Presence Of MYC Translocation Research Articles

Performance of a Liquid Biopsy Diagnostic Prediction Model for EBV-Positive Burkitt Lymphoma in Sub-Saharan Africa

Burkitt Lymphoma (BL) is classified into EBV-positive BL (EBL) and EBV-negative BL (sporadic BL), the former being more prevalent in SSA (Al-Khreisat et al 2023). Conventional diagnosis of BL depends on the morphological assessment of invasive tissue biopsy followed by immunohistochemistry (IHC) and fluorescent in situ hybridization (FISH) to reveal the pathognomonic t(8;14), or t(8;22) translocations. This is associated with significant diagnostic delay in regions with limited pathology services (Mawalla et al 2023), often necessitating initiation of treatment based on clinical or histopathological findings without immunohistochemistry (Ogwang et al 2011), with ensuing mismanagement contributing to poor survival outcomes observed in SSA compared to High-Income Countries (HIC) (Sung et al 2021). Here, we sought to explore whether liquid biopsy could be used as an initial diagnostic aid for EBL in settings without easy access to pathology using different EBV DNA parameters and the MYC-Immunoglobulin ( MYC-Ig) gene rearrangement. This prospective study collected whole blood samples prospectively from 221 participants (90 BL and 131 non-BL) enrolled in the Aggressive Infection-Related East Africa Lymphoma (AI-REAL) project (Legason et al 2022) across 5 sites in Tanzania and Uganda. The revised BL diagnostic algorithm (Naresh et al 2021) was used as a gold-standard diagnosis. CfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit (Qiagen, USA), according to the manufacturer's instructions. DNA libraries were prepared with the Thru PLEX Tag-Seq HV kit (Takara Bio Inc. Japan), and hybridization capture was performed using a custom, next-generation sequencing (NGS) panel targeting genes commonly mutated in BL and three EBV genes: EBER1, EBER2, and EBNA2. All sequencing was performed in-country (MiSeq, Illumina, CA, USA) and analyzed in a custom pipeline. Using custom scripts, EBV DNA copies per cell and EBV DNA fragment size ratio were calculated as the proportion of EBV fragments of length 180 - 200 base pairs (bp) divided by the proportion of autosomal fragments within the same size window. The diversity in the fragment size distribution of the EBV and autosomal DNA was calculated and presented as EBV entropy and autosomal entropy. Translocations were called using IgCaller (Nadeu et al 2020) and Genomic Rearrangement Identification Software Suite (GRIDSS) (Cameron et al 2017). Univariate analysis was performed to assess the association between predictor variables (clinical parameters, EBV parameters, and MYC translocation) and a diagnosis of EBL. Ten-fold cross-validation was performed on a liquid biopsy model consisting of all the liquid biopsy variables with the clinical variables included in the model as covariates. Performance metrics for the liquid biopsy model as a diagnostic aid were calculated, and variable importance was determined to assess each variable's relative influence on the model. In our cohort, EBL diagnosis was negatively associated with tumor site (neck) and not significantly associated with tumor site (jaw). A shorter duration of symptoms was associated with a diagnosis of EBL. Presence of MYC translocation t(8;14), higher EBV fragment size ratio, proportion, entropy, and copies per cell for EBER1, EBER2, and EBNA2 were all associated with a diagnosis of EBL.The combined liquid biopsy diagnostic prediction model had an estimated AUC of 90%, with sensitivity between 85%-90%, specificity between 75% - 80%, and positive and negative predictive values of 85% (Figure 1). MYC translocation t(8;14) was the most important diagnostic variable, followed by EBV entropy (Figure 2). This study demonstrates the feasibility of liquid biopsy as a non-invasive diagnostic aid for EBL in settings without easy access to pathology. To strengthen the application of this technology into clinical practice, further analysis is being conducted for weighting and assigning of scores for each predictor variable. This would assist clinicians in making a precise diagnosis, complementing existing diagnostic modalities for EBL, and reducing the rate of misdiagnosis arising from diagnosis based on clinical suspicion and tissue morphology without immunohistochemistry.

