Introduction

Studies on pairs of monozygotic twins with concordant leukemia have revealed that their leukemic cells share identical clonotypic markers indicative of an origin in one twin in utero.^{1,2,3,4,5,6,7,8} Leukemia fusion genes are ideal markers in these studies as intronic breakpoints are scattered, clone specific and stable.⁹ The prenatal interpretation of the twin data is strongly endorsed by the demonstration of leukemia fusion gene genomic sequences in neonatal blood spots of patients with leukemia.^6,10,11 These studies have therefore identified that chromosomal translocations are often early or initiating events of leukemogenesis during fetal hemopoiesis. This includes MLL gene fusions in infants,^1,2,7,11 TEL-AML1^4,5,6,8 and AML1-ETO¹⁰ for older children with acute lymphoblastic leukemia (ALL) or acute myeloid leukemia (AML), respectively.

It has been uncertain, however, if the major chromosomal subtype of childhood ALL with high hyperdiploidy (51–56 chromosomes) similarly has an origin prior to birth. Evidence compatible with an in utero origin of ALL is provided by the presence of clonotypic IGH rearrangement in archived neonatal blood spots^12,13,14 and several cases of hyperdiploid ALL have now been described as positive. These data support the likelihood that hyperdiploid ALL, like TEL-AML1-positive B-cell precursor ALL, often originates prenatally but they are indirect and ambiguous insofar as it cannot be excluded that the clonotypic IGH sequence used existed prior to the genetic events initiating hyperdiploid ALL. A more convincing case is, however, provided by Panzer-Grümayer et al,¹⁵ who described the presence of three clonotypic IGH sequences in the blood spots of a patient with hyperdiploid ALL. In this patient's cells, trisomy 14 was present and all three clonal rearrangements were present in single leukemic cells, that is, concordant with trisomy 14. In this instance, albeit in a single case, it seems very likely that hyperdiploidy itself (at least for chromosome 14) was initiated prenatally. Clearly, the case for hyperdiploidy originating in utero would be strengthened by additional evidence and we provide such data here. We describe a pair of twins with concordant ALL and high hyperdiploidy in which there is a persuasive case for an in utero origin, based on shared chromosomal gains, clonal TCRD (V2–D3) and IGH (D_H–J_H) rearrangements.

Materials and methods

Patient samples

Two female identical twins were diagnosed with ALL at the ages of 1 year and 10 months (T1) and 2 years (T2), respectively.

Clinical details

Pregnancy was uncomplicated and the birth was by Caesarean section at 38½ weeks. The twins shared a single, monochorionic placenta.

Twin 1 presented at the age of 22 months having been unwell for 5 days with right hip pain, intermittent pyrexia, listlessness and anorexia. On presentation she had splenomegaly to 2 cm and hepatomegaly to 1 cm. She was pancytopenic; hemoglobin 9.3 g/dl, white count 11 10⁹/l of which 4.7 10⁹/l were lymphoblasts, platelets 75 10⁹/l. Bone marrow aspirate confirmed ALL, immunophenotype CD10 87%, CD19 90%, CD34 86%, CD20 86%, HLA DR 91%, confirming diagnosis of B-cell precursor (common) ALL. Cerebro-spinal fluid (CSF) was clear of any leukemic infiltration. She was treated with the MRC-ALL 97 protocol, commencing treatment on the 27th February 1999 and completing treatment on the 24th April 2001.

Following her sister's diagnosis, twin 2 had monthly full blood counts performed and on the 19th April 1999 (aged 24 months) had become anemic with peripheral lymphoblasts present. Her hemoglobin was 7.1 g/dl, white cell count 15.4 10⁹/l, neutrophils 0.6 10⁹/l, blasts 71 10⁹/l and platelets 178 10⁹/l. A bone marrow aspirate performed on the 29th April showed almost complete replacement of the marrow with L1 lymphoblasts, immunophenotype identical to twin 1. CSF was clear of lymphoblasts. She was treated with the MRC-ALL 97 protocol, treatment commencing on the 26th April 1999 and finishing on the 19th June 2001. Both patients remain in clinical remission.

Cytogenetics and interphase FISH

Cytogenetic analysis of diagnostic bone marrow was performed using standard cytogenetic procedures and was reviewed by the Leukaemia Research Fund Karyotype Database in Acute Leukaemia.¹⁶ Interphase fluorescence in situ hybridization (FISH) was carried out using centromeric probes for chromosomes 6 (D6Z1), 8 (D8Z2), 9 (D9Z1), 10 (D10Z1) and 18 (D18Z1). For chromosome 21, a chromosome 21-unique sequence probe (D21S55) was used (Qbiogene-Appligene-Oncor, France). Diagnostic samples were also screened for common fusion genes using dual color probes LSI BCR/ABL and LSI TEL/AML1 ES probe kits (Vysis, UK) and a single color probe to detect rearrangements of the MLL gene (Qbiogene-Appligene-Oncor, France).

