BCR/ABL1 gene fusion is the hallmark of chronic myeloid leukemia (CML), and is generated in 5–10% of patients by a variant translocation involving 9q34, 22q11.2 and one or more additional genomic regions. The objective of the present study was to characterize, by conventional and molecular cytogenetics, 32 complex variant Philadelphia (Ph) translocations present at diagnosis in patients with CML. The chromosomes most frequently involved were 1 and 5, and the breakpoint most frequently involved was 12p13. The q-chromosome arm was more frequently involved (60%) than the p-arm. The breakpoints were located in the G-light bands in the majority of cases (85%). Additional chromosomal abnormalities were observed in 6 out of 32 (19%) patients. In conclusion, the combination of conventional and molecular cytogenetics studies has allowed us to: i) Detect and quantify the BCR/ABL1 fusion gene; ii) characterize the complex variant translocations and detect cryptic translocations; iii) confirm that the breakpoints are commonly localized in the G-light bands; (iv) confirm that the genesis of variant translocations could be via either the one-step or two-step mechanisms; and v) to report new cases of complex variant translocations.
Complex variant Ph chromosomechronic myeloid leukemiakaryotypefluorescence in situ hybridizationIntroduction
Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the proliferation and accumulation of mature myeloid cells and their progenitors (1). The hallmark of the disease is the presence of the reciprocal translocation (9;22)(q34;q11.2), resulting in a BCR/ABL1 gene fusion on the derivative chromosome 22, the so-called Philadelphia (Ph) chromosome (1). Variant translocations are identified in 5–10% of patients with newly diagnosed CML (1). The translocation can be observed either in a simple form (involving 22q11.2 and one additional breakpoint) or in a complex form, involving 9q34, 22q11.2, and at least one additional breakpoint (2,3). Although all chromosomes have been reported to participate in variants, the distribution of breakpoints clearly exhibits a non-random pattern, with a marked clustering to specific chromosome bands (2,3).
Fluorescence in situ hybridization (FISH) has been commonly used to detect the presence of the BCR/ABL1 fusion gene at disease diagnosis and also to monitor its evolution during therapy. Different FISH probes can be combined to accurately determine the complex variant translocations involving more than two chromosomes when observed by cytogenetic analysis (3).
We report herein the characterization, by conventional and molecular cytogenetics, of 32 cases with complex variant Ph translocations, diagnosed in 32 patients with CML.
Materials and methodsPatients' initial features
From 1990 to 2015, 693 patients with CML were diagnosed in three different centers: Hospital Clínic de Barcelona, Hospital del Mar de Barcelona, and Hospital Trias i Pujol de Badalona. Among these, 32 (5%) CML patients exhibited complex variant Ph translocations. The primary clinical and hematological parameters of the patients are outlined in Table I. The patients were comprised of 15 females and 17 males, ranging in age from 23 to 81 years. The ethical approval for the present study, including the written informed consent of the patients, was granted following the guidelines of the Ethics Committee of the Hospital Clínic de Barcelona, Hospital del Mar de Barcelona, and Hospital Trias i Pujol de Badalona.
Conventional cytogenetics
Bone marrow samples were processed for cytogenetic and FISH analysis. Cytogenetic studies were carried out on G-banded chromosomes obtained from 24 h un-stimulated bone marrow cultures. Karyotypes were described according to An International System for Human Cytogenomic Nomenclature (4). A300-band ideogram was considered as the standard level of resolution for the purpose of the present study. Given that three laboratories were involved in the present study, with a different chromosome quality, it was agreed that translocations with breakpoints differing in one band would be considered as the same translocation.
Molecular cytogenetics (FISH)
FISH probes were used to establish whether the BCR/ABL1 rearrangement was present, as well as its location, and to characterize the complex variant translocations. Two different FISH probes were used in order to detect the BCR/ABL1 rearrangements: LSI BCR/ABL1.ES and LSI BCR/ABL1 DCDF, as described previously (5). For the characterization of complex variant translocations, whole chromosome paint (WCP) probes for chromosomes 1, 5, 11, 12, 20 and 22; Centromeric (CEP) probes for chromosome 9.
All probes were provided by VYSIS (Abbott Products Operations AG, Allschwil, Switzerland) and the hybridization and detection were performed according to the manufacturer's protocols. Images were captured and processed with a Cytovision Ultra System (v.5.1.22; Leica Biosystems, Wetzlar, Germany).
