¹Division of Hematology-Oncology, Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA

²University of Texas Medical School, Houston, TX, USA

Correspondence to: P H Rao, Texas Children's Cancer Center, Baylor College of Medicine, 6621 Fannin St, MC 3-3320, Houston TX 77030-2399, USA; Fax: 832 825-4846

^aPresent address: Children's Mercy Hospital, Section of Medical Genetics and Molecular Medicine, Kansas City, MO, USA

We applied multicolor spectral karyotyping (SKY) to a panel of 29 newly diagnosed pediatric pre B-cell ALLs with normal and abnormal G-banded karyotypes to identify cryptic translocations and define complex chromosomal rearrangements. By this method, it was possible to define all add chromosomes in six cases, a cryptic t(12;21)(p13;q11) translocation in six cases, marker chromosomes in two cases and refine the misidentified aberrations by G-banding in two cases. In addition, we identified five novel non-recurrent translocations - t(2;9)(p11.2;p13), t(2;22) (p11.2;q11.2), t(6;8)(p12;p11), t(12;14)(p13;q32) and t(X;8)(p22.3;q?). Of these translocations, t(2;9), t(2;22) and t(12;14) were identified by G-banding analysis and confirmed by SKY. We characterized a t(12;14)( p13;q32) translocation by FISH, and identified a fusion of TEL with IGH for the first time in ALL. We identified a rearrangement of PAX5 locus in a case with t(2;9)(p11.2;p13) by FISH and defined the breakpoint telomeric to PAX5 in der(9)t(3;9)(?;p13). These studies demonstrate the utility of using SKY in combination with G-banding and FISH to augment the precision with which chromosomal aberrations may be identified in tumor cells.

Leukemia (2002) 16, 2222-2227. doi:10.1038/sj.leu.2402662

spectral karyotyping; chromosomal translocations; pediatric ALL

Introduction

Acute lymphoblastic leukemia (ALL) is the most common malignancy in children and is characterized by the accumulation of malignant immature lymphoid cells in the bone marrow and also in peripheral blood.¹ Approximately 75-90% of ALLs has clonal chromosomal aberrations, of which 50% are chromosomal translocations. The remaining 10-25% of cases show no detectable chromosomal aberrations by conventional cytogenetic methods.^2,3 However, subsequent studies using molecular cytogenetic methods such as fluorescence in situ hybridization (FISH) indicated that some of these patients harbor cryptic chromosomal translocations, ie t(12;21)(p13;q11.2). This translocation was first identified using a chromosome 12 painting probe⁴ and subsequently cloned and shown to involve TEL (ETV6) on chromosome 12 and AML1 (CBFA2) on chromosome 21.^5,6 More sensitive methods are required to detect this type of subtle translocation and one such method is multicolor spectral karyotyping (SKY)/multicolor fluorescence in situ hybridization (m-FISH).^7,8,9,10

SKY is based on the hybridization of 24 combinatorially labeled painting probes to metaphase spreads, which allows simultaneous visualization of each chromosome pair by a unique color in a single experiment.⁷ Because of the intrinsically higher resolution of SKY and its ability to detect cryptic translocations, we decided to evaluate the utility of SKY in the routine cytogenetic analysis of pediatric ALL in the setting of a clinical cytogenetics laboratory. We applied SKY to a panel of 29 pediatric pre B-cell ALLs with normal and abnormal G-banded karyotypes to determine if SKY could (1) detect all the abnormalities detected by G-banding, (2) identify cryptic translocations and (3) better define complex chromosomal rearrangements that are beyond the sensitivity of G-banding. Three novel translocations were identified by G-banding and confirmed by SKY. In addition, SKY identified another two new translocations. SKY defined all add chromosomes in six cases, a cryptic t(12;21)(p13;q11.2) translocations in six cases, marker chromosomes in two cases and in two cases refined the aberrations previously misidentified by G-banding.

