B-cell leukaemia or lymphoma with a combination of t(8;14)(q24;q32) of Burkitt leukaemia/lymphoma and t(14;18)(q32;q21) of follicular lymphoma may present clinically as de novo acute lymphoblastic leukaemia or transformation of follicular lymphoma to aggressive histology diffuse lymphoma. A number of cell lines have been reported with a complex t(8;14;18) with fusion of MYC, IGH and BCL2 on the same derivative 8 chromosome. The objective of this study was to determine the frequency and chromosomal features of this der(8)t(8;14;18) in a series of acute leukaemias and malignant lymphomas. A database of 1350 leukaemia and lymphoma karyotypes was searched for cases with structural alterations affecting both 8q24 and 18q21. A total of 55 cases were identified, of which eight revealed a complex der(8)t(8;14;18) with an MYC-IGH-BCL2 rearrangement resulting from translocation of BCL2 and MYC with a single disrupted IGH allele. Molecular cytogenetic investigation is essential to identify cases of high-grade leukaemia/lymphoma with concurrent translocations affecting the BCL2 and MYC loci.
High-grade B-cell lymphomas with concurrent deregulation of BCL2 and MYC are associated with distinct cytogenetic and morphologic features and a poor prognosis.1, 2, 3 The clonal karyotype in such cases most commonly contains the t(8;14)(q24;q32) (or light chain variant) of Burkitt leukaemia/lymphoma (BL) in combination with the t(14;18)(q32;q21) (or light chain variant) of follicular lymphoma. The cellular morphology of such cases may resemble BL, but in contrast the malignant cells typically demonstrate more nuclear irregularity and occasional admixed large centroblasts and may exhibit an atypical phenotype such as absent CD10 expression or strong Bcl-2 protein expression. Most often the proliferation rate falls short of the ∼100% characteristic of classic BL. The presence of t(14;18) and/or Bcl2 overexpression precludes a diagnosis of classic or atypical BL. Objective demonstration of MYC deregulation in such cases is important for clinical management, in light of different treatment options for cases without MYC deregulation. Reliable anti-Myc antibodies, however, do not exist for routine immunohistochemistry assessment of Myc expression in paraffin-embedded tissue. Cytogenetic analysis and fluorescent in situ hybridisation (FISH) with MYC DNA probes are the most useful tests available to assess clinical specimens for MYC rearrangements or dosage alterations.
Alternative chromosomal mechanisms leading to MYC deregulation have been described in B-cell lymphoma cell lines, including complex insertional rearrangements4 or three-way recombinations of MYC-IGH-BCL2. In the latter instance, BCL2 and MYC are deregulated by the enhancer elements of a single IGH locus, as elucidated in the DOHH2 and VAL cell lines, in which Myc expression level was shown to be increased by Northern analysis.5 The incidence and chromosomal features associated with this particular complex mechanism have not been defined in the clinical setting. To address this issue, we have identified a series of B-cell leukaemias and lymphomas with a combination of IGH-BCL2 and IGH-MYC fusions wherein the 8q24 rearrangement did not represent a standard t(8;14) or light chain variant translocation. A combination of FISH techniques including locus-specific DNA probes (L-S FISH) for the BCL2, IGH and MYC loci and multicolour karyotyping (MFISH) was used to investigate the chromosomal rearrangements associated with these clinical cases.
Methods and materials
Patients and specimens
The lymphoma database of the British Columbia Cancer Agency was searched for clinical cases that met the following criteria: (1) morphologic features of acute lymphoblastic leukaemia (ALL) or malignant (non-Hodgkin) lymphoma and (2) a karyotype at presentation or transformation that showed a structural alteration of 8q24 that did not conform to a standard or variant Burkitt-type translocation [t(8;14); t(2;8); t(8;22)] in combination with a structural alteration at 18q21. Identified cases with residual cellular material were subjected to morphology review and further molecular cytogenetic analysis.