BCR-ABL determination by the reverse transcription quantitative polymerase chain reaction (RT-qPCR)
White blood cells were isolated from peripheral blood or bone marrow samples with a lysis buffer containing 0.144 M NH4Cl and 0.01 M NH4HCO3. Total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's protocol. Reverse transcription was performed on 1 µg of RNA with the Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific, Inc.) and random hexamer primers. Briefly, 1 µg of RNA in 19 µl of RNAse-free water was incubated at 65°C for 5 min. Samples were cooled on ice and the following reagents were added to a final volume of 40 µl: 8 µl 5× RT buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM KCl, 15 nM MgCl2; Thermo Fisher Scientific, Inc.), 0.4 µl DTT (0.1 M; Thermo Fisher Scientific, Inc.); 1.6 µl dNTPs (25 mM; GE Healthcare) 1.2 µl pdN6 hexanucleotides (10X; Roche Diagnostics, Basel, Switzerland), 1.5 µl RT enzyme M-MLV (200 U/µl; Thermo Fisher Scientific, Inc.), 0.75 µl RNAsin (40 U/µl; Thermo Fisher Scientific, Inc.) and 7.55 µl RNAse-free water. Samples were then incubated at 37°C for 80 min, at 65°C for 10 min and 4°C at the end of RT step. Subsequently, qPCR was run from 2 µl cDNA as described by Van Dongen et al (6).
Results
All variant chromosome Ph translocations were complex and involved 3 chromosomes, except in case 31 where the translocation included 4 chromosomes. The karyotypes are described in Table II. The karyotypes of patients 1, 4, 5, 15, 22, 23 and 31 have already reported in a previous study (5).
The most frequent variants were t(1;9;22) (p36;q34;q11.2), t(1;9;22)(q21;q34;q11.2), t(2;9;22)(p13;q34;q11.2), t(5;9,22)(q31; q34;q11.2), t(5;9;22)(q35;q34;q11,2), t(9;22;11)(q34;q11.2;q13), t(9;22;12)(q34;q11.2;p13) and t(9;22;15)(q34;q11.2;q22), as they were identified twice.
The chromosomes included in the translocations were: 1 (n=7), 5 (n=5), 3, 11 and 12 (n=3), 2, 6 and 15 (n=2), and 7, 13, 17, 19, 20 and 21 (one each). Chromosomes 4, 8, 9, 10, 14, 16, 18, 22, X and Y were not included in any translocations. A total of 33 breakpoints were described in 32 translocations, and 17 of those were recurrent, being 12p13 (n=3), and 1p36, 1q21, 2p13, 5q31, 5q35, 11q13 and 15q22 (n=2). The q chromosome arm was more frequently involved in the translocations (n=20; 60%) than the p arm. The breakpoints were located in the G-light bands in the majority of cases (n=28; 85%), while the remaining breakpoints werein the dark bands (5q12, 17q12 and 21q21) and in the centromeric areas (1p11 and 11q11) (Table II).
Additional chromosomal abnormalities were observed in 6 out of 32 (19%) patients, including: der(22)/ der(22)/ −21,+mar/add(1)(q42), +8/dic(7;9)(q11;q11),-18 and add(20)(q13)/ +8. Clinical information on possible progression was available in 3 out of the 6 cases with additional chromosomal abnormalities, (cases 7, 15 and 30), and none of these patients were in the blast crisis phase (Table II).
FISH studies using the LSI BCR/ABL1 were performed in 23 out of 32 cases, allowing for the detection of the BCR/ABL1 fusion gene in the Ph chromosome in all cases. In the 7 cases (cases 1, 5, 14, 15, 16, 31 and 32) where WCP and CEP FISH probes were used, the complex variant translocations were confirmed. Characterization of the t(1;9;22)(q21;q34;q11.2), using G-banded karyotype and LSI and WCP FISH probes for chromosomes 1, 9 and 22 in interphase nuclei and metaphases, are shown in Fig. 1.
Molecular studies (RT-PCR) revealed e14a2 chimeric BCR/ABL mRNA in 15 cases and e13a2 chimeric BCR/ABL mRNA in 12 cases. In the remaining 5 cases the molecular studies were not performed.
Discussion
The karyotype and the combination of different FISH probes are essential to characterize complex variant Ph translocations (3,5). The use of the FISH probe for detecting the BCR/ABL1 rearrangement in interphase nuclei and metaphases is crucial to determine not only the presence of the rearrangement and its localization, but also whether further events have occurred, such as the presence of a double fusion gene, which may be relevant to interpret the clinical course and the prognosis of the disease. In our series, conventional and molecular cytogenetic studies have allowed the characterization of the 32 complex variant Ph translocations.
At present, in spite of its high genetic complexity, it is widely accepted that the clinical, prognostic and hematological features of patients with CML with complex variant translocations are not different from those with the classical t(9;22) translocation because it is accepted that the key pathological event is the formation of the BCR/ABL1 fusion gene (2).
