MYC Alterations in Diffuse Large B-Cell Lymphomas

MYC Alterations in Diffuse Large B-Cell Lymphomas Kennosuke Karube and Elias Campo MYC is a transcription factor associated with numerous physiological functions, including apoptosis, and strong oncogenic potential. MYC expression is tightly regulated in normal lymphoid cells with high levels in the initial steps of the secondary lymphoid follicle formation and in a subset of centrocytes of the germinal center light zone. BCL6 and BLIMP1 repress MYC expression in normal germinal center B and plasma cells, respectively. Paradoxically, most lymphomas with MYC genetic alterations originate from cells that usually do not express MYC, suggesting that these tumors need to develop additional oncogenic events to overcome the MYC regulatory mechanisms and also its proapoptotic function. MYC rearrangements, and to a lesser extent gene amplifications, have been detected in approximately 5% to 14% of diffuse large B-cell lymphoma (DLBCL) and these alterations are frequently associated with BCL2 or BCL6 rearrangements. The concurrent presence of these alterations confers a more aggressive behavior to the tumors with poor outcome of the patients. BCL2 and MYC protein may also be coexpressed in DLBCL independently of gene alterations and this double expression also confers poor prognosis, although not as dismal as that of double genetic hits. Additional factors may modulate the biological effect of the double hit lesions because tumors in which MYC is translocated to non-IGH partner or MYC and BCL2 protein that are expressed at lower levels may have a less aggressive behavior. Further studies are needed to define the clinical implications of MYC aberrations in DLBCL and determine the most appropriate diagnostic strategy to identify these tumors. Semin Hematol 52:97–106. C 2015 Elsevier Inc. All rights reserved.

INTRODUCTION The MYC proto-oncogene family comprises three genes encoding for the transcription factors MYC, NMYC, and MYCL that regulate multiple functions in normal cells and have a strong oncogenic potential.1 The activating mechanism and transforming properties of these genes differ in different cell lineages and tumors. MYC translocations occur almost exclusively in hematopoietic neoplasms, particularly in aggressive B-cell lymphomas and plasma cell myeloma. Gene amplifications, mutations, or dysregulation of MYC by upstream signaling pathways are also alternative oncogenic mechanisms occurring at lower frequency in these tumors. In spite of the clear implication of MYC alterations in lymphomagenesis, the understanding of its role in normal B-cell differentiation has remained elusive until recently.2,3 The development of different FISH probes and a monoclonal antibody that reliably

Hospital Clínic, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, Barcelona, Spain. The authors declare that they have no conflicts of interest or competing financial or personal relationships that could inappropriately influence the content of this article. Address correspondence to Elias Campo, MD, PhD, Department of Anatomic Pathology, Hospital Clínic, Villarroel 170, 08036-Barcelona, Spain. E-mail: [email protected] 0037-1963/$ - see front matter & 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1053/j.seminhematol.2015.01.009

Seminars in Hematology, Vol 52, No 2, April 2015, pp 97–106

recognizes MYC protein in tissues has facilitated the investigation of these alterations in lymphoid neoplasms.4,5 The ability to study large cohorts of patients with these new tools has opened new perspectives regarding the clinical implications of MYC alterations in aggressive lymphomas. Diffuse large B-cell lymphoma (DLBCL) is a very heterogeneous group of tumors with diverse clinical presentation and evolution. Although current therapeutic strategies have improved the survival of these patients, still 30% to 40% die of the disease.6 Understanding of the molecular mechanisms that contribute to this clinical heterogeneity may help to identify patients with more aggressive behavior as an initial step to design alternative therapeutic strategies. Recent studies have identified MYC gene alterations and protein expression in a subset of DLBCL with particularly aggressive behaviors. These alterations have been found frequently associated with other oncogenic abnormalities, such as BCL2 or BCL6 gene translocations and protein overexpression, revealing a more complex oncogenic scenario than initially appreciated.7 In this review, we will address the new findings on the functional role and gene alterations of MYC in normal and neoplastic lymphoid cells, the potential clinical impact of these alterations in understanding the clinical and biological heterogeneity of DLBCL, and the perspectives of MYC as a target of new therapeutic strategies for these lymphomas. 97