Morphology and immunophenotype
All tissue specimens were processed for histologic and immunophenotypic analysis using standard methods. All cases were reviewed and classified with reference to the WHO classification.6 Immunohistochemistry included CD20 (L26, Dako), CD3 (Dako), CD10 (Novocastra, clone 56C6), Bcl-2 (Dako, clone 124) and MIB-1 (Dako). Briefly, 4 μm sections of either B5-fixed or formalin-fixed paraffin-embedded blocks were stained using an automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA). Appropriate positive and negative controls were included with all analyses. Cases were considered positive for CD10 and Bcl-2 if >20% of the neoplastic cells stained positively. MIB-1 stains were scored as a percentage of the malignant lymphoid cells showing positive nuclear staining.
Cytogenetic and FISH Analysis
Cytogenetic analysis was performed as previously reported.7 G-band karyotyping was performed on 10–25 metaphases. Karyotype descriptions conform to the International System for Human Cytogenetic Nomenclature 1995.8 The DOHH2 cell line, established from a high-grade B-NHL, was used as a positive control for the FISH analyses.
The DNA probes used for L-S FISH included the following: YAC-934E1 containing the MYC gene (kindly provided by Dr Scherer, Department of Medical Genetics and Microbiology, Hospital for Sick Children, University of Toronto, Canada); BAC-367L07 containing the MYC gene; the LSI IGH/BCL2 dual fusion and IGH/MYC /CEP 8 tri-colour dual fusion translocation probes (VYSIS, Downers Grove, IL, USA). The YAC and BAC probes containing MYC were direct labelled with Cy5 (Amersham Pharmacia, Buckinghamshire, UK) by nick translation using a commercial labelling kit according to the manufacturer's protocol (VYSIS, Downers Grove, IL, USA). Three-colour FISH was performed with a cocktail containing Cy5-labelled MYC probe (BAC-367L07) mixed with the LSI IGH/BCL2 probe in a combination 40 ng MYC probe with 1 μl IGH/BCL2 probe. Hybridisation was performed according to VYSIS protocols. For interphase FISH analysis, 200 nuclei were scored for each probe used. The presence of an IGH-BCL2, IGH-MYC or MYC-IGH-BCL2 translocation was confirmed if ⩾5% of interphase nuclei showed the appropriate fusion signals. In addition, in all positive cases, two to five clonal metaphases were also examined to confirm the chromosome localisation of the probe signals.
MFISH analysis was performed on archival fixed cells accordingly to the manufacturer's protocols (MetaSystems, Altlussheim, Germany) and as previously described.9 MFISH analyses were performed on a minimum of five metaphases per case. Image capture for FISH and MFISH was performed with a Zeiss microscope (Axioplan2) equipped with the appropriate filters (DAPI, FITC, Spectrum Orange, TRITC, Cy5, DEAC) and the Metasystems ISIS imaging software programs. Established criteria were utilised for evaluating MFISH metaphases to avoid chromosomal misclassifications due to fluorescence blending.10
Identification of the study group
A total of 1350 cases of acute leukaemia and malignant lymphoma with available morphologic and cytogenetic data were identified in the lymphoma database of the BC Cancer Agency. The majority of these cases represent follicular and diffuse B-cell lymphoma or de novo B-cell ALL (154 cases), and a small minority (<2%) represent low-grade lymphoproliferative disorders such as chronic lymphocytic leukaemia. In all, 55 cases (4%) with both 8q24 and 18q21 rearrangements were identified. Of these, 24 had a standard or variant t(14;18) and t(8;14), while 31 cases revealed an atypical appearance of 8q24. Eight of the latter cases were shown to have an MYC-IGH-BCL2 fusion, and are the subject of this report (see Table 1). The remaining 23 cases were excluded due to absence of an IGH-MYC (sixteen) or IGH-BCL2 (one) fusion or due to lack of available cellular material for FISH evaluation (six). Nine patients were shown to have a t(8;9)(q24;p13), which has been previously reported in ALL in association with t(14;18).12, 13, 14, 15 Further analysis of these nine cases will be reported elsewhere.
