Detection of t(12;21) in Childhood Acute Lymphoblastic Leukemia by Fluorescence In Situ Hybridization

Detection of t(12;21) in Childhood Acute Lymphoblastic Leukemia by Fluorescence In Situ Hybridization Dionysios H. Spathas, Janet Stewart, Iain O. Singer, Anne Theriault, Mary Bovey, and J. Michael Connor

ABSTRACT: Metaphase preparations from 36 patients with acute lymphoblastic leukemia (ALL) have been retrospectively screened by fluorescence in situ hybridization (FISH) to determine the incidence of translocation (12;21) and the potential usefulness of FISH as an adjunct to conventional cytogenetic analysis. With the use of specific chromosome paints, 4 of 31 patients with B-lineage childhood ALL (13%) demonstrated rearrangements of chromosomes 12 and 21, and therefore, were considered to harbor the translocation, which had not previously been detected by conventional karyotyping. However, none of these positive cases revealed the standard reciprocal t(12;21)(p12;q22) as the sole abnormality involving chromosomes 12 and 21. The study confirms the feasibility and advantages of introducing FISH screening for t(12;21) in pediatric ALL cases and demonstrates the usefulness of FISH screening as a backup to concurrent cytogenetic analysis to resolve variant translocations and aberrant results. The presence of t(12;21) has also been correlated to clinical data to assess the prognostic significance of this translocation on its own or in association with other prognostic features. © Elsevier Science Inc., 1999. All rights reserved. INTRODUCTION Cytogenetic findings have now been established as an important factor in the diagnosis, classification, prognosis, and monitoring of leukemias [1, 2]. Moreover, correlation of specific chromosomal rearrangements with a particular type of disease may provide the means for the identification of the genes and the molecular events underlying the pathogenetic mechanisms [3–5]. In acute lymphoblastic leukemia (ALL), at least 81% of all cases appear to have a clonal chromosome abnormality by cytogenetic analysis [6, 7]. Rearrangements, such as partial deletions and translocations involving the short arm of chromosome 12, have been known for more than a decade [8, 9], and may have been underestimated because of the subtlety of the chromosomal rearrangements and the notoriously poor morphology of metaphases obtained from ALL sample preparations. The variation and breakpoints in donor chromosomes led to the postulation that translocations involving 12p are

From the Duncan Guthrie Institute of Medical Genetics, University of Glasgow (D. H. S., J. S., A. T., M. B., J. M. C.), Glasgow, United Kingdom; the Laboratory of General Biology, Medical School, University of Patras (D. H. S.), Patras, Greece; and the Department of Hematology, Southern General Hospital (I. O. S.), Glasgow, United Kingdom. Address reprint requests to: Dionysios H. Spathas, Laboratory of General Biology, Medical School, University of Patras, 261 10 Patras, Greece. Received March 11, 1998; accepted July 21, 1998. Cancer Genet Cytogenet 110:7–13 (1999)  Elsevier Science Inc., 1999. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

specific with respect to only one member of the translocation pair, namely chromosome 12 which may, therefore, carry genes relevant to leukemogenesis [9, 10]. This concept has now changed with the recent findings demonstrating the recurrent translocation (12;21) in childhood ALL [11–14] and the involvement of chromosome 21 which is also implicated in acute myeloid leukemia [15]. The new data are based on fluorescence in situ hybridization (FISH) and other molecular studies and suggest that t(12;21) occurs in high frequency but can be easily missed by conventional karyotyping. We have undertaken a retrospective study to ascertain, by FISH, the incidence of t(12;21) in a series of ALL cases that have been analyzed at the Duncan Guthrie Institute between the years 1993–95 by conventional chromosome analysis. We also aimed to determine the feasibility of introducing a FISH screening program for t(12;21) in ALL patients and to correlate this translocation with immunophenotypic and other clinical data.

MATERIALS AND METHODS Patient Characterization Thirty-six patients with ALL classified as B, pre-B, common ALL, and T-ALL were selected for this study. This group had patients with abnormal results, including acquired numerical or structural abnormalities of chromosomes 12 or 21, and patients with normal karyotypic find-

