We report three cases of T-ALL in which conventional cytogenetic analysis yielded normal karyotypes, but for which a new M-FISH technique (IPM-FISH) was able to detect a translocation. For these patients this technique highlighted a new, recurring and cryptic translocation t(5;14)(q35;q32) in childhood T-ALL which might be phenotypically restricted. The most innovative part of this technique is the use of interspersed polymerase chain reaction (IRS-PCR) painting probes that show an R-band pattern simultaneous with the combinatorial labeling. Contrary to the DOP-PCR, IRS-PCR-derived probes provide stronger hybridization signals at the telomeric ends that potentially increase the possibility of detecting cryptic translocations. All the IPM-FISH findings were validated by FISH with whole chromosome painting and unique sequence probes. These results demonstrate the efficient use of IPM-FISH as an improved, single-step method for the identification of cryptic chromosomal abnormalities. This new IPM-FISH technique is a good tool to display cryptic chromosomal abnormalities.
Novel molecular cytogenetic techniques termed multicolor fluorescence in situ hybridization (M-FISH) and multicolor spectral karyotyping (SKY) enable precise identification of complex chromosomal rearrangements.1,2,3 These methods are based on the cohybridization of 24 fluorescently labeled chromosome painting probes to metaphase chromosomes, which allows simultaneous vizualization of each chromosome pair by a unique color. We performed analysis on 60 patients with various hemopathies using a new M-FISH technique: IPM-FISH (IRS-PCR multiplex FISH).4 We report here three cases of T-ALL in which a recurring translocation t(5;14)(q35;q32), invisible by conventional cytogenetic methods, has been detected. This paper should be read in conjunction with Reference 5.
T-ALL comprises 12 to 15% of childhood acute lymphoblastic leukemia.6,7 It has clinical, immunological, cytogenetic and molecular features that are distinct from those of B-lineage ALL. The most commonly reported recurring clonal chromosome abnormalities comprise rearrangements affecting TCR loci on 7q32–36, 14q11 and 7p15. A normal karyotype has been found in 30 to 40% of patients.6,7 However, cytogenetic information on T-ALL is limited due to the often poor quality of the metaphase spreads. One concern regarding these data is the possibility that a recurring translocation may have been overlooked.
This study demonstrates the power of the IPM-FISH in resolving rearrangements totally cryptic with conventional banding techniques.
Materials and methods
To test the new IPM-FISH technique, 60 patients were selected from a group of 640 acute and chronic hematologic disorders, which occurred between January 1999 and April 2001 (24 AML, 9 T-ALL, 4 B-ALL, 11 MDS, 4 lymphomas, 4 CLL, 2 CML and 2 MPS). The main criteria for the choice of patients were: (1) the complexity of chromosomal rearrangements, leading to difficulties in interpretation and conclusion; (2) for some classical forms with identified recurrent translocation, the research of cryptic rearrangements; and (3) in some cases, with no visible abnormalities, the research of cryptic translocations.
Out of the 60 patients, 10 had normal RHG banded karyotypes (6 AML, 3 T-ALL and 1 MDS). The IPM-FISH technique identified cytogenetic abnormalities in 39 patients that had not been partially or fully characterized by standard RHG banding. Among these, three cases are noteworthy: three T-ALL which were considered normal with the RHG banding technique, showed the same cryptic translocation.
The diagnosis of T-ALL was based on morphologic and immunological features of leukemic cells. Lymphoblast morphology was studied on May–Grunwald-Giemsa stained blood and bone marrow smears and we also verified the negative staining of myeloperoxidase. Mononuclear cell fractions that consisted of over 90% leukemic cells were isolated from pre-treatment bone marrow aspirate samples by centrifugation on Ficoll–Hypaque gradients. Immunophenotyping was performed by flow cytometry (FACScalibur, Becton Dickinson, Le Pont de Claix, France) using labeled monoclonal antibodies reactive with the differentiation antigens summarized in Table 1. Patients were assigned to T cell lineage and classified using the European Group for the Immunological Characterization of Leukemias (EGIL) recommendations.8 Briefly, acute T lymphoid leukemias are defined by the expression of CD3 (cytoplasmic or membrane) and are subdivided into four subgroups, according to the degree of thymic differentiation: TI and TII (early T-ALL): cCD3+, CD1a−, sCD3−, TIII (cortical thymocyte ALL) only defined by the expression of CD1a+ (cCD3 or sCD3+); and TIV (mature T-ALL): CD1a−, sCD3+.
