Extra copies of MYC, BCL2, and BCL6 and outcome in patients with diffuse large B-cell lymphoma.
ABSTRACT: High-grade B-cell lymphoma (HGBL) with translocations involving MYC and BCL2 or BCL6 comprises ?10% of cases of diffuse large B-cell lymphoma (DLBCL) and carries a poor prognosis. The incidence, prognosis, and optimal therapy for DLBCL harboring extra copies of the genes MYC, BCL2, and BCL6, rather than their genetic translocations, are unknown. In this retrospective, single-center study we identified 144 DLBCL cases including 46 patients with classic HGBL with double-hit or triple-hit chromosomal translocations (DHL), 55 with extra copies of MYC in addition to aberrations (extra copies or translocations) of BCL2 and/or BCL6 but did not meet the criteria for HGBL (EC group), and 43 without any aberrations of MYC, BCL2, or BCL6 (wild type [WT]). Unfavorable baseline characteristics had similar frequency in the EC and WT groups, but were significantly more prevalent in the DHL group. With a median follow-up of 36 months, the 2-year event-free survival (EFS) was similar between the WT and EC groups at 77% (95% confidence interval [CI], 65-90) and 82% (95% CI, 72-93), respectively. In contrast, the 2-year EFS of the DHL group was 63% (95% CI, 51-79). The 2-year overall survival in the WT, EC, and DHL groups was 86% (95% CI, 76-97), 89% (95% CI, 81-98), and 74% (95% CI, 62-88), respectively. Among patients treated with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone), the EC group had outcomes similar to those of the WT group. Our results indicate that patients with DLBCL with extra gene copies of MYC, BCL2, and BCL6 fare differently from those with HGBL and respond well to standard R-CHOP therapy.
Project description:BACKGROUND:The poor outcome of high-grade B-cell lymphoma, with rearrangements of MYC, BCL2 and/or BCL6, also known as double-hit lymphoma or triple-hit lymphoma (DHL or THL), has been well documented, while the clinical significance of extra copies of MYC, BCL2 or BCL6 are still less well known. METHODS:In total, 130 cases of diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS) were included in our study. Fluorescence in situ hybridization and immunohistochemistry were performed in all cases to evaluate the genetic status and protein expression levels of MYC, BCL2 and BCL6. RESULTS:Among the 130 cases of DLBCL, the prevalence rates of extra copies of MYC, BCL2 and BCL6 were 10.8, 20.0 and 14.6%, respectively, and the corresponding rates of gene rearrangement were 10.0, 14.6 and 16.9%, respectively. In total, 7.7% (10/130) of patients were DHL/THL; 9.2% (12/130) of patients were DLBCL with MYC and BCL2 and/or BCL6 gene abnormalities including rearrangements or extra copies, while excluded DHL/THL. The positive protein expression rates of MYC, BCL2 and BCL6 were 46.9% (61), 75.4% (98) and 70.0% (91), respectively. Among the 51 cases with MYC/BCL2 co-expression, 14 cases showed concurrence of MYC, BCL2 and/or BCL6 genetic abnormalities, and the remaining 37 cases were classified as double-expressor lymphoma (DEL). MYC and BCL2 rearrangement and BCL2 extra copies were all associated with upregulated protein expression. Cases with concurrence of MYC, BCL2 and/or BCL6 genetic abnormalities were both associated with MYC/BCL2 co-expression. Patients with concurrence of MYC, BCL2 and/or BCL6 genetic abnormalities excluded DHL/THL had shorter OS (P?<?0.001) than patients with DLBCL with no genetic change, and showed no statistical different with patients with DHL/THL (P?=?0.419). Extra copies of MYC was independent prognostic factors for DLBCL. CONCLUSIONS:Patients with MYC and BCL2 and/or BCL6 gene extra copies might show a trend towards poor prognosis, and the detection of extra copies of MYC, BCL2 and BCL6 might deserve more attention.