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8 ings at diagnosis. Some patients with constitutional trisomy 21 were also included (cases 16, 18, 25). All patients had been referred for cytogenetic analysis and were studied at diagnosis of the disease, except patients 1, 8, 9, 13, 16, and 22, who were studied at followup. For the initial cytogenetic analysis, bone marrow samples were prepared by a direct method or with a shortterm (17 hours) culture according to standard procedures [16]. Chromosomes were described according to ISCN [17]. For immunophenotyping, leukemic cell surface characteristics were detected by standard indirect immunofluorescence assays with monoclonal antibodies to lymphoid and myeloid associated antigens [16, 18]. These included T-cell specific antigens such as CD1, CD2, CD7, and B-cell associated antigens, HLA-DR, CD10, CD13, and CD19. For the purpose of this study, a positive test result was defined as reactivity with greater than 20% of the leukemic cells. Blast cells were also tested for cytoplasmic Ig(cIg). Fluorescence In Situ Hybridization Fresh slides were prepared from bone marrow metaphase preparations stored at 2208C in methanol-acetic acid (3:1) from the material of the initial cytogenetic analysis. Commercial (Cambio) chromosome paints specific for chromosomes 12 and 21, labeled with different reporter molecules (fluorescein for chromosome 12 and biotin for chromosome 21 or vice versa) were used for dual-color hybridization. In some experiments, a chromosome 22 paint and centromeric probes specific for chromosomes 12 and 22 were also used. Briefly, slides were dehydrated by serial ethanol washings (2 minutes in 50%, 70%, 95%, and 100% ethanol) and then denatured in 70% formamide in 2 3 SSC at 708C for 4 minutes. Slides were then quenched in ice-cold 70% ethanol followed by further dehydration with ethanol series. Twelve microliters of each chromosome paint were denatured separately at 708C for 10 minutes, left to preanneal at 378C for 1 hour and then mixed and applied to each slide. Hybridization was at 408C for 16 hours under sealed coverslips. The next day, after removing the coverslip, slides were sequentially washed in 2 3 SSC (5 minutes, 428C), 50% formamide/2 3 SSC (2 3 5 minutes, 428C), 2 3 SSC (5 minutes, 428C), 4 3 SSC-Tween 20 (5 minutes, 208C), and preblocked in 15% human AB serum in 4 3 SSC-Tween 20 for 15 minutes at 378C [19]. Four hundred nanograms of avidin-rhodamine (Vector) conjugate diluted in 100 ml of the preblocking mixture, was then applied to each slide for 30 minutes at 378C, the slides were washed 3 3 3 minutes in 4 3 SSC-Tween 20, dehydrated and counterstained with DAPI (0.4 mg/ml) in mounting medium AF1 (Citifluor Ltd.). For fluorescence microscopy, a Zeiss Axioscope was used, equipped with a filter wheel and a cooled CCD camera (Photometrics KAF 1400) to collect images for documentation and further analysis using the Smartcapture software (Digital Scientific, Cambridge). RESULTS Thirty-six patients with ALL have been investigated by FISH for the presence of translocation (12;21) using com-

D. H. Spathas et al. mercial paints for the respective chromosomes. The material used in these studies originates from the initial, and in some cases, follow-up analyses of these patients. The cytogenetic classification, the immunophenotypes, and other clinical data of all patients are summarized in Table 1. Thirteen patients had additional copies or visible structural abnormality of chromosomes 12 and/or 21. Thirtyone case were diagnosed as pediatric B-lineage ALL; these included the four patients, (3, 8, 10, and 11) who proved, by FISH, to have rearrangements of both chromosomes 12 and 21, and therefore, were considered to be positive for t(12;21). In none of these positive patients did the initial cytogenetic analysis identify the translocation, nor did retrospective cytogenetic analysis, carried out on archived slides, reveal the translocation. This demonstrates the difficulty of ascertaining the t(12;21) rearrangement with standard banding techniques. The full cytogenetic findings of these positive cases are presented in Table 2. Figure 1 illustrates the relevant FISH findings and partial karyotypes. Case 3 (Fig. 1, top left) presents a composite karyotype, with most metaphases having visible add(12p11) and four copies of chromosome 21. Fluorescence in situ hybridization studies revealed the t(12;21) with both der(12)t(12;21) and der(21)t(12;21) chromosomes present. It is not clear whether the normal or abnormal chromosome 12 is involved in the rearrangement. Case 8 (Fig. 1, top right) has additional chromosomes, including an abnormal chromosome 21 with add(21)(q22), which, however, is not the one involved in the t(12;21) translocation. The distal part of this chromosome does not hybridize to either chromosome 12 or 21 paints, and its origin is unknown. In case 10 (Fig. 1, bottom left), where the cytogenetic analysis gives a normal karyotype, FISH studies have also identified metaphases with t(12;21); the derivative chromosome 12 was present in either one or two copies, while the der(21) t(12;21) was not visible. In case 11 (Fig. 1, bottom right) a t(12;22) was identified by cytogenetic analysis. Fluorescence in situ hybridization studies using paints for chromosomes 19, 12, 21, and 22, and also a centromeric probe for chromosome 12, have demonstrated a three-way translocation involving chromosomes 12, 21, and 22. In this case, the visibly altered chromosome 12 accepts a 22q fragment, while there also exists a der(21;22) and a der(21) t(12;21) chromosome. We postulate that the initial t(12;21) is followed by a second event involving the der(12) t(12;21) and one chromosome 22, which exchange fragments. This case could be comparable with the variant three-way translocations reported in the formation of the Philadelphia chromosome [20]. Correlation of the cytogenetic findings with the immunophenotypic data shows that all four t(12;21) patients had common phenotype ALL, with 3 of 4 cases (3, 10, 11) having similar immunophenotypes (DR1, CD101, CD131), while the remaining patient (8) had a CD10, CD19 immunophenotype negative for DR and CD13. Cells in all patients of the study did not express cIg. The peripheral leucocyte counts (WBC) ranged from 3.5 to 12.3 3 109/L in the t(12;21) positive group as opposed to the range of 2–687 3 109/L in the entire study.

