Detection of ETV6 and RUNX1 gene rearrangements using fluorescence in situ hybridization in Mexican patients with acute lymphoblastic leukemia: experience at a single institution

Cancer Genetics and Cytogenetics 162 (2005) 140–145

Detection of ETV6 and RUNX1 gene rearrangements using fluorescence in situ hybridization in Mexican patients with acute lymphoblastic leukemia: experience at a single institution Patricia Pe´rez-Veraa, Oreth Montero-Ruiza, Sara Frı´asa, Vero´nica Ulloa-Avile´sa, Rocı´o Ca´rdenas-Cardo´sb, Rogelio Paredes-Aguilerab, Roberto Rivera-Lunab, Alessandra Carnevalea,* a

Department of Research in Human Genetics, Instituto Nacional de Pediatrı´a Insurgentes Sur 3700-C, Col. Insurgentes Cuicuilco, Me´xico D.F. 04530, Mexico b Division of Hem-Oncology, Instituto Nacional de Pediatrı´a, Me´xico D.F. 04530, Mexico Received 28 February 2005; received in revised form 7 March 2005; accepted 30 March 2005

Abstract

The t(12;21) produces the gene fusion ETV6/RUNX1 and is a frequent rearrangement in childhood ALL, associated with a good prognosis. In Mexico its prevalence has not been reported. This study evaluated a group of consecutive Mexican children with newly diagnosed ALL, to detect the fusion using fluorescence in situ hybridization (FISH). Seventy-one bone marrow samples were analyzed with FISH, using ETV6/RUNX1 DNA probes. Abnormalities of ETV6, RUNX1, or both were found in 31 of the 71 (44%) patients. Six showed ETV6/RUNX1 fusion and 17, with extra RUNX1 copies, presented an additional chromosome 21 or dup(21)(q22). Five patients had structural changes in ETV6, and three patients showed extra copies of ETV6 and RUNX1 from polysomy of chromosomes 12 and 21. Our results revealed a fusion in 8.5% of the 71 cases analyzed. This frequency is lower than that observed in other populations (9.5–32%). The structural rearrangements resulting in RUNX1 extra copies were found in 9.8% of patients, which is close to the range reported (1.5–9.7%) by other authors. Due to the prevalence of RUNX1 overrepresentation in our population and its unknown prognostic significance, further studies should be conducted in consecutive children with ALL, to correlate this abnormality with the patients’ follow-up. Ó 2005 Elsevier Inc. All rights reserved.

1. Introduction The translocation t(12;21)(p13;q22), involving the ETV6 gene (previously TEL) located on the 12p13 band, and the RUNX1 (previously AML1) gene located on 21q22 is detected with conventional cytogenetic studies in !0.05% of the pediatric patients diagnosed with acute lymphoblastic leukemia (ALL) [1,2]. This translocation is difficult to identify, because the breakpoints are in clear bands of similar size on chromosomes 12 and 21. It is therefore considered a cryptic translocation [2]. The fusion ETV6/ RUNX1 can be detected with reverse transcriptase-polymerase chain reaction (RT-PCR) and with fluorescence in

* Corresponding author. Tel.: 152-55-1084-0900, ext. 1484; fax: 15255-1084-3883. E-mail address: [email protected] (A. Carnevale). 0165-4608/05/$ – see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2005.03.018

situ hybridization (FISH), and it may be found in <25% of the cases [2,3]. This rearrangement is frequently found in children between 1 and 10 years of age who have pre-B immunophenotype and a good prognosis. The finding is currently controversial, however, because of the late relapses that some of these patients have experienced [4–6]. Due to its frequency and influence on the patient’s prognosis, it is important to identify the ETV6/RUNX1 gene fusion. Because the prevalence in Mexico of this rearrangement has not been reported, we evaluated a group of Mexican children with newly diagnosed ALL using the FISH method. Extra-signal probes were used allowing to distinguish true fusion from false positive fusion signal [7], and to detect changes in the copy number of the genes analyzed [8], such as deletion of ETV6 or extra copies of RUNX1 [8–10].

