detected in 25% of B-precursor ALL cases; however, it is exceptionally rare beyond the age of 10 years (15 , 16) . We used the culture of single blast colonies in vitro to establish the timing of the t(12;21) during the clonal expansion of a rare case (ALL 3) of late onset t(12;21)-positive leukemia with a rapid clinical progression. We compared the results from the culture of ALL 3 with a typical t(12;21)-positive leukemia, ALL 11.

In ALL 11, the t(12;21) Is the First Clonal Event Detectable.
ALL 11 was a classical t(12;21)-positive "common" ALL (CD34+, CD10+, and CD19+), presenting at the age of 4 years and 8 months and remaining in continuous remission for 29 months. Cytogenetic analysis revealed a 46, XX, del (12)(p?12p?13), t(12;21)(p13;q22) (15 cells) karyotype of which 105 of 106 cells were t(12;21)-positive by FISH analysis. PCR analysis of the total tumor population of ALL 11 before culture revealed a single IgH rearrangement (ALL 11 IgH clone 1; Table 1Citation ). Four additional IgH rearrangements (ALL 11 IgH clones 2–5) were detected after culture through analysis of individual colonies (Fig. 4A)Citation . These were all independent in sequence (Table 1)Citation , indicating the proliferation of an immature tumor progenitor at a stage before initiation of VDJH recombination (Fig. 3)Citation . Two of the colonies carrying the major IgH rearrangement had fully recombined their second alleles independently of each other (ALL 11 IgH clones 1a and 1b; data not shown). TEL-AML1 transcripts were detected in all of the colonies analyzed (Fig. 4A)Citation . Two variants of the TEL-AML1 transcript can sometimes be identified in t(12;21)-positive leukemias, the shorter of which excludes exon 2 of AML1 (28) . In ALL 11, all colonies expressed the long version, and some colonies (colonies 3, 7, and 8) showed the presence of both fusion variants. The presence of the TEL-AML1transcript in all analyzed colonies implied a common origin of all additional IgH rearrangements. From the coincidence of immune system gene rearrangements and the expression of the TEL-AML1transcript, it was possible to suggest a natural history for ALL 11 (Fig. 4B)Citation in which the t(12;21) was the first clonal event detectable followed by IgH gene rearrangement. Minor subclones (ALL 11 IgH clones 1a, 1b, and 2–5) were generated between initiation and presentation of the disease. The major proliferating clone (ALL 11 IgH clone 1) carried the rearrangement detected from the total tumor population before culture.



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  		Fig. 4. In vitro culture system identifies the t(12;21) as the first detectable event in a typical, early onset t(12;21)-positive leukemia (ALL 11). A, PAGE of seminested/nested PCR products for TEL-AML1 and IgH expression. Arrows, two alternative TEL/AML1 transcripts, including and excluding exon 2 of AML1. Twelve representative colonies (1–12) expressed at least one of the two TEL-AML transcripts. Colonies 5, 6, and 9 did not express an IgH transcript. Variations in the size of the IgH rearrangements in the remaining colonies were apparent. Numbers 1–5 correspond to the five VDJH sequence variations observed (ALL 11 IgH clones 1–5; see Table 1Citation ). Two subclones carrying the major clonal IgH rearrangement had independently rearranged their second IgH alleles [ALL 11 IgH clones 1a and 1b; see Table 1Citation (c)]. B, a natural history for leukemia ALL 11 derived from the results in A and Table 1Citation . The t(12;21) was the first clonal event detectable, followed by IgH recombination (ALL 11 IgH clone 1). Subclones diverged continually between disease initiation and presentation (ALL 11 IgH clones 1a, 1b, 2–5).