Outcomes of Patients with Richter Transformation without Prior Chemoimmunotherapy for CLL/SLL: An International Multicenter Retrospective Study

Background: Richter Transformation (RT) refers to the development of an aggressive lymphoma in the setting of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Small molecule inhibitors (SMI), such as the Bruton tyrosine kinase inhibitors (BTKi) and BCL2 inhibitors (BCL2i), have revolutionized the treatment of CLL/SLL. Prior studies evaluated outcomes of patients (pts) with RT in an era where chemoimmunotherapy (CIT) was typically used to treat CLL/SLL. Now that SMI are standard of care, fewer pts with CLL/SLL receive CIT, therefore we sought to determine which variables predict survival in pts who developed RT without prior CIT exposure for CLL/SLL. Methods: We conducted an international multicenter retrospective study of pts from 11 academic centers. Pts with RT occurring as large B-cell lymphoma who did not previously receive CIT (e.g., fludarabine, cyclophosphamide, bendamustine, chlorambucil) for their CLL/SLL were included. We collected pt, disease, and treatment (tx) characteristics. RT was categorized into 3 groups: “Concurrent RT,” defined as RT and CLL/SLL diagnosed simultaneously (within 3 months [mos]); “RT w/o prior CLL/SLL tx,” defined as RT and CLL/SLL diagnosed >3 mos apart with the CLL/SLL never treated; and “RT with prior non-CIT tx for CLL/SLL” defined as having received prior CLL/SLL tx. Overall survival (OS) was measured from RT diagnosis (Dx) and estimated using the Kaplan-Meier method. Cox regression model was used to identify prognostic factors associated with OS. Results: 242 pts were identified. At CLL/SLL Dx, 66% of pts had unmutated IGHV, 37% had del17p/ TP53 disruption, and 20% had trisomy 12. At RT Dx, 56% of the RT with prior non-CIT tx for CLL/SLL group had a del17p/ TP53 disruption versus 27% for the concurrent RT group and 18% for the RT w/o prior CLL/SLL tx groups. Median time from CLL/SLL Dx to RT Dx was 47 mos (range 3-394) for RT w/o prior CLL/SLL tx and 46 mos (range 4-218) for RT with prior non-CIT tx for CLL/SLL. Pts with prior non-CIT tx for CLL/SLL received a median of 1 (range 1-5) prior tx. 86% of pts previously received a BTKi or a BCL2i for tx of CLL/SLL (54% BTKi only, 5% BCL2i only, 27% both). The remaining 14% had been treated with an anti-CD20 monoclonal antibody (MoAb), steroids, lenalidomide, radiation, or alemtuzumab. 85% of pts developed RT while on active tx with SMI. Median age at RT Dx was 69 years (range 37-92). At RT Dx, 38% and 21% of pts had del17p/ TP53 disruption, and trisomy 12 in CLL/SLL tissue, respectively. At RT Dx, there was no difference in highest SUV on PET (median 16, range 2.9-65.6), LDH (median 325, range 103-8162, ULN range 180-271), largest lymph node in diameter (median 4.9 cm, range 0-17.1), Ki-67 (median 80%, range 5%-100%), rate of GCB subtype (27%), presence of MYC translocation (20%), or CLL/SLL and RT clonal relationship (81%) for those pts tested amongst the 3 groups ( Table). The most common 1 st line tx for RT was CIT with or w/o radiation and/or steroids (76%), followed by CIT plus SMI (13%), SMI single agent or combination (7%), and other (4%, which includes anti-CD20 MoAb). Median follow-up from RT Dx was 42.3 mos; the median OS for the entire cohort was 25.8 mos (95% CI: 16.8-49.1). Pts with RT after non-CIT tx for CLL/SLL had a significantly worse OS (median 8.2 mos [95% CI: 5.6-14.3]) than pts with concurrent RT (median 46.3 mos [95% CI: 23.8-96.9]) and RT w/o prior CLL/SLL tx (median 63.5 mos [95% CI: 28.3-70.3]) ( Figure). From an exploratory univariable analysis, we found RT after non-CIT tx for CLL/SLL compared to concurrent RT (HR 2.32 [95% CI: 1.55-3.46]), RT Dx age (HR for 5-year increase 1.14 [95% CI: 1.05-1.23]), del17p/ TP53 disruption (HR 2.27 [95% CI: 1.45-3.56]), and LDH (HR for 2-fold increase 1.37 [95% CI: 1.20-1.58]) were prognostic for worse OS. Conclusions: This is the largest cohort of pts who developed RT having received only SMI with no CIT for their CLL reported, thus reflecting the current treatment landscape. Despite being CIT-naïve, pts in our series who received tx for their CLL/SLL and then developed RT had a short OS, underscoring the need to develop better therapies for these pts. In contrast, as in other published analyses (Wang Haematologica 2020), pts w/o prior tx for CLL/SLL had more favorable outcomes, with a median OS of approximately 5 years. Further analysis to determine PFS and OS by tx for RT for pts who have been treated with SMI for their CLL/SLL is ongoing.