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2. Design and methodology 2.1. Patients Bone marrow samples were obtained from 79 consecutive patients diagnosed with ALL during one year at the Instituto Nacional de Pediatrı´a (INP), in Mexico City. Patients were diagnosed based on the cytomorphologic classification of the French–American–British (FAB) group. This study was reviewed and approved by the Research and Ethics Committees at the INP.

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To confirm the presence of chromosome 21q duplications, some patients were analyzed using a WCP21 probe (Vysis), which hybridizes to whole chromosome 21 [14], and a 21q22.13-q22.2 probe (Vysis). The study was performed following the manufacturer’s instructions, WCP21 analysis was done in at least five metaphases per case and at least 200 nuclei or metaphases were screened with the 21q22.13-q22.2 probe.

3. Results 2.2. Conventional cytogenetics Cytogenetic studies were performed following previously reported standard methods [11,12]. Cells from bone marrow aspirates were used for a direct technique and a 24hour culture in RPMI-1640 medium. Cells were harvested using colcemid (10 mg/mL), hypotonic solution 0.075 mol/L KCl, and fixed in methanol–glacial acetic acid (3:1). Slides were prepared for cytogenetic analysis with Giemsa– trypsin banding, and the remaining pellet was stored at 4
C. Karyotypes were analyzed according to ISCN 1995 [13]. 2.3. FISH analysis It was possible to perform the FISH method with dualcolor DNA probes to detect genes ETV6/RUNX1 in 71 patients. FISH was performed on fixed bone marrow cells, following the manufacturer’s instructions [14]. An extrasignal ETV6/RUNX1 probe (Vysis, Downers Grove, IL) was used to determine the gene fusion. The ETV6 probe is labeled with SpectrumGreen fluorochrome and the RUNX1 probe is labeled with SpectrumOrange. The expected pattern for a normal cell is a two-orange (RUNX1) and two-green (ETV6) signal pattern. In cells containing the ETV6/RUNX1 fusion, the expected signal pattern is one green (native ETV6), one large orange (native RUNX1), one smaller orange (residual RUNX1), and one fused orange/ green (yellow) signal [7]. Two hundred cells, including nuclei and metaphases, were screened per patient. Cutoff values were determined in 15 control bone marrow samples (obtained from pediatric patients without leukemia, submitted to studies concerning their care). They were calculated as cutoff 5 mean 1 3 standard deviations (Table 1) [15]. As a positive control for the ETV6/RUNX1 fusion, the REH cell line (donated by Dr. Raimondi and Dr. Shurtleff) was used. In some patients, a-satellite probes 13/21 and 12 (Oncor, Gaithersburg, MD) [16] were used to determine whether the abnormal RUNX1 and ETV6 copy number were due to deletions, duplications, and extra copies or to aneuploidies as monosomies or trisomies. Hybridization was performed following the manufacturer’s recommendations. The analysis was done in at least 200 interphase nuclei or metaphases per case. To exclude false positives, cutoff values previously obtained in our laboratory for each probe were used.