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MYC FUNCTIONS AND REGULATION MYC family proteins have a similar structure containing three domains: the transactivation domain, the basic helix-loop-helix domain, and the leucine zipper domain. MYC functions as a transcriptional factor forming heterodimers with MAX via the leucine zipper domain. MYC/ MAX heterodimers bind to enhancer box (“E-box”) sequences (CACGTG) in the genome via the basic helix-loop-helix domain domain.1 This binding usually induces the transcriptional activation of MYC target genes by recruiting positive transcription elongation factor b, which in turn phosphorylates RNA polymerase II. Although MYC acts preferentially as an activator of expression, it also represses a number of genes. The formation of a protein complex with MIZ1 is a representative model of MYC-related repression. MIZ1 primarily functions as a transcriptional activator, but becomes a repressor following its association with MYC. The protein complex MYC/MAX/MIZ1 methylates the promoter regions of target genes through the recruitment of DNMT3a, resulting in transcriptional repression.8 MAD is another transcription factor that titrates out MYC and, together with MAX, recruits histone deacetylases that repress transcription. MYC regulates a broad spectrum of genes that is estimated between 10% and 15% of all human genes, including a number of miRNAs.4 This broad effect on the genome is concordant with the extensive distribution of the MYC binding E-box, but the profile of modulated genes varies in different cell types.9,10 The global effect of MYC on gene transcription has been the subject of recent studies that have provided discordant scenarios. One model proposes that MYC may act as a general amplifier of all transcribed genes of the cell by uploading to the promoters of active but not silent genes and enhancing their transcription (Figure 1).11,12 This universal amplification hypothesis explains the large number of potential MYC target genes, and the fact that these targets only partially overlap among different cell types but does not

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adequately explain the repressor function of MYC. Recent studies have questioned this “global amplifier model,” emphasizing that MYC may indeed regulate specific profiles of genes; this modulation may involve more complex mechanisms than just being uploaded to an active promoter.13,14 The transcription may be promoted by other factors such as the chromosomal tridimensional structure, MYC binding affinity, and presence of other transcription factors. This model also explains the repressor effect of MYC by binding to MIZ1 and repressing MIZ1 target genes.14 Abnormal high levels of MYC may also affect the regulation of genes by invading enhancers that may act on distant genes in the chromosomes facilitating the looping of these enhancers to active promoters. This mechanism may help to explain the differential expression of genes in tumors associated with high levels of MYC activated by oncogenic mechanisms (Figure 1).15 MYC activation has a profound influence in many different cell functions including cell proliferation and growth, DNA replication, metabolism and energy regulation, and protein and nucleotide biosynthesis, among others. Cell proliferation is modulated by the control of checkpoint genes such as GADD45 and the direct or indirect regulation of several cyclins, CDKs, and their respective inhibitors.1 Conditional myc knockout mice exhibit hematopoietic stem cell depletion, indicating that MYC may also promote stem cell self-renewal.1 MYC dysregulation also induces DNA replication stress and subsequent genomic instability, which may be related to its oncogenic potential.16 MYC may also promote oncogenesis by regulating angiogenesis through upregulation of VEGF and miR17-92 miRNA cluster.17 In contrast to the oncogenic-related mechanisms described above, MYC expression also activates cell death pathways. Concordantly with this function, B cells in which MYC expression is suppressed are resistant to apoptosis.1 The biological significance of this phenomenon is not fully understood but may be required in normal cells to prevent the potent transformation potential

Figure 1. Schema of MYC functions. Modified concept of MYC function as a universal amplifier. (Upper left): MYC binds to genes for which transcription is already active and amplifies their expression (General amplification). (Lower left): MYC also increases expression levels of specific genes with interaction of other transcription factors. (Lower right): MYC binds to enhancers and generates chromosome looping. This structural change upregulates gene expression. (Modified from Dang et al. by permission from Macmillan Publishers Ltd.15)

MYC alterations in DLBCL

of MYC and maintain its expression under strict control. MYC promotes apoptosis by the downregulation of antiapoptotic proteins such as BCL2 or BCLXL, and the induction of proapototic elements such as BIM and TP53.18 This anti-tumor effect of MYC is concordant with previous observations indicating that the sole MYC activation does not seem sufficient to develop tumors and also partially explains the need for other cooperative oncogenic events associated with MYC to promote tumor development.1