9

FISH Detection of t(12;21) in Childhood ALL Table 1 Clinical findings and data of ALL patients Patient

Age/Sex

Diagnosis

WBC (3109/L)

Immunophenotype

Cytogenetic classification

Clinical stage

1/M 7/M 2/M 5/F 2/M 13/F 2/M 7/M 2/F 2/M 8/F 1/M 1/F 2/M 13/F 3/M 11/F 4/F 7/M 2/M 4/M 4/F 4/F 8/F 4/M 2/F 11/F 16/M 24/M 14/M 50/M 79/F 4/F 23/M 26/M 16/F

Null All c ALL c ALL c ALL c ALL Null ALL c ALL c ALL c ALL c ALL c ALL c ALL Null ALL c ALL c ALL c ALL c ALL c ALL T ALL c ALL c ALL Early T ALL c ALL Null ALL c ALL c ALL T ALL c ALL c ALL c ALL ALL NHL c ALL T ALL ALL ALL

193 9.7 12.3 311 3 19.2 7.1 4.1 4 10 3.5 129.6 2 44.3 2 13.7 6.8 8.6 687 4.3 10.6 4.9 120.9 4.2 44.3 36.6 39.8 9.5 9 2.3 23.5 15.87 424 95.4 NS NS

DR, CD10, CD13, CD19, CD2, CD7 DR, CD10, CD19 DR, CD10, CD13, CD19 DR, CD10, CD13, CD19 DR, CD10, CD19 DR, CD13, CD19 DR, CD10, CD13, CD19 CD10, CD19 CD10, CD13, CD19 DR, CD10, CD13, CD19 DR, CD10, CD13, CD19, CD2 DR, CD10, CD19 DR, CD19 CD10, CD19 DR, CD10, CD19 DR, CD10, CD19 DR, CD10, CD19 DR, CD10, CD19 DR, CD2, CD7 DR, CD10, CD19 DR, CD10, CD13, CD19, CD2 CD2, CD7 DR, CD10, CD19 DR, CD13, CD19 DR, CD10, CD19 DR, CD10 DR, CD2, CD7 NS DR, CD19, CD10 DR, CD10, CD19, CD7 DR, CD10, CD19, CD7 DR, CD13, CD2 DR, CD10, CD13, CD19 NS NS NS

46Psd 46Psd 49/50 HeL 58/63 HeH 54/55 HeH 68/71 HeH 47/48 HeL 49 HeL N N 45(2X) Psd N 46/47 Psd 53 HeH 45(2X) Psd 49 HeL (Down syn) N N (Down syn) N N N 43(2X) Hypo 58 HeH N N (Down syn) 51–52 HeL 46 Psd 46 Psd 46 Psd N 42/43 Hypo 48 HeL 45(2X) Psd N 46/47 Psd N

Relapse Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Relapse Remission Diagnosis Diagnosis Diagnosis Follow-up Diagnosis Diagnosis Relapse Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Relapse Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Diagnosis Remission Diagnosis Remission

1 2 3a 4 5 6 7 8a 9 10a 11a 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

Positive immunophenotypic markers are illustrated only. Abbreviations: CD, cluster designation; ALL, acute lymphoblastic leukemia; WBC, peripheral leucocyte count at diagnosis; NS, information not supplied; M, male; F, female; NHL, Non-Hodgkin lymphoma; N, normal; Psd, pseudodiploid (46 chromosomes with structural abnormalities); HeH, hyperdiploid high (.53 chromosomes); HeL, hyperdiploid low (47–52 chromosomes); Hypo, hypodiploid (,46 chromosomes). a

Positive case.

