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Mutagenesis Advance Access published online on November 21, 2008
Mutagenesis, doi:10.1093/mutage/gen063
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© The Author 2008. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org.

Cytogenetic and molecular analysis of MLL rearrangements in acute lymphoblastic leukaemia survivors

María S. Brassesco1,2,*, Ana P. Montaldi1, Diana E. Gras1, Marjori L. Camparoto1, Nilce M. Martinez-Rossi1, Carlos A. Scrideli, Luiz G. Tone and Elza T. Sakamoto-Hojo1,3

1Departamento de Genética 2Departamento de Pediatria e Puericultura, Faculdade de Medicina de Ribeirão Preto-USP 3Departamento de Biologia, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto-USP, Universidade de São Paulo, Avenue Bandeirantes 3900, 14040-901 Ribeirão Preto, São Paulo, Brazil

The successful treatment of paediatric malignancies by multimodal therapy has improved outcomes for children with cancer, especially those with acute lymphoblastic leukaemia (ALL). Second malignant neoplasms, however, represent a serious complication after treatment. Depending on dosage, 2–12% of patients treated with topoisomerase II inhibitors and/or alkylating agents develop treatment-related acute myeloid leukaemia characterized by translocations at 11q23. Our goal was to study MLL rearrangements in peripheral lymphocytes using cytogenetic and molecular methods in order to evaluate the late effects of cancer therapy in patients previously treated for childhood ALL. Chromosomal rearrangements at 11q23 were analysed in cytogenetic preparations from 49 long-term ALL survivors and 49 control individuals. Patients were subdivided depending on the inclusion or omission of topoisomerase II inhibitors (VP-16 and/or VM-26) in their treatment protocol. The statistical analysis showed significant (P = 0.007) differences between the frequency of translocations observed for the groups of patients and controls. These differences were also significant (P = 0.006) when the groups of patients (independent of the inclusion of topoisomerase II inhibitors) and controls were compared (P = 0.006). The frequencies of extra signals, however, did not differ between groups of patients and controls. Several MLL translocations were detected and identified by inverse polymerase chain reaction, followed by cloning and sequencing. Thirty-five patients (81%) presented putative translocations; among those, 91% corresponded with t(4;11) (q21;q23), while the other 9% corresponded with t(11;X), t(8;11)(q23;q23) and t(11;16). Our results indicate an increase in MLL aberrations in childhood ALL survivors years after completion of therapy. The higher frequency in this cohort might be associated with therapy using anti-tumoural drugs, independent of the inclusion of topoisomerase II inhibitors. Even though the biological significance of these rearrangements needs further investigation, they demonstrate a degree of genome instability, indicating the relevance of cytogenetic and molecular studies during the follow-up of patients in complete clinical remission.


   Introduction
Top
 Introduction
Materials and methods
 Results
 Discussion
 Funding
 References
 
Acute lymphoblastic leukaemia (ALL) is the most common malignancy in childhood and is associated with a cure rate that varies between 78 and 85% at 5 years after the completion of therapy (1Go). Improvements in anticancer therapies have been major contributors to this long-term survival, but they have also meant the exposure of patients to high doses of partial body irradiation and combinations of drugs, which are well-known as DNA-damaging agents and whose administration is systemic (2Go).

An increasing problem for successfully cured patients, however, is the risk of secondary neoplasias attributable to genotoxic therapy, predominantly acute myeloid leukaemias (AMLs) and myelodisplastic disorders (MDSs). In general, 85% of patients with such malignancies present the same type of chromosome aberrations that are observed in de novo AML or MDS, which can be directly associated with a previous exposure to cytotoxic anti-tumoural agents (3Go).

The secondary leukaemias associated with therapy with topoisomerase II inhibitors generally occur within 3 years after therapy completion and involve monocytes with aberrations at 11q23, which are highly reproducible in culture after treatment with such drugs in a manner dependent on time and dose (4Go).