MYC REGULATION IN B-CELL DIFFERENTIATION Although MYC dysregulation was identified in aggressive lymphomas several decades ago, its normal function in the B-cell differentiation process has been only recently elucidated (Figure 2A). MYC plays a crucial role in the lymphoid follicle formation where it is transitorily expressed in different subsets of cells. MYC is not expressed in naïve B cells, but it is upregulated on antigen stimulation. This initial expression of MYC is essential for the germinal center (GC) development, but it is rapidly repressed by BCL6 in the early centroblastic expansion of the GC dark zone.4 MYC is again upregulated in a subset of centrocytes of the light zone of the GC, which is characterized by BCL6 downregulation, NFκB activation, and IRF4 expression. These light zone MYC-positive cells are poised to re-enter the dark zone and undergo further cycles of proliferation and IG somatic mutations to increase antigen selection (Figure 2B). MYC-negative centrocytes in the light zone will exit the GC to differentiate into memory B cells or long-lived plasma cells. BLIMP1 expression in these cells will repress MYC and initiate the plasma cell differentiation program. BCL6 and BLIMP1 directly bind to the MYC promoter and repress its expression in GC B and plasma cells, respectively.4

MYC DYSREGULATION IN DIFFUSE LARGE B-CELL LYMPHOMAS Recent studies using MYC FISH probes and immunohistochemistry on routine processed lymphoma tissues have expanded our view of MYC implication in other tumors beyond classical Burkitt lymphoma (BL) (Figure 3A). MYC alterations have been frequently detected in DLBCL (Figure 3B) and other aggressive large B-cell lymphomas, such as B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL (BCLU) (Figure 3C), plasmablastic lymphoma (PBL), and ALK-positive large B-cell lymphoma (LBCL). Intriguingly, most of these lymphomas originate in B cells that normally do not express MYC because they have high levels of the transcription factors BCL6 or BLIMP1 that physiologically repress MYC. Therefore, the overexpression of MYC in these tumors requires potent oncogenic events that may overcome the inhibitory effect of this

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transcription factors. On the other hand, these aggressive lymphomas also have frequent additional oncogenic alterations that seem to cooperate with MYC, probably counteracting its proapoptotic function. The oncogenic mechanisms activating MYC in lymphomas include translocations, amplifications, and point mutations (Table 1). Translocations have been identified in 5% to 14% of DLBCL.19–22 The immunoglobulin (IG) genes are the most frequent MYC translocation partner. The translocation breakpoints occur at the 50 end of MYC if the partner is an IG heavy chain gene (IGH) and the 30 end if the partner is an IG light chain gene (IGL).23,24 The IG breakpoints are concentrated in the class-switch regions, thus indicating that MYC/IG translocation occurs in the GC.23,25,26 This translocation juxtaposes MYC to the IGH or IGL enhancer, leading to constitutive MYC expression.27 Non-IG genes also act as MYC translocation partners in 35% to 53% of MYCrearranged DLBCL cases. The t(8;9)(q24;p13) translocation, which results in the juxtaposition of MYC to PAX5, has been relatively frequently reported, accounting for 20% of non-IG/MYC rearrangement.25,28 BCL6, BCL11A, ICAROS49, and BTG1 have also been found as MYC translocation partner genes.20,21,25 The MYC expression levels are higher in cases with translocation than in those without translocation, regardless of the MYC translocation partner.25 However, IG-MYC–positive lymphomas have significantly higher MYC transcript levels than those with a non-IG-MYC translocation. The mechanisms dysregulating MYC in translocations with non-IG genes are not well understood, but these observations suggest they may have a different influence in MYC transcription.25,28 In addition to translocations, MYC gains (three to four copies) and amplifications (4four copies) have been identified in 19% to 38% and 2% of DLBCL, respectively.19,29 The relevance of these alterations in the evolution of DLBCL is not yet clear. Increased MYC copy number is associated with higher levels of mRNA levels and a tendency to higher relapse rates.19,29 In one study, amplifications but not gains of MYC correlated with poor prognosis. However, the number of patients in these studies is small.19 MYC mutations have also been found in 32% of DLBCL.30 The mutation hotspots are concentrated in the activation-induced cytidine deaminase recognition motifs (RGYW) within 2 kb from the transcription start site, indicating similarities with the mechanisms of MYC rearrangement.30 In fact, MYC mutations are more frequent in MYC-rearranged cases.31 These mutations are mainly identified in the transactivation domain, which promotes the gene-transforming capacity by increasing the protein stability via reduced ubiquitin-mediated proteolysis or by inhibiting its pro-apoptotic abilities.30,32 The recent development of standardized methods for the detection of MYC protein in tumor cells by immunohistochemistry33 have revealed high levels of expression in 29% to 47% of DLBCL, a higher number than cases with gene translocations.19,34–36 Although MYC mRNA is