The study included three patients who had constitutional trisomy 21. None of these was found positive for t(12;21). DISCUSSION Fluorescence in situ hybridization has become a powerful tool in cytogenetic analysis. Recent reports have shown with this approach, that in childhood ALL, translocation (12;21) occurs in high frequency and is easily missed by conventional chromosome analysis. Using FISH, we have retrospectively studied 36 patients with ALL to determine whether any of them were positive for this translocation, who had not been identified by cytogenetic studies. Indeed, it was shown that t(12;21) was present in 4 of 31 pediatric B-lineage ALL patients (13%). The incidence of t(12;21) is reported to be at least 16% in children of Cauca-

Table 2 Chromosome studies in ALL patients (with t[12;21] shown by FISH) Patients 3a

8b 10a 11a

Karyotype 49,XY,del(1)(q?32),110,add(12)(p11),121,121[6] 50,idem,122[2] 46,XY[6] 49,XY,del(6)(q?13?23),17,18,1add(21)(q22)[6] 46,XY[34] 45,X,2X,t(12;22)(p13;q13)[2] 45,idem,add(19)(q13)[10] 45,idem,del(6)(q14–15),add(19)(q13)[2] 45,idem,del(6)(q21q?23),add(19)(q13)[1] 46,XX[2]

a

Karyotype at diagnosis.

b

Karyotype at relapse.

10

D. H. Spathas et al.

sian origin with B-lineage ALL. Thus, our results are consistent with t(12;21) being one of the most common chromosome abnormalities in childhood ALL. In addition, we have demonstrated that this translocation may occur in variant forms, which possibly represent subsequent events in the evolution of the typical translocation. In cases 3 and 8, both derivative chromosomes were observed; it is interesting to note that the transloca-

tion was accompanied by visible alterations of 12p and 21q, respectively. However, it is not clear whether the abnormal 12 was involved in case 3, while the abnormal 21 was excluded in case 8. In case 10, no visible alterations were observed in the karyotype, while FISH revealed metaphases with the der(12)t(12;21) in either one or two copies. However, the reciprocal der(21)t(12;21), thought to be crucial for leukemogenesis [14], was not evident; this

Figure 1 FISH and cytogenetic findings of t(12;21) positive cases. Top left: case 3, red 5 chrom. 21 paint, green 5 chrom. 12 paint. Top right: case 8, colors and paints same as in case 3. Bottom left: case 10, colors and paints as above. Bottom right: case 11, (a) partial image, highlighting der(21): red 5 chrom. 21 paint, green 5 chrom. 12 paint; (b) partial image, highlighting der(22); red 5 chrom. 21 paint, green 5 chrom. 22 centromeric probe; and (c) partial image, highlighting der(12): red 5 chrom. 12 centromeric probe, green 5 chrom. 22 paint.

FISH Detection of t(12;21) in Childhood ALL absence along with the presence of a second der(12) t(12;21) chromosome may suggest clonal evolution with further undefined rearrangements following the initial events. Finally, in our fourth case (11), a t(12;22) translocation was identified by cytogenetic analysis. The der(12;22) chromosome was confirmed by FISH studies; however, a der(12;21) and a der(21;22) were also observed by FISH. This suggests a three-way translocation involving these chromosomes, which may originate from events following the formation of the typical t(12;21) translocation. With one exception [21], variant forms of t(12;21) have not been reported in the literature. Our results suggest that such events do occur and can also be traced by FISH screening for t(12;21). It appears, therefore, advantageous to introduce FISH for routine screening of this translocation in certain subgroups of ALL, in addition to standard karyotyping. None of the four positives represented the groups of patients with chromosome rearrangements commonly associated with ALL, for example, t(9;22)(q34;q11) [22], abnormalities of 11q23 [23], and hyperdiploid (modal chromosome number .50) chromosomes [24]. If patients with a t(9;22), rearrangement of 11q23, or hyperdiploid karyotypes are excluded, this would decrease the work load and facilitate the introduction of routine FISH screening for the t(12;21) in the remaining cases. Alternatively, screening could be done on cases with an abnormality of chromosome 12 or chromosome 21, or a normal karyotype at diagnosis. All four of the positive cases we analyzed fell into this category. Translocation t(12;21) is now a recognized subgroup of ALL [7]. To determine whether children with t(12;21) have a different prognosis, either among themselves or in comparison with other identifiable cytogenetic groups of pediatric ALL, it is necessary to study a large series of patients. Recent reports suggest that the t(12;21) may indeed highlight a subset of childhood B-ALL cases with a favorable prognosis [25–27], although further studies are required to establish the prognostic impact of t(12;21) alone or in association with other favorable prognostic features. To this context, the presence of t(12;21) in childhood ALL has been correlated with young age (between 1–5 years), lack of hyperleukocytosis (, 20 3 109/L WBC at diagnosis), non-hyperdiploid DNA, and possibly expression of specific surface antigens. All four t(12;21) patients in this study had common phenotype ALL, with young age (2–8 years), less than 50 chromosomes, and peripheral leucocyte counts (WBC) ranging from 3.5 to 12.3 3 109/L, as opposed to a range of 2–687 3 109/L in the entire study. Three had similar immunophenotypes, positive for DR, CD10, CD13, CD19, while the fourth (case 8) had a CD10, CD19 immunophenotype, negative for DR and CD13 (see Table 2). Romana et al. [11] and McLean et al. [26] reported consistency of t(12;21) with immunophenotypes positive for DR, CD10 and CD19. Kobayashi et al. [14] reported nine t(12;21) patients; all showed expression of DR, CD10, and CD19. Four of the nine also expressed CD13. Borkhardt et al. [27] have found that in 63 cases of pediatric ALL positive for t (12;21) all but two showed CD10, CD19 positive immunopheno-