A 7-year-old boy, was referred for tiredness, fever (38°C) and bone pains. The physical examination showed multiple small adenopathies (axillar, sub-clavicular, cervical) and hepatosplenomegaly. The thorax X-ray examination revealed an enlarged mediastinum and bilateral pleural extravazation.
The peripheral blood cell count showed white blood cells (WBC) 38.8 × 109/l with 10% blast cells, 29% neutrophils, 13% immature granulocytic cells, 33% lymphocytes, and hemoglobin (Hb) 13.1g/dl, platelets: 77 × 109/l. The bone marrow was normocellular with 85% blast cells, 10% granulocytic lineage and rare megakaryocytes. The blast cells were peroxidase negative. Cytology confirmed the pleural-specific localization with 98% blast cells (Table 2).
Diagnostic immunophenotyping analysis demonstrated T-lineage lymphoblasts expressing CD1a+, CD2+, cytoplasmic CD3+ and surface negative, CD4+, CD8+, CD5+, CD7+, TCR−, CD34−, DR− and Tdt positive. Leukemic cells did not express any B- or myeloid-lineage markers. CD1a expression classified this patient in the T-III EGIL subgroup (Table 1).
Considered to be of average risk, he was included in the CLCG-EORTC 58951 protocol9 and remained disease-free 7 months after diagnosis.
A 6-year-old boy, without any pathologic antecedents, was admitted with tiredness, fever (38°C), weight loss and cough. The physical and radiological examination revealed few cervical adenopathies, hepatosplenomegaly and a mediastinal enlargement.
The blood cell count showed WBC 77.9 × 109/l with 84% blast cells, Hb 9.9 g/dl, and platelets 146 × 109/l. The bone marrow was hypercellular with 91% blast cells.
Flow cytometry demonstrated bone marrow lymphoblasts CD1a+, CD2+, cytoplasmic CD3+ and surface negative, CD4+, CD8+, CD5+, CD7+, CD34−, DR−. In addition, this T-III ALL showed a CD13 ‘aberrant’ antigen expression and an unusual Tdt negativity (Table 1).
The patient was treated according to the CLCG-EORTC 58951 study.9 The poor response at day 8 steroid treatment (more than 1 × 109 blast cells/mm3 in peripheral blood) entailed switching to the very high-risk group. He attained complete remission and remained event-free after 7 months follow-up.
A 5-year-old boy, presented with progressive weakness and fever (38.5°C). The physical examination showed small hematomas, multiple large diffuse adenopathies, hepatosplenomegaly but no mediastinal enlargement.
The peripheral blood cell count showed WBC 51 × 109/l with 85% blast cells, Hb 4.3 g/dl, and platelets 16 × 109/l. Bone marrow was hypercellular with more than 80% blast cells and 13% erythroblastic lineage.
Flow cytometry revealed a T cell proliferation CD1a+, CD2+, surface CD3+, TCRγδ+, CD4+, CD8+, CD5+, CD7+, CD34−, DR− and Tdt+. CD1a positivity assigned this patient to the T-III EGIL subgroup (Table 1).
He was included in the very high-risk group because of his poor response to steroid prephase. He was treated according to the same protocol,9 and achieved complete remission.
Blood and bone marrow cells were cultured using three different modes: a 17 h overnight incubation with colcemid at low concentration (10 μl/10 ml of RPMI 1640 medium), and 24 h and 48 h cultures with FRDU synchronization using the technique of Weber and Garson,10 in order to improve the quality of the banding. Metaphase chromosome spreads were obtained using classic procedures. After standard karyotypic analysis (RHG banding), metaphases were analyzed with FISH techniques (IPM-FISH and FISH with WCP and unique sequence probes).
The most innovative part of the IPM-FISH technique is the use of interspersed polymerase chain reaction (IRS-PCR) painting probes that show an R-band pattern simultaneous with the combinatorial labeling. This not only allows recognition of the origin of chromosomal fragments, but also definition of the breakpoints. IRS-PCR-derived probes, in contrast to the degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR), provide stronger hybridization signals at the telomeric ends that potentially increase the possibility of detecting cryptic translocations. The probes were made and provided by Genset Corporation (Evry, France) following a procedure described elsewhere.4
Briefly, painting probes were obtained by interspersed polymerase chain reaction (IRS-PCR) amplification of DNA from mono-chromosomal human/rodent hybrid cell lines. Optimized amounts of PCR products were combined, labeled with five different fluorochromes (Table 3) and hybridized.