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K. Karube and E. Campo

Figure 2. Normal B cell differentiation, MYC expression levels, and corresponding lymphoid tumors. (A) MYC is expressed in stimulated naïve B cells and in a subset of GC cells of the light zone. This expression is repressed in GC and plasma cells by BCL6 and BLIMP1, respectively. The normal counterparts of MYC-associated lymphoid tumors are generally GC B cells and plasma cells, which indicates that the loss of physiological MYC regulation is profoundly associated with lymphomagenesis. LZ, light zone; DZ, dark zone; GC, germinal center; BL, Burkitt lymphoma; DLBCL, diffuse large B-cell lymphoma; BCLU, B-cell lymphoma, unclassifiable with features intermediate between DLBCL and BL; PBL, plasmablastic lymphoma; ALKþDLBCL, ALK-positive DLBCL. (B) Immunohistochemical analysis of MYC expression in a reactive GC. Several MYC-positive cells (marked) are scattered throughout the light zone (LZ) of the GC, but not the dark zone (DZ). (Magnification, 200X.)

upregulated by low-level gains of MYC, the contribution of this alteration to protein overexpression is controversial because high protein expression is also observed in 28% to 41% of DLBCL cases without MYC abnormalities.19,29,34,35 The existence of MYC-protein–positive DLBCL cases without genetic abnormalities suggests that MYC might also be induced by other mechanisms, such as activation of signaling pathways or growth factors that regulate MYC expression.1 The detection of a high

number of MYC-positive cells correlates well with the presence of gene rearrangements, but the relationship varies among tumors. Tumors with more than 70% of MYC-positive cells usually carry gene rearrangements, but MYC translocations can also be present in up to 17% of cases with o30% of MYC-positive cells. Whether this low protein expression in gene rearranged cases is a real biological phenomenon or a technical limitation is not yet clear.37 This variability between protein expression and

Figure 3. Morphological findings and differential diagnosis of MYC-rearranged aggressive lymphomas. (A) BL is characterized by proliferating uniform medium-sized lymphoma cells with small nuclei and dispersed chromatin. Macrophages that have ingested apoptotic cells are scattered throughout to form a “starry-sky” pattern. (B) DLBCL is characterized by the proliferation of large lymphoma cells with abundant cytoplasm, large nuclei, and prominent nucleoli. (C) In BCLU, the proliferating lymphoma cells are slightly larger and feature more irregular nuclei than those in BL. No clear starry sky pattern is evident. (A-C: magnification, 400X.)

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Table 1. Pathogenic Heterogeneity of MYC-Associated Lymphoid Tumors

DLBCL

BL

Main mechanism of MYC Translocation þ dysregulation amplification MYC translocation 5% to 14%

BCL-U

ALKþDLBCL PBL

Translocation Translocation Activation by STAT3 90% to 35% to 50% 0% 100% MYC gain/amplification 21% to 38% Very rare No data 50% Translocation partners of Often non-IG (35- IG (100%) Often non-IG – MYC 53%) (38%) Concurrent BCL2 Frequent (58% to 0% 47% – rearrangement 83%) Karyotype Complex Simple Complex Complex MYC expression 29% to 47% 100% 35% 100% TCF3 or ID3 mutation Very rare 70% 21% (ID3 No data only)

Translocation 41% to 49% 20% IG (83%), non IG (17%) No data No data No data No data

Abbreviations: DLBCL, Diffuse large B-cell lymphoma; BL, Burkitt lymphoma; BCL-U, B-cell lymphoma unclassifiable with features intermediate between DLBCL and BL; PBL, plasmablastic lymphoma.

gene alterations makes it difficult to recommend the use of MYC immunohistochemistry alone as a screening method to detect gene translocations.19,35,36 The association of MYC translocation with BCL2 or BCL6 rearrangements, “double hit” (DH) is more frequent than the previous cytogenetic studies had suggested. BCL2 rearrangements have been identified in 58% to 83% of DLBCL with MYC-rearranged.19,21,38 Follicular lymphoma cases involving MYC and BCL6 rearrangements have also been reported, although at a lower frequency than with BCL2.38 DH alterations usually occur in the context of complex karyotypes, although cases with MYC rearrangements without BCL2 or BCL6 translocations also exhibit multiple chromosomal alterations, suggesting that other secondary genetic abnormalities may also cooperate with MYC.28,39