Molecular characterization of breakpoints at 11q23 led to the identification of the MLL gene (mixed lineage leukaemia or myeloid–lymphoid leukaemia), also known as ALL-1, HXR or Htrx-1, which plays an important role in gene regulation during embryonic development and the regulation of HOX genes during normal haematopoiesis (5Go). This function is subverted in leukaemias by breakage, recombination and chimeric fusion with several other genes (6Go). In contrast to the diversity of genes with which MLL translocates, however, the mapping of different breakpoints revealed that the majority of breaks occur within a 8.3-kb BamH1 delimited region known as break cluster region (BCR) (7Go), between exons 8 and 14 (8Go,9Go). This region contains eight Alu repeats, various topoisomerase II consensus sites and two scaffold attachment regions. The breakpoints in rearrangements involving 11q23 and the heterogeneity of breaks in partner genes have led to the hypothesis that the fusion products from different der(11) encoded by the 5' portion of MLL represent the most biologically relevant oncogenic proteins (10Go). Still, several questions about the clinical significance of leukaemia-associated fusions, the hallmarks of malignancy, have arisen as a result of reports about the presence of these kinds of rearrangements in non-leukaemic normal cells (8Go,10Go–14Go).

The genotoxicity of anti-cancer drugs has been evaluated by somatic cell mutation assays, most of which have been carried out in patients immediately or shortly after completion of chemotherapy. Secondary malignancies in children, however, are often related with anticancer drugs and usually occur years after therapy (15Go).

Therefore, considering that the number of patients with secondary neoplasms has increased among childhood cancer survivors, we investigated the presence of MLL gene aberrations in a cohort of patients treated for ALL and analysed years after therapy completion.


   Materials and methods
Top
 Introduction
 Materials and methods
Results
 Discussion
 Funding
 References
 
Patient samples
Fifty-two patients aged 5–22 years (average = 11.5) who were previously treated for childhood leukaemia were included in the study. These patients were diagnosed and treated at The Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, University of São Paulo, Brazil, with combined modality treatment according to three different treatment protocols proposed by the Brazilian Group of Paediatric Leukaemia Treatment (GBTLI), including vincristine, dexamethasone, daunorubicin, L-asparaginase, prednisone, methotrexate, cytosine arabinoside, 6-tioguanine, cyclophosphamide, etoposide (VP-16), teniposide (VM-26) and 6-mercaptopurine. In some cases, prophylactic cranial and/or neuroaxis irradiation was also included. The patients were classified into standard or high-risk groups according to the GBTLI criteria (Tables I and II).


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  		Table I. Characterization of patients whose treatment protocol included topoisomerase II inhibitors (VP-16 and/or VM-26)

 


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  		Table II. Characterization of patients whose treatment protocol did not include topoisomerase II inhibitors (VP-16 and/or VM-26)

 
The control group comprised 49 non-smoking healthy young subjects, aged 18 to 22 years (average = 19.9), who were not occupationally exposed and had no history of prior or concurrent malignancy. The project was approved by the Research Ethics Committee of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo, process number 1135/2000, based on the Helsinki convention criteria. All involved patients gave voluntary participation, and informed consent was obtained from each patient.

Lymphocyte culture and chromosome preparation
For lymphocyte culture preparation, the standard protocol (16Go) was used: 0.5 ml of peripheral blood was added to 10 ml RMPI 1640 medium (Sigma Chemical Co., St Louis USA) supplemented with 20% foetal calf serum, 2% phytohemagglutinin and penicillin/streptomycin. The cultures were incubated at 37°C for 72 h and treated with colchicine (0.56%) for the last 90 min before the harvesting procedure. Slide preparation was done according to the standard air dry method. Slides for fluorescence in situ hybridization (FISH) were stored at –20°C until use.

In vitro etoposide treatment
Whole-blood samples from three control individuals were cultured and treated with Nex-Vep (Bristol Myers Squibb, São Paulo, Brazil) (kindly provided by the Chemotherapy Central from the Clinics Hospital—Faculdade de Medicina de Ribeirão Preto-USP). Twenty-four hours after mitogenic stimulation, etoposide was added, and cells were incubated for 48 h at 37°C at a final concentration of 0.25, 0.5 or 1 µg etoposide/ml. One set of cultures was treated for only 1 h and subsequently washed twice in RPMI 1640 medium and subcultured in supplemented medium until harvesting (47 h recovery).

