Cryptic t(3;8)(q27;q24) and/or MYC-BCL6 linkage associated with MYC expression by immunohistochemistry is frequent in multiple-hit B-cell lymphomas

B-cell lymphomas with recurrent cytogenetic rearrangements affecting the chromosomal loci of MYC (8q24) and either BCL2 (18q21) and/or BCL6 (3q27) are referred to as doubleor triple-hit lymphomas. Most frequently, these three oncogenes are rearranged with the immunoglobulin (IG) genes, and chromosomal translocations affecting either one of the three IG loci as a partner are readily recognized by conventional G-banding. However, fluorescence in situ hybridization (FISH) using locus-specific probes with interphase nuclei to detect the rearrangements has become a substitute for time-consuming G-banding analysis. As 490% of BCL2 rearrangements involve the IG heavy chain (IGH) gene as the partner and occur downstream of the coding region of BCL2, the presence of fusion signals from the nuclei after hybridization with the BCL2-IGH dual-color, dual-fusion probe is equivalent to t(14;18)(q32;q21) translocation. On the other hand, since MYC and BCL6 involve not only IG genes but also diverse non-IG partners, and because the breakpoints on both genes distribute within a wide range, the presence of break-apart (BA) signals from the nuclei after hybridization with the MYC or BCL6 dual-color BA probe, which hybridizes to opposite sides of MYC or BCL6, does not necessarily infer equivalent significance to chromosomal rearrangements identified by conventional cytogenetic studies. To determine each partner or identify cryptic or complex translocations involving unexpected chromosomal loci, interphase FISH analysis in combination with G-banding is required. From the list of patients with B-cell lymphoma diagnosed and treated in our institution between 2008 and 2016, we identified 16 patients who had a lymphoma with recurrent chromosomal breakpoints involving multiple oncogenes, one of which was 8q24/MYC (Supplementary Table S1). Of the 16, 4 had no identifiable cytogenetic abnormalities based on G-banding at band q24 of chromosome 8 and band q27 of chromosome 3, despite the presence of BA signals of MYC and BCL6 (Supplementary Figures S1–S4). To complement the cytogenetic studies, we performed a series of interphase FISH studies using commercially available probes. Hybridization with the MYC BA probe (Vysis) revealed that red signals representing the 5′ sequences of MYC were localized at band q24 of normalappearing chromosome 8 in cases 10 and 13. In case 14, the signal was localized at the q terminal of the 10q− chromosome [add(10)(q24)]. In case 15, in which the karyotype showed tetraploidy, q24 of two chromosome 8s and the q terminal of a C-group marker were labeled with the 5′ MYC signal. On the other hand, green signals representing the 5′ MYC were localized at band q27 of normal-appearing chromosome 3 in cases 14 and 15, while the signal was localized at the p terminal of the 2p− chromosome [add(2)(p12)] in case 10, and was lost in case 13 (Figures 1a and b). Hybridization with the BCL6 BA probe (Vysis) showed that the red-labeled 5′ BCL6 signal was localized at 8q24 in cases 10 and 13, the q terminal of 10q− in case 14, and two 8q24s and the q terminal of the C-group marker in case 15. The green-labeled 3′ BCL6 was localized at 3q27 in cases 10 and 14, while the signal was not identified in cases 13 and 14 (Figures 1a and b). We then hybridized the metaphase spreads sequentially with the MYC and BCL6 BA probes, except for case 15, and confirmed colocalization of the 5′ MYC and 5′ BCL6 signals in cases 10, 13 and 14, and co-localization of the 3′ MYC and 3′ BCL6 signals in case 14 at relevant chromosomal loci (Figure 1a). In case 15, the lack of green signals but presence of four yellow signals suggested that breakage within the BCL6 occurred at a point included in the region covered by the 5′ BCL6 probe, and hybridization with another MYC BA probe (Dako) showed five yellow and two small red signals, the latter of which were localized at 3q27, indicating that breakage within the MYC was close to the 3′ end of the red-labeled 3′ MYC probe (Figure 1b). In summary, all four cases carried the 5′ MYC and 5′ BCL6 linkage, while two cases lacked the reciprocal 3′ MYC and 3′ BCL6 linkage (Table 1). Case 10 had a MYC-IGH and a BCL6-IGH linkage independently of the MYC-BCL6, and case 13 had a t(14;18)(q24:q21)/BCL2-IGH translocation. Clinical and histopathological information on the four cases is summarized in Supplementary Tables S2–S4. Case 10 presented with disseminated disease of Burkitt lymphoma/leukemia and the remaining three developed diffuse large B-cell lymphoma (DLBCL) involving the gastrointestinal tract. Immunohistochemistry of the pathological specimens showed the germinal center B-cell-like immunophenotype in all cases. Case 10 lacked the expression of BCL6 and case 15 was negative for BCL2. Case 10 was negative for the expression of immunoglobulins based on flow cytometry. Immunohistochemistry using the Y69 anti-c-MYC monoclonal antibody (Abcam PLC, Cambridge, UK) demonstrated nuclear staining in 490% of lymphoma cells in all four cases (Supplementary Figure S5). Case 10 showed a rapidly progressive and fatal course, while case 14 responded to conventional chemotherapy and remains in remission 43 years after completing the therapy. Here, we described four cases of B-cell lymphoma that carried the cryptic t(3;8)(q27;q24) and/or MYC-BCL6 linkage. As the chromosomal materials distal to band q24 of chromosome 8 and those distal to band q27 of chromosome 3 are similar in size and banding appearance, the der(8)t(3;8)(q27;q24) and der(3)t(3;8) (q27;q24) chromosomes were not reliably recognized by conventional G-banding without the aid of FISH cytogenetics using both MYC and BCL6 probes. On the other hand, we found that the MYCBCL6 linkage can occur at not only der(8)t(3;8)(q27;q24) but also independent chromosomal loci, suggesting complex translocation involving ⩾ 3 chromosomal loci or the submicroscopic exchange of chromosome materials. In the current series of multiple-hit lymphoma, the linkage accounted for 25% (4 of 16) of the cases, and BCL6 was the most frequent non-IG partner of 8q24/MYC translocation. t(3;8)(q27;q24) was first described in Burkitt lymphoma/leukemia that developed in an 11-year-old girl who was affected by ataxia telangiectasia. Subsequently, sporadic case reports Citation: Blood Cancer Journal (2017) 7, e578; doi:10.1038/bcj.2017.59