PATHOLOGICAL CHARACTERISTICS OF DLBCL WITH MYC ALTERATIONS MYC-rearranged DLBCL are morphologically indistinguishable from unrearranged cases, but they more often have a germinal center B (GCB) phenotype (19%) than non-GCB (5%), with frequent CD10 and BCL6 expression and negativity for MUM1 (Figure 3B).4,19,20,22 However, one study did not show differences in the distribution of MYC rearrangements among ABC (9%) and GCB (10%) DLBCL, and high MYC protein expression was similar in GCB (23%) and ABC (34%) DLBCL.34 Most cases with concomitant MYC and BCL2 rearrangements have a conventional DLBCL morphology, although these two translocations are found in around 47% to 78% of BCLU (see below).4,19,21 The predominant GC phenotype and frequent concurrent BCL2 rearrangement observed in MYC-rearranged DLBCL suggests that some of these cases may correspond to transformations from follicular lymphoma. Although in

most DLBCL the DH is identified at diagnosis, in up to 35% the DLBCL may be associated with a concomitant or previous history of follicular lymphoma.20 Contrary to DLBCL with MYC and BCL2 rearrangements, DH composed of concurrent BCL6 and MYC rearrangement more frequently have a non-GCB phenotype with lower expression of CD10 (21% to 66% vs.  90%) and BCL2 (22% to 64% vs.  90%) and are not associated with a previous history of follicular lymphoma. MUM1 expression was high in one study but not in another study.38,40 Interestingly, recent studies have identified that MYC translocations are significantly more common in DLBCL with immunoblastic morphology (33% to 39%) than in other cytological subtypes (7%).28,37 Contrary to other DLBCL, the MYC partner in immunoblastic DLBCL is the IGH and it is the sole translocation in 77% of the cases. These tumors also frequently express CD10 (62%), which is only detected in 15% of the immunoblastic lymphomas without MYC rearrangements.37

CLINICAL IMPACT OF MYC ALTERATIONS IN DLBCL Initial studies using FISH probes found that MYC translocations conferred a poor prognosis for patients with DLBCL.19,22,35,41–43 However, subsequent studies showing that MYC rearrangements were associated with BCL2 or BCL6 translocations (DH) in high numbers of cases (58% to 83%) suggested that the poor prognosis of MYC rearrangements could in fact caused by the synergistic action of these concomitant alterations and not by MYC aberrations alone.20,22,35,41,43 The question whether MYC alterations by themselves may influence the evolution of the tumor is still controversial. Two recent studies have shown that single MYC translocations without BCL2 or BCL6 rearrangements have a similar poor prognosis to cases with DH, even in

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R-CHOP–treated patients, and tend to occur in older patients with advanced clinical stage, higher International Prognostic Index, and more frequent extranodal involvement.19,28 However, the number of patients with isolated MYC alterations in these studies was limited, and the analysis was retrospective. In spite of the unresolved issue of the prognostic value of MYC-isolated translocations, the clinical characteristics of DH-DLBCL are reproducibly more aggressive than in tumors without MYC abnormalities.19,20,34,42,44 The impact of DH with BCL6 rearrangement is less well studied but has also been associated with aggressive clinical courses and poor prognosis, although the limited number of cases analyzed prevents definitive conclusions.38 Despite the fact that most cases with DH follow an aggressive clinical evolution, several studies have recognized some patients with relatively long survival. These infrequent cases are reported to more frequently have DLBCL rather than BCLU morphology and are typically negative or weak for BCL2 or MYC protein expression. Although the number of these cases is still low, these studies suggest that the DH alteration may be mitigated by other unrecognized factors.4,5,20,34,44 Recent studies have suggested that the MYC translocation partner genes may also influence the behavior of the tumor, with the non-IG MYC translocations exhibiting a better prognosis than MYC/IG translocations.20,21,25 In addition to the clinical impact of MYC gene alterations, high protein expression has also been related to an inferior survival. BCL2 protein is also concurrently overexpressed in about 60% of MYC-positive DLBCL cases, independently of the presence of gene rearrangement. These cases are often described as “double expression lymphoma” and have a worse prognosis than cases with overexpression of only one of the two proteins.19,33–35,43 Intriguingly, whereas the poor prognosis of double expression lymphoma cases is concordant in all studies, the independent impact of BCL2 or MYC protein expression is controversial.19,34,35 The lack of reproducibility of these studies may be partially explained by the different cut-offs and non-standardized methods used to evaluate the tumors. Thus, the immunohistochemical cutoff to consider high expression varies from 10% to 40% for MYC and from 1% to 70% for BCL2 in different studies.19,34,35 Double expression lymphoma are more common (18% to 34%) than DH DLBCL (5% to 15%), but the clinical impact of the combined genetic alterations seem to be significantly worse than just the overexpression of the two proteins.34 Patients with DH usually present with a poor performance status, rapid evolution, poor response to current therapies, and a median survival of less than 1 year.19,34–36 Although there is no consensus on how to best treat these patients, the urgent need to investigate alternative management strategies is widely appreciated.