The FISH
FISH was performed using the commercially available probes LSI MLL Break Apart Rearrangement according to the protocol of the manufacturer (Vysis, Downers Grove, IL). The probe labelled in SpectrumGreen covers a 350-kb portion centromeric to the MLL gene breakpoint region, and the SpectrumOrange-labelled probe covers a 190-kb portion telomeric to the BCR. The expected signal pattern for a normal cell nucleus is two green (yellow) orange signals. In cells harbouring MLL translocations, the green and orange signals appear separated without the yellow intersection. The advantage of this strategy is that it allows the detection of translocations irrespective of the partner involved. At least 1000 nuclei were analysed, and images were captured using the Axiovision System (Zeiss GmbH, Germany).

Translocation analysis by inverse polymerase chain reaction
Inverse polymerase chain reaction (I-PCR) was done according to Betti et al. (17Go) with few modifications. In all, 3 µg of DNA was digested with a combination of Sau3AI and XbaI (10 units each) at 37°C overnight. The addition of XbaI eliminates amplification of the native MLL gene, allowing the amplification of translocation products that lack the XbaI recognition site. After digestion, the samples were heat inactivated at 65°C for 10 min, purified to remove residual enzymatic activity by the Wizard SV Gel and PCR Clean-up System Kit (Promega Corporation, Madison, WI) and resuspended in nuclease-free water. Then, 0.5 µg of digested DNA was self-ligated in the presence of 3 units of T4 DNA ligase in a final volume of 20 µl for 16 h at 16°C. All ligation reactions were terminated at 65°C for 10 min. In all, 8 µl of ligated DNA was used in each PCR reaction. Nested primers were used for analysis of the cleavage site in exon 12 of MLL in two 28-cycle reactions at PCR temperatures of 95°C/55°C/72°C for 1 min/step.

The following primers were used: forward-1 5'-CTTTGTTTATACCACTC-3' and reverse-1 5'-TAGGGAATATAAAAGAGTGGG-3'; forward-2 5'-TTAGGTCACTTAGCATGTTCTG-3' and reverse-2 5'-CAGTTGTAAGGTCTGGTTTGTC-3'. PCR amplicons were then separated on 1% agarose gels.

Translocation sequence analysis
Individual I-PCR products were extracted from gels using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, Buckinghamshire, England). These bands were then cloned into the pGEM-T vector (Promega Corporation), transformed into pMOS Blue Escherichia coli bacteria, selected on luria broth agar plates containing 50 µg/ml ampicillin according to the manufacturer's protocol and expanded for 22 h in liquid culture. In all, 300 ng of plasmid DNA was used as a template for sequencing reactions using the Big Dye Terminator Cycle Sequence Ready Reaction Kit (Amersham Biosciences), and the products were analysed using an ABI Prism 377 DNA Sequencer (Perkin Elmer, Wellesley, MA). Quality analysis and removal of vector sequences were done using the phredPhrap software (18Go,19Go). The DNA sequences obtained were then searched against the National Center for Biotechnology Information database using the basic local alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST) (20Go).

Statistical analysis
Statistical analyses were performed using SigmaStat (Jandel Scientific, Co. CA, USA) software. All tests were carried out at a significance level of {alpha} = 0.05.


   Results
Top
 Introduction
 Materials and methods
 Results
Discussion
 Funding
 References
 
Chromosomal preparations from 46 ALL survivors and 49 control individuals were analysed using the LSI MLL (Vysis) commercial probe, which allows the detection of different rearrangements at 11q23. Patients were divided into two subgroups depending on the inclusion or omission of topoisomerase II inhibitors (VP-16 and/or VM-26) in the treatment protocol. At least 1000 nuclei were analysed per individual, and translocation frequencies varied between 0 and 0.39 translocations/100 cells for patients treated with VP-16 and/or VM-26 [n = 15; mean ± standard deviation (SD) = 0.13 ± 0.14] and between 0 and 0.4 translocations/100 cells for patients whose treatment protocols excluded these drugs (n = 31; mean ± SD = 0.09 ± 0.117). Controls presented translocation frequencies varying between 0 and 0.3 translocations/100 cells (n = 49; mean ± SD = 0.04 ± 0.06). The statistical analysis showed significant differences between the frequency of translocations between subgroups of patients and controls (P = 0.007, Kruskal–Wallis one-way analysis of variance on ranks). These differences were also significant when the frequency between patients (independently of the inclusion or not of topoisomerase II inhibitors) and controls was compared (P = 0.006, Mann–Whitney rank sum test). Patients were also compared with regard to type of treatment (with or without the inclusion of topoisomerase II inhibitors), but the frequencies between these subgroups did not significantly differ (P = 0.542, Mann–Whitney rank sum test).