Seventy-one patients were analyzed with the ETV6/ RUNX1 probes, 54 of whom also had conventional cytogenetic results. In 31 of these patients, abnormalities were observed in ETV6, RUNX1, or both: (a) 6 showed the ETV6/RUNX1 fusion, including 1 with add(21)(q22) and RUNX1 extra copies; (b) 17 showed an increase in RUNX1 copy numbers, 11 of them with polysomy 21 detected with conventional cytogenetics or FISH using a-satellite 13/21 probes and 6 with rearrangements in chromosome 21; (c) 5 showed structural changes in ETV6 and (d) 3 showed an increased number of ETV6 and RUNX1 coming from polysomy of chromosomes 12 and 21. We focused on those patients with structural abnormalities involving ETV6 and RUNX1. Table 1 shows the distribution of gender, age, leukocyte count, and immunophenotype, with the karyotype and FISH results. 3.1. Patients with gene fusion FISH analysis revealed 6 of 71 (8.5%) patients with the ETV6/RUNX1 fusion, with the ETV6 allele missing in three of them (cases 1–6; Table 1). The karyotype was abnormal in five of these; it failed in one. The cytogenetic changes observed differed among them. Patient 5 showed an add(21)(q22), and FISH detected a clone with an increased number of RUNX1 copies, in addition to gene fusion. The WCP21 probe revealed a completely stained larger chromosome 21. 3.2. Patients with increased number of RUNX1 copies and rearrangements of chromosome 21 Seven nonhyperdiploid patients (patients 5, 7–12) showed 3–6 copies of RUNX1 (Fig. 1). In some patients, this finding matched the results of the conventional cytogenetics: patients 5, 7 and 9 revealed add(21)(q22) chromosomes, with tandem duplications in the 21q22 band where RUNX1 is located. In four patients, however, there was no evidence of an abnormal chromosome 21 (cases 8, 10–12; Table 1). These patients were analyzed with the WCP21 probe, confirming the presence of a larger completely coated chromosome 21. In some cases they were also analyzed with the 21q22.13-q22.2 probe, showing extra signals on nuclei/metaphases, confirming the duplication of the region (Table 1).

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Table 1 Patients with structural rearrangements in RUNX1 or ETV6 Sex

Age, years

WBC, 109/L

Karyotype

Additional karyotype information identified with FISH or interphase FISHb

Interpretation

1

M

4

7.3

46,XY,i(6)(p10)[16]

ETV6/RUNX1 fusion and loss of one ETV6 allele

2

M

9

13.5

47,XY,1mar[3]/46,XY[22]

3

M

4

54.0

46,XY,t(3;10)(p21;q24)[16]

4

M

2

211.0

43,XY,27,215,219[cp7]/ 46,XY[5]

5

M

6

157.0

46,XY,del(6)(q24), add(21)(q22)[13]/46,XY[2]

6

M

3

7.8

Not done

7

F

12

11.7

8

M

2

1.6

46,XX,t(5;9)(qter;q22),27, ins(8;7)(p11;q11;q32), der(11)t(7;11)(q32;p15), add(21)(q22),1mar[5]/45,XX, t(5;9)(qter;q22),27, add(21)(q22)[4]/46,XX[3] 46,XY[15]

t(12;21)(p13;q22.3)(ETV61RUNX11sp;RUNX11sp),del(12)(p13)(ETV62).nuc ish 12p13(ETV61),21q22.3(RUNX12)(ETV6 con RUNX11sp)[170]/ 12p13(ETV62),21q22.3(RUNX12)(ETV6 con RUNX11sp)[3]/ 12p13(ETV62),21q22.3(RUNX12)[27] t(12;21)(p13;q22.3)(ETV61RUNX11sp;RUNX11sp),del(12)(p13)(ETV62).nuc ish 12p13(ETV61),21q22.3(RUNX12)(ETV6 con RUNX11sp)[27]/ 12p13(ETV62),21q22.3(RUNX12)[173] t(12;21)(p13;q22.3)(ETV61RUNX11sp;RUNX11sp).nuc ish 12p13(ETV62),21q22.3(RUNX12)(ETV6 con RUNX11sp)[196]/ 12p13(ETV62),21q22.3(RUNX12)[4] t(12;21)(p13;q22.3)(ETV61RUNX11sp;RUNX11sp).nuc ish 12p13(ETV62),21q22.3(RUNX12)(ETV6 con RUNX11sp)[184]/ 12p13(ETV62),21q22.3(RUNX12)[16] t(12;21)(p13;q22.3)(wcp211,ETV61RUNX11sp;RUNX11sp),dup(21)(q22) (wcp211,D21S259/D21S341/D21S3422,RUNX12).nuc ish 12p13(ETV62),21q22.3(RUNX12)(ETV6 con RUNX11sp)[167]/12p13(ETV62), 21q22.3(RUNX13)(ETV6 con RUNX11sp)[20]/12p13(ETV62), 21q22.3(RUNX12)[13].nuc ish 21q22.13|2(D21S259/D21S341/D21S3423)[14]/j21q22.13|2(D21S259/D21S341/ D21S3422)[186] nuc ish12p13(ETV61),21q22.3(RUNX12)(ETV6 con RUNX11sp)[186]/ 12p13(ETV62),21q22.3(RUNX12)(ETV6 con RUNX11sp)[9]/ 12p13(ETV62),21q22.3(RUNX12)[5] dup(21)(q22)(wcp211,D21S259/D21S341/D21S3422|3,RUNX12|4).nuc ish 12p13(ETV62),21q22.3(RUNX13-5)[110]/12p13(ETV62),21q22.3(RUNX12)[90].nuc ish 21q22.13|2(D21S259/D21S341/D21S3423|4)[134]/21q22.13|2(D21S259/D21S341/ D21S3422)[66]