Project description:Double/triple-hit lymphomas (DHL/THL) account for 5-10% of diffuse large B cell lymphoma (DLBCL) with rearrangement of MYC and BCL2 and/or BCL6 resulting in MYC overexpression. Despite the poor prognosis of DHL, R-CHOP chemotherapy remains the treatment backbone and new targeted therapy is needed. We performed comprehensive cytogenetic studies/fluorescence in situ hybridization on DLBCL and Burkitt lymphoma cell lines (n = 11) to identify the DHL/THL DLBCL in vitro model. We identified MYC/IG in Raji and Ramos (single hit); MYC/IG-BCL2 (DHL) in DOHH2, OCI-LY1, SUDHL2, and OCI-LY10; MYC/IG-BCL2/BCL6 (THL) in VAL; and no MYC rearrangement in U2932 and HBL1 (WT-MYC). Targeting MYC in the DHL/THL DLBCLs through bromodomain extra-terminal inhibitors (BETi) (JQ1, I-BET, and OTX015) significantly (p < 0.05) reduced proliferation, similar to WT-MYC cells, accompanied by decreased MYC but not BCL2 protein. Moreover, BETi suppressed MYC transcription and decreased BRD4 binding to MYC promoter in DHL cells. CD47 and PD-L1 are immunoregulatory molecules often expressed on tumors and regulated by MYC. High levels of surface CD47 but not surface PD-L1 was observed in DHL/THL, which was reduced by JQ1 treatment. BETi in combination with Pan-HDAC inhibitor had a limited effect on survival of DHL/THL, while combination of BETi and BCL2 inhibitor (ABT-199) had a significant (p < 0.005) inhibitory effect on survival followed by BCL-XL inhibition. Overall, the data suggests that MYC-expressing DLBCLs are probably addicted to the MYC-oncogenic effect regardless of MYC rearrangements. In summary, we identified an in vitro model for DHL/THL DLBCLs and provide evidence for the therapeutic potential of BET inhibitor alone or in combination with BCL2 inhibitor.
Project description:Genomic alterations and protein expression levels have been established as prognostic factors for survival in patients with diffuse large B-cell lymphoma (DLBCL). In particular, double-hit DLBCL (DHL), which exhibits translocations in MYC and BCL2 and/or BCL6, is known to be associated with a poor prognosis. However, the clinical significance of gene alterations and protein expression levels for MYC, B-cell lymphoma (BCL)2, and BCL6 are unclear. In this study, we analyzed 61 adult patients diagnosed with DLBCL without DHL, who were treated with rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisolone, or similar regimens. There were no differences in the distribution of MYC expression rates among the different MYC gene statuses. In log-rank tests, MYC translocation was a prognostic factor for overall survival (OS; P = 0.011), whereas BCL2 and BCL6 translocation were not prognostic indicators (P = 0.999 and P = 0.925, respectively). Although the expression levels of MYC and BCL6 were not significantly associated with OS, the expression of BCL2 was a prognostic factor for OS (P = 0.027). Furthermore, copy number gains in the MYC, BCL2, and BCL6 genes did not affect OS. MYC translocation (hazard ratio, 4.769; range, 1.518-14.98; P = 0.007) and BCL2 protein expression (hazard ratio, 3.072; range, 1.002-9.413; P = 0.049) were independent prognostic factors for survival in multivariate analyses. In conclusion, MYC translocation and BCL2 expression may need to be investigated at the initial diagnosis to predict prognosis in patients with DLBCL.
Project description:Double/triple hit lymphoma (DH/TH), known as high-grade B-cell lymphoma (HGBL), is an aggressive diffuse large B cell lymphoma (DLBCL), defined as having concurrent MYC, BCL2, and/or BCL6 gene rearrangements. While gene rearrangements represent significant genetic events in cancer, copy number alterations (CNAs) also play an important role, and their contributions to rearrangements have yet to be fully elucidated. Using FISH and high-resolution CNA data, we defined the landscape of concurrent gene rearrangements and copy gains in MYC, BCL2, and BCL6, in a cohort of 479 newly diagnosed DLBCL. We also show that concurrent translocations and copy number alterations, in combinations similar to DH/TH, identify a unique subset of DLBCL, alternative DH/TH, that have survival outcomes similar to DH/TH DLBCL patients.