 
In ALL 3, the t(12;21) Is Preceded by Clonal Expansion with TCR{delta} Rearrangement.
ALL 3 presented at the age of 15 years and 6 months and showed an aberrant immunophenotype with coexpression of both lymphoid and myeloid markers (CD10+, CD19+, CD38+, and 54% cells with CD13+). Cytogenetic analysis identified the presence of three related cells lines [46, X, -X, t(3;12)(p2;p1), del(6)(q13q21), t(12;21)(p13;q22), +16 (9 cells)/47, X, -X, del(11)(q21q23), t(12;21)(p13;q22), +16, +mar (10 cells)/?94, XX, -X, -X, del(11)x2, t(12;21)x2, +16, +16, +mar, +mar inc[?]/46, XX (1 cell)]. All cells (50 of 50) were t(12;21) positive by FISH analysis. The patient died only 7 months into treatment. A single clonal TCR{delta} rearrangement, but no IgH rearrangement, could be detected in the total tumor population before culture (Table 1)Citation . After culture in vitro, three variations of TCR V{delta}2-D{delta}3 sequence (ALL 3 TCR{delta} clones 1–3) were identified (Fig. 5ACitation and Table 1Citation ). This suggested the proliferation of a very immature, nonlineage-committed tumor progenitor, which transformed before the initiation of IgH recombination (Fig. 3)Citation . When TEL-AML1 gene expression was simultaneously analyzed with the TCR{delta} rearrangements, colonies were found to carry either the short transcript (colonies 2 and 10), the long transcript (colonies 3, 5, 6, and 8), both short and long transcripts (colony 7), or no transcript at all (colonies 1, 4, and 9; Fig. 5ACitation ). The TEL-AML1-negative colonies, however, all carried the major TCR{delta} gene rearrangement (ALL 3 TCR{delta} clone 1). The suggested natural history specific to ALL 3 (Fig. 5B)Citation is that clonal expansion of blasts with the major TCR{delta} rearrangement (ALL 3 TCR{delta} clone 1) occurred first, followed by the t(12;21), and lastly, in a minor subset of blasts, by modification of the existing TCR{delta} rearrangement (ALL 3 TCR{delta} clones 2 and 3).



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  		Fig. 5. In vitro culture system identifies clonal proliferation with TCR{delta} rearrangement preceding the occurrence of the t(12;21) in an exceptionally rare, late onset t(12;21)-positive leukemia (ALL 3). A, PAGE of seminested/nested PCR products for TEL-AML1 and TCR{delta} expression in ALL 3. Arrows, two alternative TEL/AML1transcripts, including and excluding exon 2 of AML1.Ten representative colonies (1–10) each show the presence of a TCR V{delta}2-D{delta}3 rearrangement. However, only 7 of those (Lanes 2, 3, 5–8, and 10) carried a TEL-AML1 transcript. Colonies 2 and 10 carried the short transcript; colonies 3, 5, 6, and 8 carried the long transcript; and colony 7 carried both the long and short TEL-AML1 transcripts. Colonies 1, 4, and 9 did not carry a TEL-AML1 transcript. Numbers 1–3 under the gel correspond to the three TCR{delta} sequence variations (ALL 3 TCR{delta} clones 1–3; see Table 1Citation ) observed in individual colonies. B, a natural history for leukemia ALL 3 derived from the results in A and Table 1Citation . TCR V{delta}2-D{delta}3 recombination was the first clonal event (ALL 3 TCR{delta} clone 1), followed by t(12;21) and, in a subset of blasts, by subsequent TCR V{delta}2-D{delta}3 subclone modification (ALL 3 TCR{delta} clones 2 and 3).

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we investigated the pathogenesis of pediatric B-precursor ALL by analyzing the stage of B-cell development at which tumor transformation takes place and by relating the occurrence of the t(12;21) to that stage. We developed an in vitro culture system to allow the proliferation of B-cell progenitors and the formation of single cell-derived colonies. DNA or cDNA from single blast colonies was subsequently used to detect clonal markers, either IgH or TCR{delta} rearrangements, and in two cases, the TEL-AML1 fusion transcript.