Lymphomagenic Roles of MYC, E2F and Mir-150-5p in Plasmablastic Lymphoma: Therapeutic and Prognostic Implications

Introduction: Plasmablastic lymphoma (PBL) is an uncommon non-Hodgkin lymphoma which is characterized by an aggressive clinical behavior, very poor prognosis and which mostly affects immunosuppressed individuals. Although different studies have described genetic alterations in PBL, its transcriptome and epigenome have been scarcely studied. Our goal was to describe the gene and miRNA expression profiles and to identify new therapeutic targets and new prognostic factors in PBL. Methods: Formalin fixed paraffin embedded (FFPE) samples from 66 patients with PBL and samples from 14 reactive adenopathies, as a control group, were included for miRNA and gene expression studies (microarrays and RNA-seq, respectively). Enrichment analyses of miRNAs and genes differentially expressed, using hallmark data set, were carried out. The role of miR-150-5p in PBL lymphomagenesis was studied in vitro upon introduction of specific miRNA mimic in the PBL-1 cell line (Mine S, Sci Rep. 2017). Expression of the miRNA and its target genes was analyzed by RT-qPCR and Western Blot. Its role in cell proliferation, cell cycle progression and apoptosis induction, was assessed respectively by MTT assay and flow cytometry. Results: Patients with PBL showed 36 differentially expressed miRNAs compared to controls. Two expression profiles of these miRNAs were identified in PBL, related with the presence of MYC-translocation. The functional enrichment analysis of genes targeted by deregulated miRNAs revealed an enrichment of MYC and E2F targets, genes involved in G2M checkpoint and mTORC1 signaling pathway. This latter observation was confirmed by gene set enrichment analysis (GSEA) of genes differentially expressed in PBL (Fig.1). Different genes are shared among the top 3 enriched hallmark gene sets (Fig.2), this data suggests that both MYC and E2F are involved in the deregulation of cell cycle in PBL. Additionally, the upregulation of E2F1, E2F3 and E2F8 correlates significantly with an increased expression of MKI67 (Ki67), indicating that E2F exerts a crucial role in the abnormal proliferation in PBL. To identify deregulated miRNA-target interactions related with the top 3 enriched hallmark gene sets, we studied the correlation between the most differentially expressed miRNAs (miR-148a-3p, -4417, -150-5p, -342-5p, -342-3p, -3609) and their differentially expressed target genes. Only miR-150-5p showed a significant negative correlation with the anti-apoptotic gene BIRC5, coding for survivin, in PBL patients (p. 0.0063) (Fig.2). Of interest, high expression of BIRC5 and low expression of miR-150-5p correlated with high expression of MKI67 (p. 0.0006 and p. 0.04, respectively) indicating that the deregulation of miR-150-5p and BIRC5 may favor the proliferation of PBL cells. Low expression of miR-150-5p (lower than median= 6.56) was associated with shorter overall survival (p <0.0001), suggesting that this miRNA may represent a potential prognostic factor in PBL. We further studied the role of miR-150-5p in PBL lymphomagenesis by mimicking this miRNA in PBL-1 cell line. Upregulation of miR-150-5p decreased the expression of survivin at both mRNA and protein level, promoted apoptosis and reduced viability and proliferation, pointing out a pro-apoptotic role of miR-150-5p via survivin inhibition. Consistent with the known role of survivin in the control of mitosis, the upregulation of miR-150-5p and the consequent reduction in survivin levels produced a slight arrest of cells in G1 cell cycle phase and lower numbers of cells in G2-M phase. Finally, a positive tendency between BIRC5 and E2F3 expression was observed (p. 0.06), being this latter gene negatively correlated with miR-150-5p (p. 0.017). Indeed, a greater expression of E2F3 was encountered in those patients with low expression of miR-150-5p. Thus, these results suggest that E2F3 could repress miR-150-5p and mediate apoptosis via BIRC5 induction. Conclusions: We identified miR-150-5p as a new promising prognostic factor, and MYC and E2F as important regulators of global gene expression in PBL. Among these genes, E2F3 could represent a lymphomagenic-driver in PBL by repressing miR-150-5p and inducing BIRC5 expression. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Efficacy of Salvage Chemotherapy in Diffuse Large B Cell Lymphoma with Primary Treatment Failure According to Putative Cell of Origin