9

M

12

69.9

46,XY,add(21)(q22) [8]

10

F

12

2.3

46,XX[20]

11

F

2

68.0

47,XX,t(1;19),1mar[20]

12

F

15

8.3

46,XX[20]

Case no.a

ETV6/RUNX1 fusion

ETV6/RUNX1 fusion and duplication 21q22 with 2 extra copies of RUNX1

ETV6/RUNX1 fusion and loss of one ETV6 allele Duplication 21q22 with 2-4 extra copies of RUNX1

Duplication 21q22 with 2 extra copies of RUNX1 Duplication 21q22 with 2 extra copies of RUNX1 Duplication 21q22 with 2-5 extra copies of RUNX1 Duplication 21q22 with 2-4 extra copies of RUNX1 Duplication 21q22 with 2 extra copies of RUNX1

(Continued )

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dup(21)(q22)(wcp211,RUNX12).nuc ish 12p13(ETV62),21q22.3(RUNX13)[46]/12p13(ETV62),21q22.3(RUNX12)[154] dup(21)(q22)(wcp211,D21S259/D21S341/D21S3422,RUNX12).nuc ish 12p13(ETV62),21q22.3(RUNX13)[108]/12p13(ETV62),21q22.3(RUNX12)[92].nuc ish 21q22.13|2(D21S259/D21S341/D21S3423)[73]/21q22.13|2(D21S259/D21S341/D21S3422)[127] dup(21)(q22)(wcp211,D21S259/D21S341/D21S3422|4,RUNX12|5).nuc ish 12p13(ETV62),21q22.3(RUNX13|6)[170]/12p13(ETV62),21q22.3(RUNX12)[30].nuc ish 21q22.13|2(D21S259/D21S341/D21S3423|5)[138]/21q22.13|2(D21S259/D21S341/ D21S3422)[62] dup(21)(q22)(wcp211,D21S259/D21S341/D21S3422|3,RUNX12|4).nuc ish 12p13(ETV62),21q22.3(RUNX13-5)[81]/12p13(ETV62),21q22.3(RUNX12)[119].nuc ish 21q22.13|2(D21S259/D21S341/D21S3423|4)[47]/21q22.13|2(D21S259/D21S341/ D21S3422)[153] dup(21)(q22)(D21S259/D21S341/D21S3422,RUNX12).nuc ish 12p13(ETV62),21q22.3(RUNX13)[41]/12p13(ETV62),21q22.3(RUNX12)[159].nuc ish 21q22.13|2(D21S259/D21S341/D21S3423)[44]/21q22.13|2(D21S259/D21S341/D21S3422)[156]

ETV6/RUNX1 fusion and loss of one ETV6 allele ETV6/RUNX1 fusion

Patient 12 presented a 13/21 a-satellite normal pattern. All patients had a pre-B cell immunophenotype, except numbers 11 and 13, who presented with T-ALL. Interphase FISH results are highlighted in boldface type. Cutoff values: ETV6/RUNX1 5 2%; 3  RUNX1 5 7.3%; 4  RUNX1 5 0.5%; 1  ETV6 5 7.7%; 3  21q22.13|2 5 2.8%; 4  21q22.13|2 5 1%. a

b

46,XY,t(2;19)(p23;p13),t(9;12) (q22;p13)[15]/46,XY[3] 5 M 17

26.8

47,XY,121[9]/46,XY[44] 5 M 16

2.3

48,XX,121,1mar[4]/46,XX[9] 12 F 15

80.3

15 M 14

13.6

51,XY,14,16,118,121, 121[11]/46,XY[9]