Materials and methods

Patient samples

A total of 86 cases of pre-B ALL was diagnosed at the Texas Children's Hospital between March 1998 and January 2000. All bone marrow aspirates of newly diagnosed patients have routine cytogenetic analysis by G-banding and selected FISH studies (eg TEL/AML1). Cases with adequate metaphase cells remaining after routine cytogenetic analysis were subjected to SKY analysis in a blinded fashion. A total of 29 cases had SKY analysis based on the availability of cytogenetically prepared cell pellets. The ages of these patients ranged from 21 days to 16 years with a mean of 5.96 years. Among the 29 patients, 14 were male and 15 were female.

Cytogenetic analysis

Metaphase spreads from bone marrow aspirate specimens were obtained from unstimulated short-term cultures grown in the clinical cytogenetics laboratory at The University of Texas Medical School (Houston, TX, USA). Chromosome preparations were made using standard protocols. All remaining cell pellets were stored in methanol/acetic acid at -20°C. Clonal chromosomal abnormalities identified by G-banding were described according to the International System for Human Cytogenetic Nomenclature (ISCN).¹¹

Spectral karyotypic analysis

Slides for SKY were freshly prepared using cell pellets provided by the clinical cytogenetic laboratory after routine cytogenetic analysis had been completed. The cocktail of human chromosome paints was purchased from Applied Spectral Imaging (ASI, Carlsbad, CA, USA). Hybridization and detection were carried out according to the manufacturer's protocol with slight modifications. Chromosomes were counter-stained with 4',6-diamidino-2-phenylindole (DAPI). For each case, a minimum of five metaphases was analyzed using SKY View 1.2 software (ASI). Breakpoints on the SKY-painted chromosomes were determined by comparison of corresponding DAPI banding of the same chromosome with the G-banded karyotype of the same specimen. By this method, we were able to define the breakpoints on add and der chromosomes but were unable to assign the precise breakpoints of chromosomal segments from partner chromosomes that generated the add or der chromosomes.¹² A breakpoint was considered recurring if identified in two or more cases. SKY was performed with encoded samples without prior knowledge of the clinical cytogenetic diagnosis in each case. Comparison with routine cytogenetic results was carried out only after all SKY analysis had been completed.

FISH analysis

Locus-specific probes (MLL, TEL, TEL/AML1) and painting probes for chromosome X (WCP X) were obtained from Vysis (Downer's Grove, IL, USA). The chromosomal arm probe for the short arm of chromosome X (CAP X) was purchased from AL technologies (Arlington, VA, USA). The cosmid yIgH6-9 (VH) probe located near the telomeric end of 14q (gift from Dr WM Kuehl, National Cancer Institute, Bethesda, MD, USA) and YAC 788C3 containing PAX5 at 9p13 were labeled by nick translation using spectrum red dUTP (Vysis, Downer's Grove, IL, USA). Probe hybridization and detection were performed as recommended by the manufacturer's protocols with some modifications.

Results

We analyzed chromosomal rearrangements and breakpoints in a panel of 29 newly diagnosed pre-B ALLs by G-banding and SKY. Of these 29 cases, only one case had apparently normal karyotype by G-banding (case 22), three cases with numerical chromosomal changes only (cases 3, 19 and 29), three cases with both numerical and structural changes (cases 11, 13 and 20) and the remaining 23 cases with structural chromosomal aberrations only. By SKY, we confirmed G-banded karyotypes in 66% and refined in 34% of the total chromosomal aberrations. SKY defined add marker chromosomes in six cases, cryptic translocations in six cases, precisely identified the misidentified chromosomal aberrations by G-banding in two cases, and the marker chromosomes in two cases.

Analysis of structural rearrangements

The comparison of partial karyotypes of G-banding, SKY and FISH is presented in Table 1. By SKY, we confirmed the numerical chromosomal changes identified by G-banding in three cases (3, 19 and 29) and could not detect any structural changes. In case 22, G-banding analysis detected no chromosomal aberration but SKY identified del(4)(q?).

The additional chromosomal material was detected on Xp (case 7), 1p (case 13), 6p (case 25), 12p (cases 2, 8), 16p (case 17) and 20p (case 5) by G-banding analysis (Table 1; Figure 1a) In case 7, the additional material on Xp was identified as der(X)t(X;8)(p22.3;q?). FISH with an Xp painting probe confirmed this translocation. Cytogenetic analysis identified a del(6)(q13q21) and add(20)(p13) in case 5. SKY analysis of the same patient confirmed the del(6), refined the add(20)(p13) to der(20)t(X;20)(?;p13) and identified an additional translocation - der(12)t(12;21) (p13;q22). FISH using a TEL/AML1 specific probe and WCP X probe confirmed these translocations. The additional chromosomal material on 1p, 6p and 16p was identified as del(1)(p13), t(6;8)(p12;p11) and der(16) t(X;16) (?;p13.3), respectively.