The specific probes allowed for the detection of extra signals (only signals with the green–yellow–red pattern were considered), with frequencies varying from 0 to 0.98 signals/100 cells (mean ± SD = 0.28 ± 0.28) for the first group of patients (exposed to topoisomerase II inhibitors) and from 0 to 0.69 signals/100 cells (mean ± SD =0.16 ± 0.16) for the second group. For the control group, the extra signal frequencies varied between 0 and 0.79 signals/100 cells (mean ± SD = 0.18 ± 0.19). Statistical analysis did not show differences in extra signal frequency between groups (P = 0.43, Kruskal–Wallis one-way analysis of variance on ranks) (Table III).


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  		Table III. Mean frequency of MLL translocations and extra signals obtained for ALL survivors and healthy individuals

 
The analysis of variables (such as sex, risk of relapse and inclusion of radiotherapy) that could influence translocation and extra signal frequency did not show differences between groups. The results for patients treated for ALL during childhood were also analysed with regard to the time in complete clinical remission (CCR), which varied between 5 and 192 months. The statistical analysis performed for patients divided into two subgroups, <60 months and >60 months after completion of therapy, did not show an influence of the time in CCR since the frequency of translocations and extra signals did not significantly (P > 0.05, Kruskal–Wallis one-way analysis of variance on ranks) differ between subgroups.

In order to identify translocations involving the MLL gene, I-PCR was performed. Here, the proposed translocation region was digested by restriction enzymes, self-ligated and amplified using divergent primers. This technique was initially used for etoposide-treated cells, which served as positive controls. Several translocations involving the MLL gene were seen in cultured cells, appearing as multiple bands with different molecular weights (Figure 1).


Figure 1
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  		Fig. 1. Partial gels showing putative MLL translocations detected by I-PCR in peripheral blood lymphocytes treated with VP-16 (0, 25, 0, 5 and 1 µg). (A) Samples digested with Sau3A1 (the arrow shows germ line MLL gene); (B) Samples digested with a combinations of Sau3A1 and XbaI. {Phi}X174 = molecular weight marker; w = negative control; P = 1-h pulse treatment

 
Individual PCR products were cut, extracted from gels and subsequently cloned into the pGEM-T vector. A total of 60 clones were recovered. Further sequencing of PCR products confirmed the veracity of the rearrangements, showing MLL fusions with several partners such as t(3;11), t(7;11), t(8;11), t(11;12), t(11;16), t(11;17), t(11;19) and t(11;22). The most frequent rearrangements, however, corresponded to t(4;11) and t(9;11) with 65 and 12%, respectively.

Samples from 43 long-term ALL survivors were also analyzed by I-PCR. Thirty-five patients (81%) presented putative translocations that appeared as three, two or single bands of different molecular weights (Figure 2). The control group was also analysed by this technique, showing putative MLL rearrangements in 49% of individuals. The presence of two or three putative translocations was statistically higher in the group of patients (P < 0.05) when compared with the control group (data not shown). A total of 33 clones were obtained; among these, 30 translocations corresponded to t(4;11) (q21;q23), while the remaining three corresponded to t(11;X), t(8;11)(q23;q23) and t(11;16). The results were also analysed with regard to time in CCR, which did not influence the results. Similarly, analysis considering other variables (e.g. sex, risk of relapse and inclusion of radiotherapy) did not influence the number of translocations between patients. Examples of the sequenced breakpoint junctions are shown (Figure 3).


Figure 2
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  		Fig. 2. Partial gel showing putative translocations at 11q23 (MLL) detected by I-PCR in peripheral lymphocytes from long-term ALL survivors. {Phi}X 174 = molecular weight marker; w = negative reaction control; Co+ = positive control; ‘*’ indicates patients treated with topoisomerase II inhibitors.

 


Figure 3
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  		Fig. 3. MLL translocations detected by I-PCR. Individual break junctions are flanked by the germ line MLL sequence and sequences at chromosome 4. Break site refers to the position in the MLL gene (GenebankTM accession number Y373585) fused to a partner. Grey boxes indicate short microhomologies between MLL and AF4 (4q21).