Although all chromosomes have been described in the complex variants, some regions are more frequently involved (3). In our series of experiments, the chromosomes most frequently involved were chromosomes 1 and 5, while the more frequent breakpoint was 12p13. The q chromosome arm participated more frequently (60%) than the p-arm. It may be hypothesized that the longer the arm, the higher the probability of recombination. Chromosomes 4, 8, 9, 10, 14, 16, 18, 22, X and Y were not identified in our translocations. All 32 variant translocations identified in our experiments have been previously described in complex variant Ph translocations (3,7).
The present study observed that the breakpoints of the variant 9,22 translocations locate preferentially, with 85% of them in the G-light bands (CG-richest areas). Fisher et al (8), reported this association in relation with that the CG richness areas reflect increases in the density of the CpG islands, genes, repetitive elements, and recombination.
Variant translocations may be caused by different mechanisms. Some variants are originated by multiple simultaneous breaks (one-step) and some arise as a result of two, or even more, genetic events in close succession (two-step or multiple-step) (9–11). In our series, the complex variant t(9;22;V) was identified in 30 out of 32 cases at the time of diagnosis suggesting that the t(9;22;V) originated in a stem cell, probably as the result of a one-step translocation. In two cases (cases 5 and 7), a two-step translocation could explain the complex variant translocations. In case 5, the insertion of material from chromosome 1 into the der (9) involved a second breakpoint in 9q34. In case 7, the identification of the two cell lines, t(9;22) and t(1;9;22), at the time of diagnosis suggests a two-step translocation.
Clonal evolution typically coincides with or precedes the accelerate phase or blast crisis of CML (9,10). Therefore, an inherent implication of the two-step mechanism is that variant translocations might be associated with a poorer prognosis (9,10).
Secondary abnormalities in the chronic phase of CML have been reported between 10–20% of cases, with the frequency being similar in t(9;22) or its variants. In our series, 19% of the cases have a secondary abnormality, which is concordant with the reported rates (1).
In conclusion, the combination of conventional and molecular cytogenetics studies has allowed us: i) To detect and quantify the BCR/ABL1 fusion gene; ii) to characterize the complex variant translocations and detect cryptic translocations; iii) to confirm that the breakpoints are commonly localized in the CG-richest regions of the genome; (iv) to confirm that the genesis of variant translocations could be via either the one-step or two-step mechanisms; and v) to report new cases of complex variant translocations, which can involve new breakpoints that can eventually be recurrent and important for the understanding of this leukemia.
Acknowledgements
Not applicable.
Funding
No funding was received.
Availability of data and materials
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
Authors' contributions
DC conceived and designed the study. JG, BE and DC were responsible for the data acquisition, selection and analysis. AA, CG and MLG were responsible for the analysis and interpretation of the conventional (karyotype) and molecular (FISH and RT-qPCR) studies. MN and FC were responsible for the analysis and interpretation of data, and critically revised the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The study protocol was approved by the Ethics Committee of the Hospital Clínic de Barcelona, Hospital del Mar de Barcelona and Hospital Trias i Pujol de Badalona. Written informed consent was obtained from all patients.
Patient consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