Morphology and immunophenotype
Of the eight study cases, five presented initially as a follicular lymphoma, grade 1, with subsequent transformation to high-grade lymphoma, NOS. Three cases presented as B-cell lymphoblastic leukaemia/lymphoma that on review were classified as a high-grade lymphoma, NOS. All testable cases strongly expressed Bcl-2 protein and/or CD10. The proliferative rate as assessed by MIB-1 immunostaining was notably less than the ∼100% characteristic of classic BL, typically in the range of 70–90% of the cells (see Table 1).
Cytogenetic and FISH analyses
DOHH2 cell line studies
The cytogenetic and molecular features of this cell line have been previously characterised and shown to contain a der(14)(14pter->14q32.3::8q24->8qter), a der(8)(8pter->8q24::14q32.3::18q21->18qter) and a der(18)(18pter->18q21::14q32->14qter).5, 16 In these studies, exon 1 of MYC was shown to be retained on the der(8), but recombined with the 5′ IGH enhancer and Jh-BCL2 fusion element that had been translocated from the pre-existing der(14), while exons 2 and 3 of MYC were translocated to the der(14) adjacent to the residual 3′ IGH enhancer. Overexpression of Myc was demonstrated by Northern analysis. In the current study, MFISH and L-S FISH with a three-colour hybridisation cocktail including the t(14;18) probe and aqua-labelled MYC probe was used to confirm these gene recombinations on the der(8) and der(14) chromosomes in DOHH2 cells (data not shown). The der(18) by MFISH did not show evidence of chromosome 14 material attached to 18q21 (which is the usual result for t(14;18) with the MFISH reagent used in this analysis, presumably due to the minimal amount of chromosome 14 material translocated to the der(18) and/or due to deficiencies in the probe reagent for such telomeric sequences).
Cytogenetic and FISH analysis of the clinical cases
The G-band/FISH-revised karyotypes are presented in Table 2. Cases 1–7 were shown to have an MYC-IGH-BCL2 triple fusion on the der(8) chromosome. In cases 1–6, the der(8) was similar in size to a normal chromosome 8 but showed a heavier G-band at 8q24 than occurs normally or with a standard t(8;14) (see Figure 1). This dark band was shown by MFISH to be derived from chromosome 18. The presence of IGH on the der(8) was not evident by MFISH analysis, but was confirmed in all cases by LS-FISH. The intensity of this IGH signal was variable between cases, however, most likely due to differing IGH breakpoint sites and the small amount of IGH sequence sandwiched between BCL2 and MYC. MFISH also did not detect chromosome 14 material on the der(18) in any of the clinical cases (as discussed above). Case 7 showed a translocation of a large segment of the der(14), with breakpoint at 14q?22–24, onto the der(8) giving rise to a much larger der(8) and a smaller der(14). Case 8 showed two copies of the MYC-IGH-BCL2 fusion inserted into a der(9) and a der(13), respectively, with the q arm of both chromosome 8 homologues retaining normal morphology.
Cases 1–5 showed L-S FISH features on the der(8), der(14) and der(18) that were identical to the DOHH2 cell line. Cases 6–8 showed different variations on this theme. In case #6, the der(14) had a standard IGH-BCL2 fusion, whereas the der(8) had a MYC-IGH-BCL2 triple fusion at 8q24. MFISH showed that both the der(14) and the der(8) were translocated with chromosome 18 material. (A comprehensive analysis including G-band and MFISH features of a cell line (Tat-1) derived from this case has previously been reported.11) In this case, it appears that a duplication of the IGH-BCL2 fusion and one or both IGH enhancers was translocated onto the der(8) 3′ of the MYC locus. An additional copy of this entire BCL2-IGH-MYC fusion element was inserted into a der(13) chromosome. This case also contained an unbalanced der(1)t(1;8)(p36;q22), resulting in a total of five copies of MYC. Of note, a similar der(1)t(1;8)(p36;q22) was also present in case #3, which contained a total of four copies of MYC. Case #7 showed a translocation between 8 and 14 with a more proximal breakpoint at 14q?22–24, and L-S FISH confirmed a MYC-IGH-BCL2 fusion on the der(8) and a weak MYC probe signal on the der(14), implying a complex rearrangement or inversion to generate the triple fusion on the der(8). Case #8 did not show alteration of 8q, but showed two copies of a MYC-IGH-BCL2 fusion, one within a complex der(9) and another within a complex der(13). This case showed the usual configuration of t(14;18). The rearrangements involved in cases 6–8 apparently involved complex mechanisms, the nature and sequence of which could not be fully determined by this investigation.