 

   Discussion
Top
 Introduction
 Materials and methods
 Results
 Discussion
Funding
 References
 
During the past 30 years, improvements in therapy for childhood leukaemias have significantly increased cure rates (21Go) by the development and use of integrated treatments that include radiation and multidrug therapy (22Go). Both modalities enhance the risk of subsequent complications, however, which may reduce patients’ quality of life and survival (23Go). Compared with the normal population, individuals with a history of childhood cancer have a 10- to 20-fold increased risk for the development of secondary neoplasias attributed to radiotherapy and chemotherapy (3Go). Recently, a higher risk of therapy-related leukaemias among childhood ALL survivors has been reported (24Go).

A great variety of balanced translocations involving 11q23 have been demonstrated in therapy-related acute myeloid leukaemia (t-AML) and therapy-related myelodisplastic disorders (t-MDS), which have been significantly associated with exposure to topoisomerase II inhibitors (25Go). In the present study, we performed a screening of numerical and structural aberrations involving the MLL gene (on 11q23 band) in long-term ALL survivors compared to control individuals, as evaluated by FISH on interphase nuclei using a dual-colour DNA probe. The statistical analysis showed significant differences when comparing patients (independently of the inclusion of VP-16 and/or VM-26 in treatment) and control individuals, which could indicate the influence of drugs used in chemotherapy.

According to Pui and Relling (26Go), the involvement of other topoisomerase II inhibitors such as anthracyclines (doxorubicin, 4-epi-doxorubicin), mitoxanthone, dactinomycin and dipiperazine derivatives in the development of t-AMLs has been observed at lower frequencies. Moreover, there are reports of t-AMLs showing aberrations at 11q23 in patients whose treatment did not include topoisomerase inhibitors but did include alkylating agents or anti-metabolites (27Go–29Go). Additionally, it has been shown that MLL rearrangements can occur following in vitro treatment with drugs that do not interact with topoisomerase II such as methotrexate and cytosine arabinoside (30Go), campthotecin, 5-fluoroacyl and vinblastine (31Go). Thus, exposure to other drugs present in the treatment protocol could influence the frequency of MLL aberrations in treated patients.

The probes used in the present study also allowed the detection of extra signals. Recently, an increase in copy number of 11q23 (with amplification of MLL through double minutes, homogeneously stained regions or rings) has been reported as a new cytogenetic anomaly in myeloid malignancies (32Go,33Go). Nevertheless, it has been suggested that independent of the amplification pattern, MLL dosage could evoke phenotypic effects similar to those observed for MLL fusions (34Go). Karenko et al. (35Go) showed an elevated frequency of numerical rearrangements of chromosome 11 in survivors of cutaneous T-cell lymphoma compared with control individuals. The statistical analysis of our results, however, did not show an increased frequency of extra signals in ALL-treated patients when compared to the control group.

Treatment of paediatric ALL consists of several doses of anticancer drugs given weekly or daily for 2–3 years (36Go), which can increase the frequency of chromosome rearrangements (2Go). Mutational studies of the FMS proto-oncogene demonstrated the acquisition of mutations in patients treated with alkylating agents and radiation (37Go). The absence of such mutations in biopsies at initial diagnosis suggested that they were somatically acquired after treatment, and they appeared with increased frequency in relation to the normal population that was not exposed to cytotoxic drugs. Similarly, studies of hypoxanthine phosphoribosyl transferase mutations demonstrated a significantly elevated frequency in patients treated for pre-B ALL, even 2 years after completion of therapy (15Go).

Besides MLL aberrations, several other gene fusions have been identified in patients with therapy-related leukaemias such as t(9;22) (38Go), t(3;21) (39Go), translocations that involve RUNX1 (40Go) and balanced translocations with ETO and EVII (41Go), in addition to t(14;21), t(17;21), t(1;21), t(15;21) and t(3;21) (42Go). Furthermore, aberrations at 11q23 have been found during treatment of primary cancer, as observed in case of t(11;17) MLL–GASP (43Go). These observations demonstrate the susceptibility of these genes to damage, pointing to the possibility of these translocations being initial events in leukaemogenesis (44Go).