Hybridization on metaphase chromosomes was performed with minor modifications, as described by Cherif et al.11 After pre-treatment and denaturation of the slide, hybridization was carried out at 37°C, in a humid chamber, for at least 48 h. Post-hybridization washes were performed as described. Biotinylated probes were detected with Cy7-conjugated avidin and biotinylated goat anti-avidin (Vector Laboratories, Burlingame, CA, USA). Chromosomes were counterstained with DAPI. For image acquisition, an epifluorescence microscope Leica DMR-XA equipped with Leica special filters (Leica, Wetzlar, Germany) was used, according to Speicher et al1 and Eils et al.2 Images were acquired using a cooled charged device (CCD) camera (Sensys, Photometric Perceptive Scientific, Chester, UK) on a vario-TV adapter with a magnification factor of 0.8 to 1.63. Image analysis was made using M-FISH software from Leica (Leica Q-FISH and Leica MCK). For each patient, a minimum of 15 metaphase cells were analyzed.
FISH with WCP and unique sequence probes
Each case was analyzed using a dual-color chromosome paint, following the protocol recommendations by the manufacturer (Coat; Appligene Oncor, Illkirch, France). Chromosome 5 probe was labeled with Spectrum Green (SG) and chromosome 14 probe was labeled with Spectrum Orange (SO).
In order to confirm and precisely define the breakpoints, unique sequence probes were also hybridized: BAC 45L16 (5q35)(kindly provided by R Berger, U434 INSERM-CEPH, Paris, France) and IgH (14q32) (LSI IGHc/IGHv dual-color break apart probe; Vysis, Downers Grove, IL, USA). The BAC 45L16 was labeled with digoxigenin and detected with a rhodamine anti-digoxigenin antibody. IgH probe (14q32) was used according to the manufacturer's instructions (Vysis). For each probe and each patient, a minimum of 20 metaphase cells was analyzed.
Results and discussion
The patients were studied with the IPM-FISH technique. For the three cases, the leukemic cells demonstrated a reciprocal, balanced translocation between the long arms of chromosomes 5 and 14 (Figure 1a). This was the sole cytogenetic abnormality identified for these patients. This translocation t(5;14) was cryptic with conventional cytogenetic technique (RHG banding) (Figure 1b). All IPM-FISH findings were validated by traditional fluorescence in situ hybridization (whole chromosome painting (Figure 1b) and unique sequence probes (Figure 2)). For the three patients, hybridization with BAC 45L16 (5q35) showed signals on chromosome 5, der(5) and der(14) (Figure 2a). These results indicated that the translocation breakpoint on chromosome 5 occurred at 5q35 within BAC 45L16. The study with the IgH probe showed that, for the three cases, the breakpoint was proximal to the IgH locus (Figure 2b).
In cases 1 and 2, the karyotype was 46,XY,t(5;14)(q35;q32), in case 3, the patient had loss of normal chromosome 5 and had a duplication of the derivative chromosome 5; his karyotype was 46,XY,der(5)t(5;14)(q35;q32),t(5;14)(q35;q32).
According to the EGIL scoring system, the three patients with t(5;14)(q35;q32) were assigned to the same T-III immunological subgroup which comprises about 50% of pediatric and adult T-ALL. The T-III subgroup is defined by the expression of CD1a regardless of reactivity with other T cell markers including CD3 in the membrane.8 In fact, this subgroup contains many immunological inter-patient differences.12 Our three patients illustrate this heterogeneity well: even though all expressed CD1a, CD2, CD5, CD7, CD8 and CD4, flow cytometry revealed many differences: patient 1 and 2 were cytoplasmic CD3+ but surface negative in contrast to patient 3 who expressed surface CD3 and TCRγδ. Patients 1 and 3 were Tdt+ as are more than 95% T-lineage ALL whereas patient 2 failed to express Tdt and his leukemic blasts showed an aberrant myeloid-lineage CD13 expression. Considering our small number of cases, it is impossible to reach a conclusion about t(5;14) frequency in T-ALL. Therefore, the study of additional cases might allow a new subdivision of the T-ALL group in cytogenetic subgroups. To our knowledge, this translocation t(5;14)(q35;q32) has not been previously identified as a recurring translocation in T-ALL, either in pediatric cases or in adult forms.