The combination of BCL2 and MYC rearrangements resulting from t(14;18) and t(8;14) or variants in the same karyotype is well recognised. This combination was first recognised by Mufti et al17 and its association with a poor outcome was identified by Kramer et al.3 In our institution, 24 such cases have been identified among 1350 leukaemia and lymphoma karyotypes. A listing of reported cases up to 1996 is contained in the report by Berger et al.18 In such cases, the IG heavy or light chain genes have been implicated in the deregulation of BCL2 and MYC. Participation of the light chain genes is particularly common in this setting, evident in one or other of the participating translocations in 11 of the 24 cases that we have identified. The malignant cells can have variable morphology and immunophenotype, and have been reported in the literature as either precursor B-lineage ALL, lymphoblastic lymphoma, Burkitt-like lymphoma, and ‘blastic or blastoid’ transformation of FL and DLBCL. These acute B-cell leukaemias/lymphomas with Myc and Bcl2 deregulation have morphological and phenotypic features in the grey zone between classic BL and diffuse large B-cell lymphoma (DLBCL). A specific designation for this type of B-cell malignancy is currently not included in the WHO classification system. These cases can be distinguished from atypical BL, which is used to denote cases believed to be part of the clinical and biological spectrum of BL.6 The distinction between these two entities is aided by immunohistochemistry, which shows Bcl-2 overexpression in the majority of these aggressive leukaemias/lymphomas in contrast to its absence in virtually all cases of BL. Despite the common feature of MYC deregulation, BL will respond in many cases to high-dose chemotherapy and bone marrow transplantation, whereas the outcome for patients such as those described in this report is dismal, with at best a short-term response to aggressive therapy (Kuruvilla J et al. Blood 2003; 102: 392a, abstract).2, 3 The atypical morphologic features and aggressive behaviour of these high-grade B-cell lymphomas may be attributable to concomitant Bcl2 and Myc overexpression.
Cytogenetic evidence of an atypical alteration of 8q24 in combination with t(14;18) has been identified in a number of anecdotal reports, in which the 8q24 alteration has been described as a der or add(8)(q24) or as der(8)t(8;14;18).16, 19, 20, 21, 22, 23, 24 The molecular structure of the t(8;14;18) rearrangement has been studied in detail in the cell lines DOHH2, VAL and ROS-50.5 These studies have demonstrated that concurrent deregulation of BCL2 and MYC was achieved by translocation with a single IGH allele, whereby separation of the 3′ and 5′ IGH enhancer elements occurs with relocation of the BCL2-5′ IGH enhancer fusion to the der(8), and translocation of MYC adjacent to the 3′ IGH enhancer element that is retained on the der(14). Interestingly, in the VAL cell line, three copies of the BCL2-IGH-MYC fusion element were evident, one on the der(8) and two copies on an isoderivative 8q. The cytogenetic description that has commonly been used for this complex translocation, t(8;14;18), does not address the complexity of the rearrangement and does not conform to standard ISCN usage. The proper description requires use of the long or short form description for each derivative chromosome. The short description would be as follows: der(8)t(8;14)(q24;q32)t(14;18)(q32;q21), der(14)t(8;14)(q24;q32), der(18)t(14;18)(q32;q21) (personal communication, Dr Felix Mitelman).