The correct identification of different rearrangements can be relevant in early detection at diagnosis and prognosis of human neoplasias, principally in therapy-related leukaemias. MLL is considered a promiscuous gene, mainly because of its participation in >50 different translocations (34Go,45Go–47Go). The identification of these aberrations by classic methodologies is difficult because they present a known 5' sequence, but the 3' end could be a myriad of translocation partners (48Go,49Go). I-PCR eliminates this problem by the amplification of circularized fragments, amplifying any segment flanking a known DNA sequence. Among the different translocations detected in etoposide-treated lymphocytes, several partners were identified, including t(3;11), t(4;11), t(7;11), t(8;11), t(11;12) and t(11;16), demonstrating the diversity of chromosome regions with which 11q23 translocates. Although translocations involving these chromosomes have been reported in patients, the sequences analysed in the present study did not show identity with those gene partners. Nonetheless, the most frequent translocations detected by this technique corresponded with MLL–AF4 and MLL–AF9.

Similarly, this technique allowed the detection of putative translocations in 35 ALL patients (81%); among these, a total of 33 clones were obtained, and sequence analysis demonstrated that 30 corresponded to t(4;11)(q21;q23).

AF4–MLL makes up 50% of translocations in infants with leukaemia (50Go). This translocation has also been found in most normal infants analysed and in monozygotic twins with concordant leukaemia (51Go). Moreover, it is the only translocation involving MLL that has been detected in bone marrow samples from normal children. The most frequent MLL translocations in therapy-related leukaemias, however, are t(9;11)(q13;q23) and t(11;19), which in some cases make up 48% of translocations (52Go).

According to Slany (34Go), from a clinical point of view, even though MLL shows vast recombinogenic potential, only five translocations occur in more than two-thirds of clinical cases with MLL rearrangements: t(4;11) MLL–AF4, t(9;11) MLL–AF9, t(10;11) MLL–AF10, t(11;19) MLL–ENL and t(11;19) MLL–ELL. The other fusion partners are found in a limited number of patients, suggesting that these genes are rarely hit by translocation events or that the fusion proteins are less potent in transformation, and consequently, other cooperative events would be necessary to cause leukaemia.

An interesting aspect of our results is the presence of microhomologous regions detected at the breakpoint junctions in most of the translocations analysed. The cloning of breakpoints between RUNX1 and ETO and in MLL, AF4 and AF9 by Xiao et al. (53Go) revealed frequent deletions and microhomologies at the break sites, without site-specific recombination or homologous recombination. This suggests recombination of sequences followed by non-homologous repair. Additionally, there is evidence of the involvement of DNA-protein kinase in the repair of MLL (17Go). These heterogeneous breaks were observed in the sequences analysed in this study; however, even though the breakpoint sites varied among different rearrangements, they were limited to few bases. Of note, some of the rearrangements detected by I-PCR did not appear in frame after sequencing, suggesting that they could be irrelevant, but indicators of instability.

The results obtained by the analysis of translocations by FISH and by molecular analysis were compared (30 patient samples were submitted to both techniques) and in general, the results were concordant. While 19 patients were positive for MLL translocations by FISH and by I-PCR, the other 11 presented putative translocation bands, but the translocations were not detected by FISH. Interestingly, these patients presented extra signals, as visualized by the cytogenetic analysis, suggesting that the bands observed could represent other types of rearrangements.

The risk estimates for therapy-related leukaemias after treatment with epipodophylotoxins are variable. According to Felix (54Go), between 2 and 12% of patients treated with epipodophylotoxins develop theraphy-related acute myeloid leukaemia with aberrations at 11q23 or, less frequently, t(8;21), t(3;21), t(8;16), t(15;17), t(9;22) or inv(16). Unlike t-LMAs associated with alkylating agents and radiotherapy, the t-LMAs associated with topoisomerase II inhibitors are overt and appear within 30–34 months after completion of therapy.

For the group of patients analysed by cytogenetic and molecular analyses, the time following completion of therapy was variable (5–193 months), with a mean of ~4 years. For statistical analysis, these patients were divided into two subgroups, <60 months and >60 months (time in CCR). There were no differences between the subgroups, even though a decrease in aberration frequency would be expected over with time. According to the literature, the inclusion of radiotherapy in different treatment regimens is associated with an increase (two to three times) in the risk of developing solid tumours (55Go). Among the ALL patients analysed in the present study, 13 (28%) underwent different doses of cranial or neuroaxis {gamma}-radiation, independent of the inclusion of VP-16 or VM-26 in treatment. These patients did not show increased MLL translocation frequency, however, compared with patients who were only treated with chemotherapeutic combinations.