Of 12 B-precursor ALL bone marrow samples subjected to the culture conditions, 7 diagnostic samples were analyzed for the presence of clonal immune system gene rearrangements. The culture system improved the sensitivity of detection of minor leukemic subclones compared with conventional PCR analysis of the total tumor population before culture. In particular, detection of minor subclones with independent IgH rearrangements was improved, presumably because of the preferential proliferation of immature blasts in vitro. By comparing the relatedness of the IgH or TCR{delta} sequences from the individual colonies, we were able to determine precisely the level of maturity of the progenitor targeted for transformation. Two leukemias were found to be derived from a mature tumor progenitor with IgH genes fully recombined, one from an intermediate progenitor with partially rearranged IgH gene and four from a very immature tumor progenitor with nonrearranged IgH genes. Therefore, the in vitro culture system allowed us to conclude that subclonal diversity, indicated by the presence of multiple antigen receptor gene rearrangements, may be much more common than estimated previously (7 , 10) and that transformation in B-precursor ALL may occur either before or after initiation of VDJH recombination. Such variation in the timing of the transforming factors may reflect distinct pathogenic mechanisms in the development of pediatric B-precursor ALL, although how and why such biological variations should occur still remains to be answered.

We used the culture system to analyze TEL-AML1 expression with respect to the clonal expansion of IgH or TCR{delta} gene rearrangements. The classical t(12;21)-positive leukemia (ALL 11) showed extensive clonal diversity in the culture system with five subclonal IgH rearrangements detected. Two of the subclones (ALL 11 IgH clones 1 and 3), which included the major proliferating clone (ALL 11 IgH clone 1), had no inserted nucleotides at the D-JH join, consistent with a fetal origin (36) . Detection of the TEL-AML1 transcript in all colonies confirmed the common clonal origin of all independent, additional IgH rearrangements detected in this leukemia in vitro. Therefore, the results were consistent with the t(12;21) being the earliest detectable clonal event in ALL 11.

We then analyzed an exceptionally rare t(12;21)-positive leukemia (ALL 3) which presented very late (aged 15 years and 6 months) and showed an aggressive clinical course. We hypothesized that this leukemia might have a different pathogenesis, compared with the typical standard-risk t(12;21)-positive leukemia (ALL 11). ALL 3 lacked a detectable clonal IgH rearrangement both before and after culture. The colonies did possess, however, one of three variations of the dominant TCR{delta} rearrangement. Although TCR V{delta}2-D{delta}3 immune system gene rearrangements in ALL are generally considered to be monoclonal and, therefore, stable markers of malignancy, our results support the finding made previously by Steenbergen et al. (37) that multiple subclonal V{delta}2-D{delta}3 rearrangements may be generated and selected throughout the disease. The aberrant coexpression of lymphoid and myeloid markers in the leukemic lymphoid-committed blasts and the lack of a clonal IgH rearrangement in ALL 3 suggested transformation of an early tumor progenitor at a stage before lineage commitment. The majority of the colonies were positive for the TEL-AML1 transcript and showed subclonal occurrence with either the long, the short, or both versions together. However, in ALL 3, we identified colonies positive for the common TCR{delta} rearrangement that were negative for the TEL-AML1 transcript.

The possibility that the TEL-AML1 transcript was undetectable in some colonies in ALL 3 as a consequence of the effects of culturing in vitro seemed unlikely. The level of TEL-AML1 expression in the other t(12;21)-positive leukemia (ALL 11) was not affected in any way by the culture system. All colonies in ALL 11 showed equally amplifiable levels of the TEL-AML1 transcript. It was also unlikely that subclonal evolution affected, specifically, the level of TEL-AML1 transcript expression in some colonies of ALL 3, whereas in others, expression remained abundant. Probably, the most important factor explaining this unusual biological finding is that ALL 3 was a clinically distinct form of t(12;21)-positive ALL and so the alternative natural history we have proposed for this leukemia is feasible.