Detection of allele-specific TCRD gene rearrangements

DNA was extracted from bone marrow slides and peripheral blood and PCR amplification of TCRD rearrangements was performed as described previously.^17,18 Nested PCR was performed with 1 l of the primary PCR product as template. Products were size separated on 1% agarose-gel electrophoresis, excised, extracted and cloned using the pSTBlue-1 Perfectly Blunt™ Cloning Kit (Novagen, Cambridge, UK). Random colonies were picked, and clonal inserts were directly sequenced. Sequences were compared by BLAST search with the human germ-line sequences deposited on the GenBank. Allele-specific oligonucleotides (ASO) probes were designed, for each TCRD rearrangement of T2, with the junctional region at the center. PCR products were spotted onto Immobilon-NY⁺ membrane (Millipore, Watford, UK), crosslinked by UV light exposure, hybridized with -³²P-dATP-labeled ASO probes and washed as described before.¹⁹

Detection of IGH and TCR gene rearrangements

PCR amplification of IGH (complete and incomplete), IGK-Kde and TCRG gene rearrangements and heteroduplex analysis of products were performed as described previously.^19,20,21,22 Mixing of PCR products followed by heteroduplex analysis was used for assessing whether the identified rearrangements were identical^23,24 PCR products were sequenced and the template DNA used was either the PCR product or a homo- (or hetero)duplex band excised and eluted from a polyacrylamide gel. Sequences were identified using DNAPLOT software (W Müller and H-H Althaus, University of Cologne, Germany) by searching for homology with all known human germ-line sequences obtained from the VBASE directory of human Ig genes (http://www.mrc-cpe.cam.ac.uk/i
mt-doc/) and IMGT (http://imgt.cnusc.fr:8104/).

Results

Cytogenetics and interphase FISH

G-banded chromosomal analysis revealed a high hyperdiploid karyotype for T1: 56,XX,+X,+4,+8,+9,+10,+14,+17,+ 18,+21,+21. There was no evidence of a TEL-AML1 fusion signal, but one or two additional signals of AML1 were observed in two populations (with one or two extra signals), confirming the additional copies of chromosome 21.

Cytogenetic analysis failed on T2 due to the absence of metaphases. FISH analysis using the BCR-ABL and TEL-AML1 probes showed no evidence of fusion signals but an extra copy each of ABL and AML1 signals indicated that additional chromosomes 9 and 21 were detected. This was confirmed by the results obtained with the centromeric probes for chromosomes 9 and 21. An extra copy of chromosomes 8, 10 and 18 was identified by the appropriate centromeric probes, as observed by G-banded analysis in T1. No information was available for T2 on chromosomes 4, 10, 17 or X due to limited material.

Common disomies between the twins were observed for chromosomes 6, 11 (from the MLL probe), 12 (from the TEL signals of the TEL-AML1 probe) and 22 (from the BCR signals of the BCR-ABL probe).

Detection of allele-specific TCRD gene rearrangements

TCRD gene rearrangements occur in approximately 90% of the cases of B-cell precursor ALL. The V2–D3 rearrangements occur at an early stage following DJH rearrangement, and shortly before or after the first or initiating leukemogenic mutation.¹⁸

The amplification products of both patients for the TCRD region were cloned and sequenced. Two sequences were identified consistently for T2, indicating a biallelic rearrangement for the TCRD gene (Figure 1), with distinct 7 and 6 base pair N regions, respectively. A range of sequences was obtained for T1, suggesting oligoclonality for the rearrangements, which is a relatively common observation.²⁵ DNA extracted from a T1 bone marrow slide (99% blasts) was analyzed by dot-blot experiments, using ASO probes designed for each of the T2's TCRD rearrangements. It was found that one of the two rearrangements was present and therefore shared by the twins (Figure 1).

Figure 1.

Analysis of the TCRD gene rearrangements in the leukemic cells of the twins concordant for hyperdiploid ALL. (a) Schematic representation of the two V2–D3 rearrangements identified for T2, with V2, N and D3 nucleotide sequences shown. The sequence of both probes is represented by the underlined sequence. (b) PCR analysis of the patients' diagnostic DNA, with specific V2–D3 primers. (c) Dot-blot analysis of T1's diagnostic DNA with the specific probe T2_ASO_1 probe designed from T2's identified rearrangements. M, molecular weight marker; T1 and T2 patients; H, blank control with no template; C, control samples from other patients; N16, Nalm16 cell line. Sensitivity was determined to be 10 ng of template DNA, by using a dilution series of T2 diagnostic DNA.