Current conventional cytogenetic techniques (RHG banding) are unsuitable to detect cryptic abnormalities, especially in T-ALL, for which the quality of the metaphase spreads is often poor. The recent study of Schneider et al6 included 343 cases of T-ALL studied with conventional cytogenetic methods (G-banding). They highlighted two new recurring chromosome aberrations that had not been previously described: del(1)(p22) and t(8;12)(q13;p13) and 10 aberrations which were previously only reported once in T-ALL. They also reported 43.2% (153 cases) normal karyotypes, an identical rate to those previously reported.13,14
It is more than likely that among these ‘normal’ karyotypes, some were carrying cryptic chromosomal abnormalities, not seen because of the technique limitations, like poor metaphase quality or translocations implicating bands of the same intensity (like the t(12;21)(p13;q22) in B-ALL). As already suggested in 1985 by Williams et al15 most, if not all ALL, are probably carriers of chromosomal abnormalities.
Advances in fluorescence technology also include the SKY technique.3 Spectral karyotyping (SKY) is a technique that uses the same combinatorial approach to probe production as M-FISH, but detection of the combinations of fluorochromes corresponding to each chromosome is different. M-FISH uses specific narrow bandpass filters for each fluorochrome1 and SKY uses a Fourier transform spectrometer to analyze the fluorochromes simultaneously.3 To date, three studies have used SKY in patients with ALL, to identify cryptic chromosomal abnormalities and characterize the nature of complex chromosomal rearrangements.16,17,18 Rowley et al16 analyzed 15 cases of T-ALL, in 1999, with the SKY technique. Out of these 15 cases, 10 had random abnormalities and five had a normal karyotype in a bone marrow sample obtained at diagnosis. They did not identify any recurring translocation.
Mathew et al17 analyzed 30 pediatric patients with ALL (28 B-lineage ALL and two T cell ALL). His study is the first to have detected abnormalities in pediatric patients with ALL in whom no chromosomal abnormalities have been found by conventional cytogenetics. It also showed that SKY may not detect inversions and may fail to determine the balanced origin of subtle translocations. Actually, some cryptic rearrangements, such as the der(21)t(12;21) translocation, remain undetected.18 This lack of detection is probably due to the absence of subtelomeric sequences in commercially available probes. It is expected that probes enriched with subtelomeric sequences will increase the resolution power of SKY.
IPM-FISH allows a global genome approach simultaneously with the R-band pattern. Moreover, the M-FISH technique allows, for each metaphase, analysis of images corresponding with each individual fluorochrome. The ‘filter by filter’ analysis of chromosomes enables a better and more precise characterization of rearrangements, even very small ones. This new IPM-FISH technique constitutes a good tool to display cryptic chromosomal abnormalities. In fact, the IPM-FISH system allows a global genome approach simultaneously with R-band pattern and permits determination of the balanced origin of subtle translocations.
In conclusion, t(5;14)(q35;q32) has been identified as a new, recurring and cryptic translocation in childhood T-ALL. However, the small number of patients in our series makes it impossible to determine the frequency of this translocation in T-ALL at the present time. Moreover, the study is too recent to be able to judge the prognostic significance. It needs to be further assessed in larger numbers of patients. Cloning of the t(5;14)(q35;q32) breakpoint and characterization of the relevant genes at 5q35 and 14q32, together with clinical and immunophenotypic characterization of additional similar cases of T-ALL will be important in advancing our understanding of the significance of these hemopathies.
IPM-FISH system is patented under FR 2 784 683 and 3 PCT WO 00/22164 patents.
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We thank Dr R Berger and M Busson-Le Coniat for providing the BAC 45L16. This work was supported by grants from La Ligue contre le Cancer du Bas Rhin.
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Hélias, C., Leymarie, V., Entz-Werle, N. et al. Translocation t(5;14)(q35;q32) in three cases of childhood T cell acute lymphoblastic leukemia: a new recurring and cryptic abnormality. Leukemia 16, 7–12 (2002). https://doi.org/10.1038/sj.leu.2402347
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