Efficacy of Salvage Chemotherapy in Diffuse Large B Cell Lymphoma with Primary Treatment Failure According to Putative Cell of Origin

Real-World Outcomes of Patients with Diffuse Large B-Cell Lymphoma Receiving Second Line Therapy in the United States

Background: In the rituximab era, outcome data from patients (pts) receiving second-line therapy (2L) for diffuse large B-cell lymphoma (DLBCL) are derived mostly from randomized controlled trials or selected cohorts from specialized care centers. As new salvage strategies emerge, there is a need to reevaluate the landscape of 2L DLBCL (treatments and outcomes) in real-world (rw) patient cohorts. We investigated the timing of 2L in a cohort of US pts with DLBCL, the utilization of autologous stem cell transplantation (ASCT) as consolidation, and the overall survival (OS) in these pts. Methods We included patients from the Flatiron Health electronic health record (EHR) derived, de-identified database, with a first incident DLBCL diagnosed after January 2011, and with documented 2L therapy receipt (n=524). All pts received ≥4 doses of an anthracycline-based immune-chemotherapy in the frontline therapy (1L) setting and initiated 2L active immune-chemotherapy (anti-CD20 monoclonal antibody monotherapy was excluded) prior to December 2019 to allow for at least 6 m of potential follow-up through May 31 2020. Patients with central nervous system disease at diagnosis were excluded. Real-world overall survival (rwOS) probabilities, indexed to the start of 2L, were estimated in the entire cohort using the Kaplan-Meier method. Hazard ratios (HR) comparing rwOS were estimated using Cox regression models. Baseline covariates included demographic information, DLBCL histologic subtypes, cell of origin (COO) classified by the Hans' algorithm, cytogenetics, and the time interval in months (m) from end of 1L therapy to initiation of 2L therapy. Analyses were also re-indexed to 1 and 3 years post-2L, comparing unadjusted rwOS in pts who had received ASCT on or prior to these timepoints versus those who did not. Results In the study sample of 524 pts (57% men), median age at 2L initiation was 68 years (range 59 to 76) and 63% had stage III or IV disease at diagnosis. Initiation of 2L happened within 6 m of 1L completion for a majority of pts, 276/524 (53%), and within 2 months for one quarter, 137/524 (26%); for the rest, 92 pts (18%) initiated between 6-&amp;lt;12 m after 1L completion, 72 pts (14%) between ⩾12-24 m and 84 pts (16%) after 24 m. Consolidation with ASCT was documented for 115 pts (22%) and 17 pts (3%) received chimeric antigen receptor T-cells therapy. Unadjusted median rwOS in the entire cohort was 22 m [95% CI: 17, 38] (Figure 1A). This varied by interval between 1L completion and 2L initiation; for pts with shorter interval (&amp;lt;12 m) from 1L, median rwOS was 17 m [95% CI: 12, 30], while it was 43 m [95% CI: 22, 54] for those with a ⩾12 m interval (Figure 1B). The presence of MYC translocation alone or in combination with other cytogenetic abnormalities was associated with a shorter unadjusted median rwOS vs. absence of MYC: 9.3 m [95% CI: 8.2, 32, n=33] vs. 26 m [95% CI: 18, 43, n=491] (Figure 1C). In the adjusted model controlling for baseline covariates, older age (⩾ 70) appears to be associated with increased hazard of death (HR: 1.48 [95% CI: 1.14, 1.91]). Unadjusted rwOS analyses at 1 year following start of 2L showed better survival for patients receiving ASCT versus those who did not (HR: 0.40 [0.21, 0.76]). This association was sustained at 3 years (HR: 0.17 [0.04, 0.75]. Conclusion In a contemporary cohort of rw pts with DLBCL receiving 2L immune-chemotherapy, the majority did not receive ASCT. In this setting, age ⩾70, shorter intervals between 1L completion and 2L initiation, presence of MYC translocations, and no ASCT were factors associated with shorter survival. It may be important to address the implications of these clinical features in the investigation of 2L therapy options for pts with DLBCL. Disclosures Sawas: Daiichi Sankyo: Speakers Bureau; Affimed: Research Funding; Roche: Current equity holder in publicly-traded company; Flatiron Health: Current Employment; Gilead: Speakers Bureau; Seattle Genetics: Speakers Bureau. Lau:Flatiron Health, Inc: Current Employment; Roche: Current equity holder in publicly-traded company. Thomson:Flatiron Health, Inc: Current Employment; Roche: Current equity holder in publicly-traded company. Maignan:Flatiron Health, Inc: Current Employment, Current equity holder in publicly-traded company; Roche: Current equity holder in publicly-traded company.