Full figure and legend (74K)

Detection of other Ig and TCR gene rearrangements

We next assessed the twins' leukemic cells DNA for clonal IGH, IGK and TCRG rearrangements by heteroduplex analysis and sequencing.^19,20,21,22 Three clonal rearrangements were identified (Table 1). In one, the twins' leukemic cells shared a common or 'stem' DJ joining sequence but with different V regions. The second rearrangement is indicative of an original single VDJ rearrangement in the twins followed by a subsequent partial V region replacement (of VH7.4.1 by VH2.5) in twin 2. A third clonal rearrangement (VH3.7.4–DH3.16-JHbc) was restricted to twin 1. Note that the shared V–D–J junctions include common N regions nucleotides (underlined in Table 1). IGK-Kde and TCRG rearrangements in the twin samples appeared to be extensively polyclonal and were not analyzed further.

Table 1 -  Analysis of the IGH sequences in the leukemic cells of Twins 1 and 2.

Full table

Discussion

Although many monozygotic twin pairs concordant for leukemia have now been described (reviewed in Greaves et al²⁶), this is the first reported pair with high hyperdiploid ALL and evidence for a monoclonal, in utero origin. The evidence, based on sharing of chromosomal gains and identical TCRD and IGH genomic sequences, including N nucleotides, is equivalent to that we described previously for a pair of twins with T-ALL/NHL that had a common TCRB gene rearrangement.³

Clearly, both twins had high hyperdiploid karyotype, one identified by cytogenetics and one by interphase FISH. The common additional chromosomes included 8, 9, 10, 18, 21, 21 (+21) and the common disomies were observed for chromosomes 6, 11, 12 and 22. Although chromosomes 10, 18 and 21 are frequently gained in high hyperdiploidy (found in 55, 67 and 86% of hyperdiploid karyotypes, respectively) chromosomes 8 and 9 are gained much less often (30 and 15%, respectively). The twins also showed disomy for chromosome 6, which is a common trisomy in hyperdiploid ALL, found in approximately 74% of the cases.^27,28 Therefore, the simultaneous occurrence of these rare numerical observations in both twins is also a significant finding.

It is likely therefore that in this twin pair with high hyperdiploid ALL, the leukemic clone originated as a single transforming event in utero. Neonatal blood spot data has previously suggested a prenatal origin of hyperdiploid ALL, in a nontwin setting.^12,13,14 However, these data alone are not unequivocal, the reason being that the clonal IgH sequence present in both the neonatal blood spot and the leukemic cells at diagnosis could have been present in a normal clone of B-cell progenitors prior to the molecular change (hyperdiploidy itself?) that initiated leukemogenesis and this might have provided the positive signal in the blood spots. In twin pairs with hyperdiploidy, the sharing of IgH/TCR clonal sequences is only possible if the leukemia originated from the same progenitor cell prenatally. Although our twin pair shared a similar, if not identical, high hyperdiploid karyotype with a rare combination of chromosomal gains, it is unclear from these data alone whether hyperdiploidy itself, perhaps via some form of mitotic, chromosomal nondisjunction, was itself a prenatal and initiating event. Panzer-Grümayer et al¹⁵ have provided, however, evidence based upon neonatal blood spots that trisomy 14 in a case of hyperdiploid ALL was prenatal in origin. Three different clonal IGH rearrangements were present in single isolated leukemic cells and in the patient's neonatal blood spot. Further confirmation of the timing and contribution of hyperdiploidy to the natural history of childhood leukemia might be provided by discovering the molecular mechanism underlying hyperdiploidy.

If hyperdiploid ALL is commonly initiated prenatally, then it is likely that, as with TEL-AML1,^8,29 this initial transforming event requires one or more additional genetic hits for overt leukemogenesis. This conclusion is based upon the observation that the concordance rate for childhood ALL in monozygotic twins is 10% not 100%²⁶ and TEL-AML1 itself is not leukemogenic in vivo^30,31 (Ford Anthony M and Mel F Greaves, unpublished). Based again on findings with TEL-AML1, it can be anticipated that the prenatal initiation of hyperdiploid ALL results in a preleukemic clone of B-cell precursors still retaining B-cell differentiation capacity.³² The necessary postnatal events for hyperdiploid ALL are not yet characterized but they might include kinase activating mutations³³ and promotion by infectious exposures.^29,34,35

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Acknowledgements

This work was supported by the Leukaemia Research Fund programme grants (CJH and MG), the Foundation for Science and Technology, Portugal (PRAXIS XXI/BD/19575/99) (ATM) and the Dutch Cancer Society/Koningin Wilhelmina Fonds (Grant SNWLK 2000-2268) (VHJV and JJMO).