ReferencesHeimSMitelmanFCancer Cytogenetics: Chromosomal and Molecular Genetic Aberrations of Tumor Cells4thWiley-BlackwellNew Jersey201510.1002/9781118795569JohanssonBFioretosTMitelmanFCytogenetic and molecular genetic evolution of Philadelphia-chromosome-positive chronic myeloid leukaemiaChronic Myeloproliferative Disorders. Cytogenetic and molecular genetic abnormalitiesBainBJKargerBasel44612003MitelmanFJohanssonBMertensFMitelman Database of Chromosome Aberrations and Gene Fusions in Cancerhttp://cgap.nci.nih.gov/Chromosomes/MitelmanMcGowan-JordanJSimonsASchmidMISCN 2016: An International System for Human Cytogenomic NomenclatureKargerBasel2016CostaDCarrióAMadrigalIAriasAValeraAColomerDAguilarJLTeixidoMCamósMCervantesFStudies of complex Ph translocations in cases with chronic myelogenous leukemia and one with acute lymphoblastic leukemiaCancer Genet Cytogenet1668993200610.1016/j.cancergencyto.2005.08.02416616117Van DongenJJMMacintyreEAGabertJADelabesseERossiVSaglioGGottardiERambaldiADottiGGriesingerFStandardized RT-PCR analysis of fusion gene transcripts from chromosome aberrations in acute leukemia for detection of minimal residual disease. Report of the BIOMED-1 Concerted Action: Investigation of minimal residual disease in acute leukemiaLeukemia1319011928199910.1038/sj.leu.240159210602411Atlas of Genetics and Cytogenetics in Oncology and Haematologyhttp://atlasgeneticsoncology.orgMarch262018FisherAMStrikePScottCMoormanAVBreakpoints of variant 9;22 translocations in chronic myeloid leukemia locate preferentially in the CG-richest regions of the genomeGenes Chromosomes Cancer43383389200510.1002/gcc.2019615884100GorusuMBennPLiZFangMOn the genesis and prognosis of variant translocations in chronic myeloid leukemiaCancer Genet Cytogenet17397106200710.1016/j.cancergencyto.2006.10.00617321324BennourASennanaHLaatiriMAElloumiMKhelifASaadAMolecular cytogenetic characterization of variant Philadelphia translocations in chronic myeloid leukemia: Genesis and deletion of derivative chromosome 9Cancer Genet Cytogenet1943037200910.1016/j.cancergencyto.2009.05.01019737651BennourASaadASennanaHChronic myeloid leukemia: Relevance of cytogenetic and molecular assaysCrit Rev Oncol Hematol97263274201610.1016/j.critrevonc.2015.08.02026412717
From patient 4: (A) Giemsa banded karyotype. The der(1),der(9) and der(22) chromosomes are indicated by arrows. (B) Interphase FISH, showing a normal red signal (ABL1; 9q34), normal green signal (BCR; 22q11.2), yellow signal (BCR/ABL1 rearrangement; Ph), and one green and red signal that was half the size of a normal signal (BCR; der(1) and ABL1; der(9), respectively). (C) Metaphase FISH showing chromosome 1 and der(1) in green, the centromere of the chromosome 9 and the chromosome 22 in red. The der(9) shows red in the centromere, and attached to 9q34 green material from chromosome 1 and red material from chromosome 22. FISH, fluorescence in situ hybridization.
Main clinical and hematological data of 32 CML patients at the time of diagnosis.
No.
Age
Palpable spleen
WBC (×109/l)
Hb (g/l)
Platelets (×109/l)
Therapy
Survival (months)
1
27
NO
67
97
745
HU, IFN,ALLO-SCT
9
2
81
NO
18.5
112
884
IMATINIB
60
3
NA
NA
NA
NA
NA
NA
NA
4
38
YES
57.9
150
237
HU, IFN, ALLO-SCT
22
5
51
YES
254
90
498
HU, IFN, ALLO-SCT
23
6
NA
NA
NA
NA
NA
NA
NA
7
67
YES
15
111
1,075
IMATINIB, DASATINIB
NA
8
39
NO
41
145
126
IMATINIB, DASATINIB
55
9
23
NA
49
95
349
NA
NA
10
NA
NA
NA
NA
NA
NA
NA
11
NA
NA
NA
NA
NA
NA
NA
12
67
NO
42.6
146
326
IMATINIB
108
13
72
YES
7.9
124
249
IMATINIB, DASATINIB, BOSUTINIB
108
14
27
NO
24
133
378
DASATINIB, PONATINIB, ALLO-SCT
36
15
36
YES
537
93
503
HU, IFN ALLO-SCT
24
16
NA
NO
NA
NA
NA
NA
NA
17
48
YES
234
68
69
HU, DASATINIB, BOSUTINIB
24
18
61
YES
73
93
30
IMATINIB, DASATINIB, ALLO-SCT
12
19
50
NO
149
95
466
IMATINIB
120
20
45
YES
93
111
250
IMATINIB, DASATINIB ALLO-SCT, PONATINIB
24
21
53
NO
18
108
208
IMATINIB, NILOTINIB
96
22
43
NA
12
116
1294
HU, IFN
121
23
34
YES
228
90
297
HU, IFN, ALLO-SCT
27
24
39
NA
12.9
81
16
IMATINIB, ALLO-SCT DASATINIB
60
25
74
NO
27
142
347
IMATINIB, DASATINIB
59
26
41
NO
178
108
282
IMATINIB
96
27
50
NO
88
144
336
IMATINIB
132
28
44
YES
130
110
394
NILOTINIB
32
29
42
NA
126
116
260
IMATINIB
108
30
50
NA
21.6
110
72
NA
NA
31
38
YES
212
92
122
HU, IMATINIB
10
32
64
NA
6.7
171
206
IMATINIB, NILOTINIB
144
Patients 1, 4, 5, 15, 22, 23 and 31 have been investigated in a previous study (5). WBC, white blood cell count; Hb, hemoglobin; HU, hydroxyurea; IFN, alpha-interferon; ALLO-SCT, allogeneic stem cell transplantation; NA, not available.
Karyotype, chromosomal region of the additional chromosome/s involved in the complex variant Ph translocation and its location in a G-light band for the 32 patients with CML.