Project description:Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma (NHL), representing approximately 25% of diagnosed NHL. DLBCL is heterogeneous disease both clinically and genetically. The 3 most common chromosomal translocations in DLBCL involve the oncogenes BCL2, BCL6, and MYC. Double hit (DH) DLBCL is an aggressive form in which MYC rearrangement is associated with either BCL2 or BCL6 rearrangement. Patients typically present with a rapidly growing mass, often with B symptoms. Extranodal disease is often present. Though there is a paucity of prospective trials in this subtype, double hit lymphoma (DHL) has been linked to very poor outcomes when patients are treated with standard R-CHOP. There is, therefore, a lack of consensus regarding the standard treatment for DHL. Several retrospective analyses have been conducted to help guide treatment of this disease. These suggest that DA EPOCH-R may be the most promising regimen and that achievement of complete resolution predicts better long-term outcomes.
Project description:Genomic rearrangements in the MYC locus occur in ?12% of lymphomas with diffuse large B-cell lymphoma (DLBCL) morphology and are associated with inferior outcome. Previous studies exploring MYC rearrangements have primarily used fluorescence in situ hybridization (FISH) assays to characterize break-apart status but have rarely examined breakpoint location, and in some cases have not examined partner identity. We performed targeted sequencing of MYC, BCL2, BCL6, and the immunoglobulin (IG) loci in 112 tumors with DLBCL morphology harboring MYC rearrangement. We characterized the location of the MYC rearrangement at base pair resolution and identified the partner in 88 cases. We observed a cluster of breakpoints upstream of the MYC coding region and in intron 1 (the "genic cluster"). Genic cluster rearrangements were enriched for translocations involving IGH (80%), whereas nongenic rearrangements occurred mostly downstream of the MYC gene with a variety of partners, including IGL and IGK Other recurrent partners included BCL6, ZCCHC7, and RFTN1, which has not previously been described as a MYC partner. We compared 2 commercially available FISH break-apart assays for the MYC locus and observed discordant results in 32% of cases examined, including some with MYC-IGL and MYC-IGK rearrangements. In cases of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement (HGBL-DH), so-called "double-hit" lymphomas, the majority of MYC rearrangements had non-IG partners (65%), with breakpoints outside the genic cluster (72%). In patients with de novo HGBL-DH of DLBCL morphology, MYC-IG rearrangements showed a trend toward inferior time to progression and overall survival compared with MYC-non-IG rearrangements. Our data reveal clinically relevant architecture of MYC rearrangements in lymphomas with DLBCL morphology.
Project description:High-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements (HGBL-DH/TH) is a newly defined entity in the latest World Health Organization Classification. Accurate diagnosis would appear to mandate fluorescence in situ hybridization (FISH) for all tumors with diffuse large B-cell lymphoma (DLBCL) morphology. We present the results of FISH, cell-of-origin, and immunohistochemistry (IHC) testing from 1228 DLBCL biopsies from 3 clinical trials and a population-based registry. HGBL-DH/TH made up 7.9% of the DLBCL, confined primarily to the germinal center B-cell-like (GCB; 13.3%) compared with activated B-cell-like (ABC; 1.7%) subtype (P < .001). HGBL-DH/TH with BCL2 rearrangement is a GCB phenomenon with no cases observed in 415 ABC DLBCL. A screening strategy restricting FISH testing to tumors of GCB subtype (by Lymph2Cx or Hans IHC) plus dual protein expression of MYC and BCL2 by IHC could limit testing to 11% to 14% of tumors, with a positive predictive value of 30% to 37%; however, this strategy would miss approximately one-quarter of tumors with HBGL-DH/TH with BCL2 rearrangement and one-third of all HGBL-DH/TH. These results provide accurate estimation of the proportion of HGBL-DH/TH among tumors with DLBCL morphology and allow determination of the impact of various methods available to screen DLBCL tumors for FISH testing.