11 types, with coexpression of CD13 in 15 of these cases. A pattern is thus emerging of t(12;21) association with early pre-B or pre-B ALL based on the expression of DR, CD10, and CD19, with occasional variants, such as case 8 in our study. This patient is currently in second remission and his karyotypic findings also include a 6q deletion and additional chromosomes (7 and 8). These findings probably represent evolution of the abnormal clone identified at diagnosis, 7 years ago, which, at that time, presented only a del(6q) and add(21)(q22). These abnormalities may therefore represent the earliest events in the leukemogenesis, although their simultaneous occurrence in the original abnormal clone cannot be inferred. One case has been reported in the literature where a 6q deletion was identified at diagnosis and a different clone with t(12;21) appeared in relapse [28]. The remaining three of our positive patients are currently in first remission. The clinical significance of our findings is not yet known because the follow-up time (4 years for the oldest case in the study) has been too short to permit assessment of survival or treatment responses. Borkhardt et al. [27], indicate that the presence of t(12;21) identifies a subgroup of B-cell precursor ALL with a good outcome and that, in addition to well-established clinical features such as age, chromosome number, and white blood count, it may help to enroll such ALL patients in less intensive chemotherapy regimens. Our patients may fall into this group, since all four presented young age, low leukocyte count, and moderate chromosome number. The observed variant forms of t(12;21) may, however, complicate this issue. Three ALL patients with constitutional trisomy 21 were included in this study; these were negative for t(12;21), confirming similar reports [29]. These findings do not shed light on the etiologic association between Down syndrome and acute lymphoblastic leukemia, which remains unknown. It is established [30, 31] that the t(12;21)(p12;q22) results in gene fusion between the TEL gene on chromosome 12 and the AML1 gene on chromosome 21. AML1 is believed to code for a transcription factor [32] and is known to be also involved in the formation of fusion transcripts in acute myeloid leukemia [33, 34]. The TEL-AML1 fusion gene is located on the derivative chromosome 21. Although chromosome rearrangements associated with the t(12;21) have been found to be heterogenous, and at times complex, molecular studies have shown fusion of the sequences on the der(21)t(12;21)(p12;q22) chromosome to be consistent [14]. This suggests that the TEL-AML1 gene fusion transcript from the derivative 21 may be critical to leukemogenesis. Evidence for this is also provided by recent molecular studies which show that the fusion transcript codes for a 93 Kda protein, which dominantly interferes with AML1 dependent gene regulation. As a result of the translocation, AML1B, which is the main AML1 transcript and normally an activator of transcription, is converted to a repressor [35, 36]. The reciprocal product of the AML1-TEL fusion on chromosome 12 is absent in most patients [35, 37] and is not considered to play a role in leukemogenesis; however, deletions of the normal TEL allele have been identified in most patients with t(12;21),

12 and may provide a further proliferation advantage to leukemic cells [26, 37, 38]. Acute lymphoblastic leukemia accounts for one fourth of all childhood cancers and affects approximately 2000 children each year in the United States alone [39]. It is hopeful that elucidation of the molecular events underlying the pathogenetic mechanisms will contribute toward the prevention and therapeutic management of the disease. The valuable contribution and encouragement of Dr. Elizabeth Boyd throughout this project is gratefully acknowledged. We also wish to acknowledge the expert typing assistance of Mrs. A. Labropoulou.

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