The induction of leukaemia-associated fusion genes such as t(8;21) AML1–ETO, t(9;22) BCR–ABL and t(6;9) DEK–CAM (associated with AML and chronic myeloid leukaemia) by high doses of ionizing radiation has been reported in vitro (56Go). Several authors have demonstrated that the production of unstable aberrations (rings and dicentrics) after radiotherapy decreases with time, and sequential cytogenetic studies of peripheral lymphocytes in patients treated for different cancers have shown a constant decline in chromosomal aberrations (57Go, 58Go). In recent years, treatment directed to the central nervous system has undergone modifications, with a tendency toward reducing doses and the irradiated area using dose rationing regimens (59Go). Currently, ALL patients undergo doses of 18 (classified as standard risk of relapse) and 24 Gy (higher risk of relapse), according to the GBTLI protocol (60Go). Nonetheless, several epidemiological studies have demonstrated that the risk of t-LMAs or t-MDSs is predominantly associated with chemotherapy and not radiotherapy, principally emphasizing the relationship between specific drugs and chromosome rearrangements (61Go), as verified for topoisomerase II inhibitors associated with 11q23 aberrations.

The investigation of rearrangements that could represent a risk factor for the development of secondary neoplasias is important for cancer survivors since strategies in cancer treatment are continuously evolving. It is necessary to perform follow-up with patients for the evaluation of late effects. Chromosomal rearrangements such as RUNX1–RUNX1T1, PML–RARA, CBFB–MYH11, MLL–MLLT1 and BCR–ABL1 have been observed in non-Hodgkin lymphoma patients after high-dose therapy (62Go). Previously, we also reported elevated frequency of ETV6–RUNX1 gene fusion in ALL patients (63Go), as well as BCR–ABL and IGH–MYC in patients treated for lymphoproliferative malignancies during childhood (Camparoto ML, Brassesco MS and Sakamoto-Hojo ET unpublished data), indicating late effects of anticancer treatment. Although the MLL aberrations detected in the present study appear at low frequency in ALL survivors, there were significant differences when compared to the control group. None of these patients have developed t-AML–MDS with a median 4 years of clinical follow-up.

Even though the observed therapy-induced genetic damage is not predictive of an increased risk of developing secondary neoplasias, it represents a persistent genetic alteration.


   Funding
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
References
 
Fundação de Amparo à Pesquisa do Estado de São Paulo (02/13317-8 and MSB fellowship 03/01915-0); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior; Conselho Nacional de Desenvolvimento Científico e Tecnológico.


   Acknowledgments
 
We are grateful to all donors and their parents whose cooperative spirit was remarkable. We also thank Pedro Alejandro Vozzi for assistance in statistical analysis and Sueli A. Neves, Luiz A. da Costa Jr and Mendelson Mazucato for technical assistance.

Conflict of interest statement: None declared.


   Notes
 
* To whom correspondence should be addressed. Hospital das Clínicas da Faculdade de Medicina de Rebeirão Preto-USP, Laboratório de Pediatria-Bloco G, Avenue Bandeirantes 3900, Bairro Monte Alegre, CEP, 14048-900, Ribeirão Preto, São Pauto, Brazil. Tel: +55 16 36022651; Fax: +55 16 36022700; Email: marsol{at}rge.fmrp.usp.br


   References
Top
 Introduction
 Materials and methods
 Results
 Discussion
 Funding
 References
 

    1. Pui CH, Cheng C, Leung W, et al. Extended follow-up of long-term survivors of childhood acute lymphoblastic leukemia. N. Engl. J. Med. (2003) 379:640–649.

    2. Byrne J. Long-term genetic and reproductive effects of ionizing radiation and chemotherapy agents on cancer patients and their offspring. Theratology (1999) 59:210–215.[CrossRef]

    3. Lönning L, Zimmermann M, Reiter A, Kaatsch P, Henze G, Riehm H, Schrappe M. Secondary neoplasms subsequent to Berlin-Frankfurt-Münstr therapy of ALL in childhood: significantly lower risk without cranial radiotherapy. Blood (2000) 95:2770–2775.[Abstract/Free Full Text]

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Received on August 10, 2008; revised on October 3, 2008; accepted on October 28, 2008.


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