LMO2 Negative Expression Predicts the Presence of MYC Translocations in Aggressive B-Cell Lymphomas: Erratum.

LMO2 Negative Expression Predicts the Presence of MYC Translocations in Aggressive B-Cell Lymphomas: Erratum.

LMO2 Negative Expression Predicts the Presence of MYC Translocations in Aggressive B-Cell Lymphomas

LMO2 Negative Expression Predicts the Presence of MYC Translocations in Aggressive B-Cell Lymphomas

Pathology and classification of aggressive mature B-cell lymphomas.

Pathology and classification of aggressive mature B-cell lymphomas.

Identification of Anti-Lymphoma Biomarkers of Response to the Anti-CD37 Antibody Drug Conjugate (ADC) IMGN529

Background IMGN529 is an antibody drug conjugate (ADC) consisting of an anti-CD37 antibody with direct anti-tumor activity conjugated via a thioether linker to the cytotoxic maytansinoid antimicrotubule agent DM1. IMGN529 has shown pre-clinical (Deckert et al, Blood 2013) and clinical activity in lymphoma (Stathis et al, ASH 2014; NCT01534715). Here, we assessed the anti-tumor activity of IMGN529 on a large panel of B cell and T cell human lymphomas to identify potential biomarkers of response.Methods Fifty-four lymphoma cell lines [diffuse large B cell lymphoma (DLBCL), n.=27; mantle cell lymphoma (MCL), n.=10; anaplastic large T-cell lymphoma, n.=5; marginal zone lymphomas, n=6, others, n=6] were exposed to increasing doses of IMGN529 or to the unconjugated DM1 for 72h. Cell proliferation was measured using the MTT. Apoptosis induction was defined by at least 1.5-fold increase in caspase 3/7 signal activation with respect to controls using the Promega ApoTox-Glo Triplex Assay. CD37 surface expression was assessed by cytofluorimetry. Gene expression profiling (GEP) was done with the Illumina HumanHT-12 Expression BeadChips on untreated cell lines followed by GSEA (NES > |2|, P<0.05, FDR<0.25) and limma t-test (FC> |1.2|; P< 0.05; top 200 up and top 200 down).Results. The IMGN529 median IC50 in the 54 cell lines was 780pM (95%C.I., 263pm-11.45nM). Activity was stronger (P<0.001) in B cell lymphoma cell lines (n= 46; median IC50=450pM; 95%C.I., 150-800pM) than in T cell lymphoma cell lines (n=8; median IC50=22.5nM; 95%C.I., 14-40nM). The median IC50 for DM1 was 30pM (C.I.95%, 20-40pM) with no differences between B and T cell lymphoma origin. IMGN529 induced apoptosis in 33/54 (61%) lymphoma cell lines. Surface CD37 expression was higher in cell lines derived from B than from T cells (P< 0.0001): IMGN529 IC50 values, but not of DM1, were negatively correlated with surface CD37 expression across all cell lines (R=-0.39; P= 0.018), but not within the individual B or T cell subgroups. Among B cell lines, DLBCL cell of origin, TP53 status or the presence of BCL2 translocation did not affect the sensitivity to IMGN529, while IC50s were higher in the presence of MYC translocation (P= 0.043). No association was seen between IMGN529-induced apoptosis or the sensitivity to DM1 with DLBCL cell of origin, TP53 status or the presence of BCL2 or MYC translocations.We then compared the baseline gene expression profiling of DLBCL cell lines that were highly sensitive to IMGN529 (IC50< 800pM; "S") versus less sensitive/resistant DLBCL cell lines (IC50>10nM, "R"), separately for germinal center B cell type (GCB) (S, n=11; R, n=8) and for activated B cell like (ABC) (S, n=4; R, n=3). In both DLBCL groups, MYC targets, genes involved in unfolded protein response, glycolysis and DNA repair were enriched in transcripts more expressed in R than S cell lines. Transcripts associated with low sensitivity included CD44, VIM, ANXA2, BCL2, ANXA2P1, HSP90B1, NFKBIZ, CDK6, BIRC5 in GCB and HSPA1B, HSP90AA1, CADM1, CD86, TUBB2A, TUBG1, NOTCH1 in ABC cell lines. HEBP1, PHB, PSME3, RNU6-15, RPL13 were more expressed in both GCB and ABC R. Genes involved in PI3K/AKT/mTOR, hypoxia, INF-gamma, TNFA signaling via NFKB and in complement were more expressed in S than in R cell lines. Genes associated with sensitivity to IMGN529 comprised: CD37 (IMGN529 target), CD79A, CHI3L2, FAM117B, LPAR5, NFATC1, PTPN22, RBM38, SGPP1, SLC6A16 in both GCB and ABC cell lines; BASP1, CXCR5, BIK, LY86, TLR10, CD86, LCK, CD22, PTPN22, BCL6, PIK3IP1, CDKN2A in GCB; AFF3, PIM1, MGMT, PDE4B, NFKBIE, SYK, FOXO1in ABC.Conclusions. IMGN529 showed a very strong anti-tumoral activity in pre-clinical lymphoma models. High expression of CD37 and mostly genes involved in BCR signalling were associated with sensitivity to IMGN529. Conversely, the presence of MYC translocation, a high expression of MYC targets and of genes known to be involved in drug resistance (BCL2, BIRC5, CDK6, heat-shock proteins, annexins, proteasome and tubulin components) appeared to negatively affect the response to the ADC but also represent therapeutic targets for novel combinations to be explored. DisclosuresRossi:Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Janseen: Honoraria. Sloss:Immunogen Inc: Employment.