In case 11, SKY refined the misidentified translocation t(1;13)(q21;p13) by G-banding analysis to der(20)t(13;20) (q11;q12) (Figure 1b). Conventional G-banding analysis identified mar chromosomes in two cases (2 and 15). The derivation of these markers was identified as inv(7)(q31q36) in case 2 and del(9)(q11) in case 15 by SKY analysis (Figure 1c). We identified two translocations involving 9p13 region, ie t(2;9)(p11.2;p13) and der(9)t(3;9)(?;p13). Further FISH analysis using a YAC clone containing PAX5 revealed a rearrangement of PAX5 locus in a case with t(2;9)(p11.2;p13) (case 10) (Figure 2a) and defined the breakpoint in der(9)t(3;9)(?;p13) telomeric to PAX5 (case 14).

Identification of 12p abnormalities

Conventional G-banding analysis identified a t(12;21) (p13;q22) translocation in one patient (case 4) but SKY detected this cryptic translocation in six more cases (5, 6, 7, 12, 17, 24). The der(21) was detected by SKY in all the cases except case 8 and this may be due to the sensitivity of the method. FISH using a TEL/AML1 dual-color probe confirmed the presence of a t(12;21)(p13;q22) translocation in all seven cases. In four out of six cases, G-banding reported a del(12)(p13) and analysis of the remaining two cases (5 and 17) revealed no chromosome 12 abnormality. In addition G-banding detected add material at 12p13 in two cases (2 and 8). By SKY, it was possible to identify the additional chromosomal material in case 2 as der(12)t(7;12)(?;p13) and in case 8 as der(12)t(4;12)(q?;p13). No TEL gene rearrangements were detectable by FISH analysis on metaphase spreads from these cases. However, TEL was deleted in case 8. Both conventional and spectral karyotypic analysis identified a t(12;14)(p13;q32) translocation (case 20). FISH using TEL (12p13) and IGH (14q32) probes confirmed the reciprocal nature of the translocation (Figure 2b).

Discussion

Non-random chromosomal changes play an important role in diagnosis and prognosis for preB ALL in children.³ Yet the most frequent abnormality, t(12;21), is cryptic to routine cytogenetic analysis and can only be reliably detected by FISH. Thus it is conceivable that by combining more sensitive molecular cytogenetic techniques with G-banding karyotyping in the routine analysis of leukemic blasts, new recurrent translocations with prognostic value can be discovered.³ We applied SKY to a panel of 29 newly diagnosed pediatric pre-B cell ALLs with normal and abnormal G-banded karyotypes to identify cryptic translocations and define complex chromosomal rearrangements. As illustrated in Table 1, SKY confirmed the chromosomal abnormalities detected by routine cytogenetics in all but one case. No chromosomal aberration was detected by G-banding analysis in case 22, but SKY/DAPI identified del(4)(q?) in this patient. This is probably due to the different clone identified by SKY/inverted DAPI in this patient. In this study, we identified five novel non-recurrent translocations - t(X;8)(p22.3;q?), t(2;9)(p11.2;p13), t(2;22) (p11.2;q11.2), t(6;8) (p12;p11) and t(12;14)(p13;q32). Of these translocations, three, t(2;9), t(2;22) and t(12;14), were identified by both G-banding and SKY. The other two were identified by SKY only. The significance of these translocations and their breakpoints are not yet known in ALL.