MYC ACTIVATION IN OTHER AGGRESSIVE LARGE B-CELL LYMPHOMA In addition to DLBCL, MYC dysregulation occurs frequently in other aggressive B-cell lymphomas (Table 1).

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The mechanism and molecular context in which this dysregulation occurs differs among entities. In contrast to DLBCL, in which MYC translocations appear in the context of complex karyotypes, MYC rearrangement in BL is usually the sole chromosomal alteration.45–47 In nearly all cases, IGH and IGL are the translocation partners of MYC.7 Genome-wide sequencing studies have shown that somatic mutations of TCF3, a gene associated with cell proliferation through activation of the PI3K pathway, or its inhibitor ID3 occur in approximately 70% of cases, in contrast to DLBCL, which rarely features mutations in these two genes.48–53 BCLU is a provisional category in the World Health Organization 2008 lymphoma classification created to identify tumors that cannot be reliably classified as DLBCL or BL.5,7 This category reflects a biological gray zone also recognized by molecular studies that have shown a subset of aggressive lymphomas with an intermediate gene expression profile between tumors classified as molecular Burkitt and clear DLBCL.5,46,54 These tumors are considered biological aggressive,20,44,55–57 but a recent study has shown that the outcome of these patients may be similar to that of DLBCL.58 MYC rearrangement has been reported in 30% to 90% of BCLU cases and 47% to 78% also carry BCL2 or BCL6 rearrangements.5,20,44,46,55–57,59,60 Similar to DLBCL, the partner of MYC in these translocations is frequently a non-IG gene (38%).46,57 Intriguingly, a recent study has detected the typical BL mutations in ID3 in 21% of BCLU cases, suggesting that BCLU may include a group of cases more related to BL than DLBCL.61 The study of the mutational profile of these tumors may be an important new tool to understand the ontogeny and clinical relevance of this heterogeneous group of tumors. PBL is an aggressive tumor characterized by the proliferation of large cells generally with immunoblastic morphology but a plasma cell-related phenotype. These tumors occur in immunocompromised or elderly patients and are frequently positive for EBV. MYC rearrangement has been found in 40% to 50% of the cases with the IG as major translocation partner (83% of cases) and associated with strong protein expression.62–64 These rearrangements seem to be more common in HIV-related PBL than in other subgroups of patients and are associated with a shorter overall survival.65 Some cases of PBL with MYC rearrangements have some overlapping features with plasma cell myeloma, such as monoclonal serum immunoglobulins and lytic bone lesions.66 However, the EBV infection and the immunocompromised status of the patients make these cases closer to PBL. ALK-positive large B-cell lymphoma is an aggressive tumor characterized by ALK rearrangement and protein expression.67 Similar to PBL, these cases express a plasma cell-related phenotype with negative mature B-cell markers and expression of BLIMP1 and XBP1. In spite of the BLIMP1 expression, MYC protein is strongly expressed in all cases without apparent genetic alterations.68,69 STAT3

MYC alterations in DLBCL

upregulation mediated by activated ALK is considered to contribute to the upregulation of MYC in these tumors.69 Primary mediastinal large B-cell lymphoma is a distinctive entity showing a localized mediastinal mass as a principal clinical finding.7 Genetic abnormalities of MYC are not common in this lymphoma. MYC rearrangements and mutations have been reported in 0% to 6% and 19% of primary mediastinal large B-cell lymphoma cases, respectively.70,71 The mutations are located in non-coding regions and, thus, their significance is not clear.70,71 JAK2 dysregulation, a common feature of this lymphoma, may upregulate MYC expression in vitro.72