Project description:A proportion of MYC translocation positive diffuse large B-cell lymphomas (DLBCL) harbour a BCL2 and/or BCL6 translocation, known as double-hit DLBCL, and are clinically aggressive. It is unknown whether there are other genetic abnormalities that cooperate with MYC translocation and form double-hit DLBCL, and whether there is a difference in clinical outcome between the double-hit DLBCL and those with an isolated MYC translocation. We investigated TP53 gene mutations along with BCL2 and BCL6 translocations in a total of 234 cases of DLBCL, including 81 with MYC translocation. TP53 mutations were investigated by PCR and sequencing, while BCL2 and BCL6 translocation was studied by interphase fluorescence in situ hybridization. The majority of MYC translocation positive DLBCLs (60/81?=?74%) had at least one additional genetic hit. In MYC translocation positive DLBCL treated by R-CHOP (n?=?67), TP53 mutation and BCL2, but not BCL6 translocation had an adverse effect on patient overall survival. In comparison with DLBCL with an isolated MYC translocation, cases with MYC/TP53 double-hits had the worst overall survival, followed by those with MYC/BCL2 double-hits. In MYC translocation negative DLBCL treated by R-CHOP (n?=?101), TP53 mutation, BCL2 and BCL6 translocation had no impact on patient survival. The prognosis of MYC translocation positive DLBCL critically depends on the second hit, with TP53 mutations and BCL2 translocation contributing to an adverse prognosis. It is pivotal to investigate both TP53 mutations and BCL2 translocations in MYC translocation positive DLBCL, and to distinguish double-hit DLBCLs from those with an isolated MYC translocation.
Project description:Concomitant deregulation of MYC and BCL2 comprises clinically significant, yet poorly characterized biological high-risk feature in diffuse large B-cell lymphoma (DLBCL). To interrogate these lymphomas, we analyzed translocations and protein expression of BCL2, BCL6, and MYC; correlated the findings with comprehensive mutational, transcriptomic, and clinical data in 181 patients with primary DLBCL; and validated the key findings in independent data sets. Structural variations of BCL2 were subtype-specific and specifically increased BCL2 expression. Molecular dissection of MYC deregulation revealed associations with other lymphoma drivers, including loss of TP53, and distinctive gene expression profiles. Double protein expression (DPE) arose from heterogeneous molecular backgrounds that exhibited subtype-dependent patterns. In the germinal center B-cell (GCB) DLBCL, concurrent alterations of MYC and BCL2 loci gave rise to the majority of DPE DLBCLs, whereas among the activated B-cell (ABC) DLBCLs, concurrent alterations were infrequent. Clinically, DPE DLBCL defined a prognostic entity, which was independent of the International Prognostic Index (IPI) and cell of origin, and together with the loss of TP53 had a synergistic dismal impact on survival. In the DPE DLBCL, the loss of TP53 was associated with a chemorefractory disease, whereas among the other DLBCLs, no correlation with survival was seen. Importantly, BCL6 translocations identified non-GCB lymphomas with favorable BN2/C1-like survival independent of IPI and concurrent DPE status. Taken together, our findings define molecular characteristics of the DPE in DLBCL, and recognize clinically feasible predictors of outcome. Given the emerging taxonomical significance of BCL2, BCL6, MYC, and TP53, our findings provide further depth and validation to the genomic classification of DLBCL.
Project description:"Double-hit" lymphoma (DHL) is a high-grade B-cell lymphoma that harbors concurrent MYC and BCL2 or BCL6 rearrangements. Because cases of MYC/BCL6 DHL are uncommon, most reported conclusions have been based on cases of MYC/BCL2 DHL. Lack of experimental MYC/BCL6 DHL models continues to hinder the pathophysiologic and therapeutic investigations of this disorder. We herein describe a novel MYC/BCL6 DHL cell line, designated DH-My6, carrying both the MYC-IGH and BCL6-IGH fusion genes. Interruptions of MYC and BCL6 expressions using short interfering RNAs and chemical inhibitors led to significant attenuation of DH-My6 cell growth. Greater antitumor effects were found when the cells were treated with a combination of MYC and BCL6 inhibitors. Moreover, the PLK1 inhibitor volasertib and the HDAC inhibitor vorinostat synergized strongly when combined with the bromodomain inhibitor JQ1. DH-My6 is a new well-validated MYC/BCL6 DHL cell line that will provide a useful model for studies of the pathogenesis and therapeutics for the less common DHL tumor type. The rationale for approaches targeting both MYC and BCL6, and in combination with PLK1 or HDAC inhibitors for superior suppression of the aggressive MYC/BCL6 DHL warrants further in vivo testing in a preclinical model.