This study has confirmed that translocation of BCL2 and MYC with a single IGH allele is a relatively rare event, identified in eight of 1350 acute leukaemia and malignant lymphoma karyotype analyses performed by our program. Five of the eight cases showed a similar pattern to that seen in the cell line DOHH2. Three cases showed a more complex version of this rearrangement, involving sequential duplication and translocation to generate a MYC-IGH-BCL2 fusion at one or more sites. In two of these extraordinary cases, a duplication of this triple fusion element was inserted within a complex der(13). In addition, unbalanced translocations of MYC to other chromosomes were detected, resulting in extra dosage of MYC in addition to the MYC-IGH-BCL2 fusions.
This unusual combination of rearrangements is relatively common in high-grade B-cell lymphomas, especially in those cases with atypical features of BL. The presence of this type of rearrangement may be suspected by the unusual dark G-band appearance of the terminal band at 8q24.2–24.3 which actually represents band 18q22. However, any structural alteration of 8q24 in the clinical setting of an aggressive B-cell lymphoma should be suspected of harbouring a simple or complex IGH-MYC translocation. It is possible that additional cases in our database may have contained cryptic or masked versions of either an IGH-BCL2 or IGH-MYC fusion and escaped inclusion in the study group. The possibility of IG light chain involvement in triple gene fusions must also be kept in mind. From the clinical and morphologic perspective, the identification of both BCL2 and MYC translocations, in simple or complex form, in a lymphoma with aggressive histology favours a diagnosis of transformed follicular lymphoma or de novo ALL/high-grade B-cell lymphoma, NOS, over classical Burkitt lymphoma. In light of the poor prognosis associated with this combination of translocations, accurate and timely recognition may be useful to guide clinical management. Consequently, the identification of a high-grade B-cell leukaemia/lymphoma by the pathologist should trigger appropriate cytogenetic and/or FISH investigations to confirm or exclude these clinically important genetic alterations. Equally important, an 8q24 structural alteration detected by chromosomal banding may not actually represent a MYC rearrangement, and requires confirmation by FISH analysis.
In summary, this study confirms that a significant proportion of high-grade B-cell leukaemia/lymphomas are characterised by concurrent BCL2 and MYC rearrangements with IG gene(s) that may involve simple or complex mechanisms. All cases of high-grade B-cell leukaemia/lymphoma, NOS, as defined in this study, should be suspected of having Bcl2 and Myc translocations. Additional investigation by cytogenetic analysis, FISH and immunohistochemistry may be helpful to determine the structural and functional status of the BCL2 and MYC genes. A combination of FISH probes for IGH, BCL2 and MYC labelled with three colors is especially useful to identify these chromosomal alterations. The possibility of cryptic or complex rearrangements involving the IG heavy and light chain genes and MYC should be kept in mind when interpreting atypical karyotypes or FISH signals. Efficient methods to detect MYC deregulation at the protein and/or RNA level that are applicable in the clinical laboratory, such as quantitative RT-PCR, are required for the assessment of Bcl-2 and Myc expression level in B-cell malignancies.
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We would like to acknowledge the cytogenetic technologists of the Cancer Genetic Laboratory, Department of Pathology and Laboratory Medicine, of the BC Cancer Agency. The high-quality cytogenetic and molecular cytogenetic analyses performed by this group have made this study possible. This research was also supported in part by NIH Grant #UO1-CA84967-1 (JMC, RDG, DEH). Stevan Knezevich is the first recipient of the Patricia Mansion Memorial Fellowship of the Lymphoma Foundation Canada (formerly the Lymphoma Research Foundation Canada).
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B-Lymphoblastic Lymphomas Evolving from Follicular Lymphomas Co-Express Surrogate Light Chains and Mutated Gamma Heavy Chains
The American Journal of Pathology (2016)
The cooperating mutation or “second hit” determines the immunologic visibility toward MYC-induced murine lymphomas