Therefore, in addition to the clinical distinction between the two t(12;21)-positive leukemias, ALL 3 and ALL 11 were also biologically distinct. They transformed at different stages of B-cell differentiation; in ALL 11, the t(12;21) appeared to be the first clonal event, whereas in ALL 3, clonal proliferation, marked by a TCR{delta} rearrangement, preceded the t(12;21). Importantly, in both ALL 3 and ALL 11, the t(12;21) occurred at a stage before the initiation of IgH recombination. This is consistent with the notion that the VDJ recombination machinery does not play a role in generation of the t(12;21) (38) . Although the t(12;21) may not be the initiating event in ALL 3, it appeared to confer some proliferative advantage because both in vitro culture and cytogenetic studies suggested the presence of the t(12;21) in the majority of leukemic cells.

Rafi et al. (39) recently described a rare case of adult t(12;21)-positive B-precursor ALL where FISH analysis revealed that only a very small subset of leukemic blasts (12%) were positive for the t(12;21). Wiemels et al. (40) reported a pair of twins sharing a clonotypic t(12;21) breakpoint where one twin presented typically at the age of 5 years, but curiously, the second twin did not present with leukemia until 9 years later at the age of 14. The role of the t(12;21) in the pathogenesis of ALL in the second twin is unclear. Together, these findings suggest the existence of a biologically distinct subgroup of t(12;21)-positive leukemias and support our observations that the natural history of ALL 11 and ALL 3 is indeed different.

The proposed natural history for ALL 3 is also in agreement with recent studies suggesting that other nonrandom chromosomal translocations may not always be the primary etiological event in leukemogenesis. Uckun et al. (41) showed that in some infant ALLs, only a small proportion of tumor cells carried the t(4;11) (causing an MLL-AF4 fusion), inferring that the t(4;11) might not always be the initiating event in this tumor type. The two subgroups of infant ALL, one with the t(4;11) uniformly present in the tumor population and the second, with the t(4;11) present in only a proportion of tumor blasts, were clinically distinct.

In summary, we have investigated the relative timing of molecular events occurring in the development of B-precursor ALL using an in vitro system. We demonstrated that initial clonal expansion occurs during various stages of early B-cell differentiation but most frequently before IgH VDJ recombination. In addition, analysis of in vitro blast colonies allowed us to place the occurrence of the t(12;21) after initiation of TCR{delta} rearrangement in a t(12;21)-positive leukemia with an unusually late onset. Whether the presence of clonal proliferation before the occurrence of the t(12;21) in B-precursor ALL is also associated with typical tumors or is restricted to those with late onset remains to be established by in vitro analysis of additional cases.


   ACKNOWLEDGMENTS
 
We thank Dr. J. Pound for his invaluable expertise and advice. We also thank Birmingham Regional Cytogenetics Laboratory (Birmingham Women’s Hospital, United Kingdom) for providing the cytogenetic and FISH analyses and P. Short and J. Jesson (Birmingham Children’s Hospital, United Kingdom) for the flow cytometry data. Finally, we thank Dr. G. Stewart for useful discussions throughout this study.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by the Zara Bannister Fund, from Birmingham Children’s Hospital, United Kingdom, the Leukaemia Research Fund, the Kay Kendall Leukaemia Fund, and the Cancer Research Campaign. Back

2 To whom requests for reprints should be addressed, at the CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. Phone: (44) 121-414-7403; Fax: (44) 121-414-4486; E-mail: t.stankovic{at}bham.ac.uk Back

3 The abbreviations used are: ALL, acute lymphoblastic leukemia; ASO, allele-specific oligo hybridization; IL, interleukin; CD40LMF, CD40 ligand-transfected murine fibroblast; Flt3L, FMS-like tyrosine kinase 3 ligand; MF, murine fibroblast; IgH, immunoglobulin heavy chain; FISH, fluorescence in situ hybridization; TCR, T-cell receptor; D-JH, recombination of diversity and joining segments of the heavy chain gene; VDJH, recombination of variable, diversity, and joining segments of the heavy chain gene. Back

Received 6/ 4/01. Accepted 10/ 3/01.


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Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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