A significant number of cases of pediatric ALL has been shown to have 12p abnormalities. The majority of these 12p abnormalities is associated with a t(12;21)(p13;q22) translocation (25%) involving the fusion of Ets-related transcription factor TEL/ETV6 on 12p13 to the transcription factor AML/CBFA2 on 21q22.^5,6 This translocation was not easily identifiable by conventional G-banding methods due to the similar banding pattern on these chromosomes. By SKY, we are able to identify this translocation in seven cases compared to one case by G-banding. Furthermore, this translocation was confirmed by dual-color FISH using TEL/AML1 probe in all seven cases. Since its initial cloning, ETV6/TEL has been found to be rearranged in translocations involving more than 30 other chromosomal bands with 12 different genes. These include ARNT (1q21),¹³ ARG (1q25),¹⁴ MDS1/EVI1 (3q26),¹⁵ BTL (4q11q12),¹⁶ ACS2 (5q31),¹⁷ PDGFRB (5q33),¹⁸ STL (6q23),¹⁹ JAK2 (9p24),²⁰ ABL (9q34),²¹ CDX2 (13q12),²² NTRK3 (15q25),²³ AML1/CBFA2 (21q22)⁵ and MN1 (22q11).²⁴ In the present study, SKY identified three cases with different partner chromosomes, ie der(12)t(7;12)(q?;p13), der(12)t(4;12)(q?;p13) and t(12;14)(p13;q32). The TEL gene is not found to be rearranged in case with der(12)t(7;12) and is found deleted in der(12)t(4;12). In contrast, one study has shown the involvement of ETV6/TEL in patients with t(7;12)(q36;p13) translocation in six out of seven cases.²⁵ The BTL¹⁶ gene on 4q11-q12 and HLBX²⁶ gene on 7q36 might be involved in these translocations. However, FISH analysis on metaphase spreads from the patient with t(12;14) has shown the fusion of TEL with IGH on der(12) and der(14). This translocation has not been reported previously in ALL (http://cgap.nci.nih.gov/Chromosomes/Mitelman).

As expected, SKY was able to refine the cytogenetic diagnosis of 10 out of 29 cases, defining all add chromosomes in six cases, marker chromosomes in two cases and refining the misidentified aberrations by G-banding in two cases. However, these refinements did not alter the clinical diagnosis of preB-ALL or the therapeutic decisions. SKY has also been utilized recently to detect chromosomal complexity in other hematological and solid malignancies.^{27,28,29,30,31,32,33,34} SKY analysis of cases of T cell leukemia,²⁹ acute leukemia³⁰ and myelodysplastic syndrome^31,32 with normal karyotypes failed to detect any cryptic translocations but did refine most of the chromosomal rearrangements. However, one study on acute myeloid leukemia identified cytogenetic aberrations in 7% of karyotypically normal patients.²⁹ Our previous SKY analysis on pediatric ALLs identified three cryptic translocations in 10% of the cases with normal karyotypes.³⁴ In the present study, we identified a rearrangement of PAX5 locus in a case with a t(2;9) translocation by FISH. In addition, FISH defined the breakpoint in a der(9)t(3;9)(?;p13) translocation telomeric to PAX5. This gene has been previously mapped to 9p13 and shown to be involved in lymphoplasmocytic lymphoma with t(9;14)(p13;q32).³⁵ The t(2;9)(p11.2;p13) may be a variant translocation involving the encoding immunoglobulin kappa chain complex. The PAX5 gene belongs to the PAX gene family, which is characterized by the presence of paired domains and plays a critical role in fetal development and organogenesis.³⁶ The gene encodes for a 50 kDa B cell-specific transcription factor, BSAP (B cell-specific activator protein).³⁷

In conclusion, our study demonstrates the power of SKY in identifying complex chromosomal rearrangements in ALL. It can be considered as part of the routine cytogenetic workup of newly diagnosed ALL.

Acknowledgements

This study was partially supported by a research grant from the Fleming-Davenport Foundation.

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Figure 1 Identification of all add chromosomes (a), misidentified chromosomes by G-banding (b) and marker chromosomes (c) by SKY. For each image, left to right is inverted DAPI and classification color.

Figure 2 (a) Spectrum red-labeled PAX5 (9p13) hybridized on to metaphase preparation from case 10. The FISH signal can be seen on both der(2) and der(9) chromosomes. (b) Metaphase spread from case 20 was hybridized with TEL (green) and IGH (red) probes. Fusion signal was identified by yellow color on der(12) and der(14).

Table 1 Summary of G-banding, SKY and FISH results from patients with pre-B ALL