Loss of one allele ETV6

nuc ish 12p13(ETV61),21q22.3(RUNX12)[100]/12p13(ETV62), 21q22.3(RUNX12)[100] del(12)(p13)(ETV61).nuc ish 12p13(ETV61),21q22.3(RUNX13-4)[163]/12p13(ETV61), 21q22.3(RUNX12)[5]/12p13(ETV62),21q22.3(RUNX12)[32] del(12)(p13)(ETV61).nuc ish 12p13(ETV62),21q22.3(RUNX13)[111]/ 12p13(ETV61),21q22.3(RUNX13)[41]/12p13(ETV62),21q22.3(RUNX12)[148] del(12)(p13)(ETV61).nuc ish 12p13(ETV61),21q22.3(RUNX13)[135]/12p13(ETV61), 21q22.3(RUNX12)[19]/12p13(ETV62),21q22.3(RUNX12)[46] t(9;12)(q22;p13)(ETV62),21q22.3(RUNX12)(ETV61sp con?).nuc ish 12p13(ETV62),21q22.3(RUNX12)(ETV61sp con?)[131]/ 12p13(ETV62),21q22.3(RUNX12)[69] 4 M 13

880.0

Not done

Interpretation Additional karyotype information identified with FISH or interphase FISHb Karyotype WBC, 109/L Age, years Sex Case no.a

Table 1 Continued

Loss of one allele ETV6 and extra copies of RUNX1 by aneuploidy Loss of one allele ETV6 and extra copies of RUNX1 by aneuploidy Loss of one allele ETV6 and extra copies of RUNX1 by aneuploidy Translocation of one allele ETV6

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3.3. Patients with structural abnormalities in ETV6 The loss of one allele ETV6 was detected in 4 patients (cases 13–16); one of them (case 13, Table 1) whose conventional cytogenetic study failed and three patients (cases 14, 15, and 16) with polysomy 21 had 3–4 copies of RUNX1. These patients were analyzed and the ETV6 deletion was confirmed with the presence of two signals using the a-satellite probe for chromosome 12. In patient 17, a three-signal pattern of the ETV6 gene was detected, produced by the translocation t(9;12)(q22;p13), which was revealed by conventional cytogenetics.

4. Discussion ETV6 and RUNX1 genes have been involved in multiple rearrangements associated with different types of leukemia. The t(12;21), which produces the ETV6/RUNX1 fusion, is particularly frequent in the ALL identified in up to 25% of the patients [3,10]. When looking for this abnormality with FISH, several authors have reported other rearrangements involving these genes as the deletion of ETV6 or the tandem duplication of RUNX1, which has been related to clinical characteristics and the disease’s natural history [8,9,17,18]. Because the prevalence of these abnormalities is unknown in Mexican children who have ALL, 71 consecutive pediatric patients with ALL at the diagnostic stage were studied using the FISH technique. The prevalence of ETV6/RUNX1 gene fusion was found to be 8.5%. This percentage is low, compared with that observed in the majority of populations analyzed using RTPCR, FISH, or both. These range from 9.5% in a Korean population [7] to 32% in Italian and German patients [19]. The lower prevalence of this translocation in other populations has been related to the higher frequency of T cell ALL [20], because this immunophenotype is uncommon in patients with the translocation [4]. However this argument does not explain the results. In our Institute, as in the analyzed patients, the B-cell immunophenotype is the most frequent and is found in 75% of children with ALL [21,22]. Three patients with the gene fusion also had ETV6 loss, which has been previously described in pediatric ALL as a secondary change frequently associated with the translocation [17]. In the present study, the ETV6/RUNX1 gene fusion was not found in karyotypes with hyperdiploidy O50 chromosomes, t(9;22), t(1;19) or with 11q23 rearrangement, as it has been reported by other authors [23]. Currently all patients are being treated and none has relapsed; however, not enough time has elapsed to be able to reach a conclusion about their prognosis. In some patients without the ETV6/RUNX1 fusion, we observed losses of ETV6 signal and a t(9;12) that produced an extra signal of the gene. Several studies have shown that