MYC AS A POTENTIAL THERAPEUTIC TARGET Although various MYC-targeting therapies have been assessed in experimental studies, no direct MYC inhibitors are currently in clinical trials. MYC knockout mice exhibit embryonic lethality resulting from developmental defects in multiple organs.73 This important role of MYC in both normal and tumor cells causes difficulties in the development of MYC-targeting drugs. Several drugs that target MYC-induced oncogenic pathways rather than MYC itself have been developed. One representative example is the drugs that target the bromodomain and extra-terminal (BET) family genes. As described in the previous section, MYC globally increases histone lysine acetylation. The acetylated histone induces transcriptional activity by recruiting proteins of the BET family, such as BRD2, BRD3, and BRD4.74 Importantly, asymmetric loading of BRD4 has been observed at enhancer sites in DLBCL cell lines and primary lymphoma cells. Several BET inhibitors have been developed. JQ1 inhibits the transcriptional activation of MYC by preventing BRD4 from binding to chromatin and suppresses the proliferation of DLBCL cell lines and xenografted tumor cells.75 BET inhibitors seem to synergize with histone deacetylase inhibitors, suggesting that a combination of these drugs may be a potential alternative to counteract MYC activation.76

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Other alternatives to indirectly target MYC are being explored.77 Recently, several drugs that target MYC-associated tumors have been developed according to the concept of “synthetic lethality” (Figure 4). This idea aims to target genes or pathways that are required to maintain cell function only under conditions of a particular oncogenic dysregulation but not in normal cells.78 Then the suppression of this gene would not be critical to the fate of a normal cell but would lead to the death of tumor cells exhibiting this particular oncogenic activation. This idea has been explored for MYC dysregulation and several potential targets have been identified that include CDK1, Aurora kinase, SUMO activating enzyme, and DR5. Several drugs specific for these targets are currently in clinical trials.78

CONCLUSION MYC dysregulation is one of the most characterized oncogenic events in DLBCL because of a better understanding of its molecular mechanism and improvement of identification techniques such as immunohistochemistry and FISH. Several studies have recognized the aggressive clinical behavior of cases with the concomitant presence of MYC and BCL2 or BCL6 translocations. However, this poor outcome seems to be modulated by multiple factors such as morphology, MYC or BCL2 protein expression, and MYC translocated partner. The relative contribution of each of these aspects to the prognosis is not yet well characterized and needs further studies. MYC and BCL2 proteins may also be concomitantly expressed, but their immunohistochemical detection is not equivalent to the genetic DH because these later patients behave significantly worse than the tumors with only double expression. The recognition of lymphomas with DH is critical to improve the management of these patients. However, there is not yet a clear consensus on how to identify these tumors. The use of a systematic FISH analysis in all DLBCL seems to be an intense effort. A combined strategy beginning with an immunohistochemical

Figure 4. Synthetic lethality. According to this concept, gene A exhibits a synthetic lethal interaction with MYC. MYC is expressed but well-regulated in normal cells and no damage occurs even if gene A is inhibited. On the other hand, gene A is essential for the survival of a cancer cell with MYC dysregulation and, therefore, the inhibition of gene A induces tumor cell death. Under this condition, gene A could be a good drug target in MYC-dysregulated tumors.

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screening for BCL2 and MYC protein expression followed by FISH analysis in all cases with BCLU morphology and in DLBCL with double expression, germinal center phenotype, or high proliferation may be helpful.4,5 Further studies are needed to clarify the role of the features that modulate the behavior of aggressive lymphomas carrying a double genetic hit and the best strategies to detect these tumors. The identification of these patients should lead to the design of new therapeutic strategies that may overcome the dismal prognosis of most of these patients.

Acknowledgments The authors are deeply grateful to Daniel Martínez for his advice and to Steven H. Swerdlow for his inspiring discussions and comments. The authors are supported by grants from the Comisión Interministerial de Ciencia y Tecnología (CICYT; grant no. SAF12-38432), and the Generalitat de Catalunya (2009SGR992). E.C. is an ICREA-Academia researcher of the Generalitat de Catalunya and K.K. receives a research fellowship from the Uehara Memorial Foundation (Japan).

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