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Fig. 1. (A) Metaphase and nucleus with extra copies of RUNX1 gene (red signals) on der(21) indicated by arrowhead (O). (B) Metaphase of patient showing more copies of the same gene, indicated by arrowhead (O). Asterisk (*) indicates normal chromosomes 21.

ETV6 takes part in several changes that can be detected using the FISH method [8,24,25]. Our results showed 7 of 71 (9.8%) patients with dup(21)(q22) and overrepresentation of RUNX1, confirmed with FISH using the probe WCP21 (which revealed larger chromosomes entirely composed of chromosome 21 material) [18,26,27], or studying the region 21q22.13-q22.2 [28]. It has to be noted that in only three of the seven patients did the conventional cytogenetics reveal a chromosome 21 larger than its homologous representing tandem triplications and quadruplications of 21q22, similar to those described by other authors [9,18,26–28]. The inconsistency between the result of a normal karyotype with conventional cytogenetics and the demonstration of 21q22 duplication with FISH could be an effect of suboptimal chromosome quality of leukemic cells. The duplication may also be cryptic, as shown by Mikhail et al. [20], who reported RUNX1 amplification using RT-PCR and FISH, in chromosomes of apparently normal size; it has also been observed that leukemic cells can have different numbers of gene copies [9,20]. In ALL, patients with overrepresentation of RUNX1 and recurrent translocations are uncommon. To our knowledge, only 2 patients described by Mikhail et al. [20] and by Ma et al. [29] presented the t(12;21). In this study we found the overrepresentation of RUNX1 in one patient with the t(12;21) and in another with a t(1;19). We also detected 2 patients with a monosomy of chromosome 7 (27). This aneuploidy was reported previously by Harewood et al. [18] in patients with extra RUNX1 and recently, Heerema et al. [30] found 4 of 75 patients with 27, three showed add(21)(q22), and one i(21)(q10)2 [18,30]. These findings suggest that both abnormalities could be related. The frequency of RUNX1 extra copies by tandem duplications is unknown and the patients described to date are from different Institutions [8,9,18,20,26–29,31]. Harewood et al. [18] described 20 patients with RUNX1

tandem amplifications that were collected during 6 years, and estimated that the frequency in children with ALL was 1.5%. On the other hand, Mikhail et al. [20] found four children (9.7%) out of 41 patients with ALL, revealing that there may be variations in the frequency between different populations. In this study, tandem duplication producing RUNX1 extra copies was seen in 9.8% of consecutive Mexican children with ALL. Most of them were over 12 years old, had low leukocyte counts and most of them had pre-B immunophenotypes. These characteristics are similar to those of other patients with the same rearrangement [8,9,18,26,27]. In summary, using the FISH method, we found the prevalence of the t(12;21) lower than expected and we detected other rearrangements involving the RUNX1 and ETV6 genes. It is important to consider that the small number of cases studied could bias the incidence of these abnormalities. The RUNX1 overrepresentation was found at a frequency similar to that of the gene fusion. Due to its prevalence in our population and because its significance in the evolution of the disease is unknown [18], further studies should be conducted in patient carriers of this rearrangement. Acknowledgments O. M.-R. and V.U.-A. were supported by the SNICONACyT (Mexico) scholar fellowship (Ayudante de Investigador). References [1] Rubnitz JE, Look T. Molecular genetics of acute lymphoblastic leukemia. In: Pui C-H, editor. Childhood leukemias. New York: Cambridge University Press, 1999:197–217.

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