Comprehensive MYC and BCL2 Genetic Profiling in De Novo Diffuse Large B-Cell Lymphoma Demonstrates Clinically Relevant Genetic Alterations According to Cell of Origin Subtype

Background:MYC and BCL2 are critical driver genes for non-Hodgkin lymphoma including diffuse large B-cell lymphoma (DLBCL). However, the clinical impact of MYC and BCL2 genetic alterations, apart from translocations, has not been comprehensively investigated using high-resolution genetic assays, such as next generation sequencing and high-resolution SNP arrays. Moreover, correlations with cell of origin (COO) subtype, determined by gene expression profiling, have not been widely studied in a large homogeneously treated cohort. We determined the frequency and clinical impact of MYC and BCL2 genetic aberrations in DLBCL in a large population-based cohort uniformly treated with R-CHOP.Methods: We analyzed 347 newly diagnosed de novo DLBCL cases that were treated with R-CHOP in BC. Comprehensive clinical annotation was available through the BCCA Lymphoid Cancer Database. Deep targeted re-sequencing of the coding exons of MYC and BCL2 was performed using a Truseq Custom Amplicon assay (Illumina) on the Miseq platform. High-resolution copy number analyses were performed using Affymetrix SNP 6.0 arrays. Immunohistochemical staining and break-apart FISH assays for MYC and BCL2 were performed on tissue microarrays (n=332). Dual positivity for MYC (cut off; 40%) and BCL2 (cut off; 50%) proteins identified a dual protein expresser (DPE) phenotype. COO classification was achieved using the Lymph2Cx assay based on NanoString technology in 299 patients and the Hans algorithm in 32 cases with low tumor content (<40%).Results: COO determination revealed 193 cases to be GCB subtype, 107 cases ABC/non-GCB and 30 were unclassifiable. Using next generation sequencing, 310 SNVs were detected in MYC (29/347; 8% of cases) and BCL2 (88/347; 25% of cases), with mean redundant coverage depths of 633-fold. All MYC and 98% of BCL2 mutations were misssense mutations. Analysis of copy number alterations using GISTIC 2.0 revealed significant focal gain/amplification (gain/amp) peaks affecting 8q24, including the MYC locus (68/341; 20% of cases) and 18q21, including the BCL2 locus (82/341; 24% of cases). MYC and BCL2 translocations were detected in 38/283 (13%) and 90/300 (30%) of tumors, respectively.MYC gain/amp, BCL2 mutation, BCL2 translocations and double MYC/BCL2 translocation (DHIT) were seen significantly more often in GCB-DLBCL (26% vs 10%, P =0.001; 35% vs 11%, p<0.001; 44% vs 6%, p<0.001; and 13% vs 0%, p<0.0001, respectively), and BCL2 gain/amp was observed more commonly in ABC-DLBCL (44% vs 12%, p<0.001). MYC translocations were significantly associated with MYC protein expression in ABC, GCB and all cases (p<0.0001). On the other hand, BCL2 protein expression was significantly associated with BCL2 mutation and translocation in GCB-DLBCL (both, p<0.0001), and BCL2 gain/amp in ABC-DLBCL (P =0.0037).With a median follow up of 6.5 years for living patients, the presence of MYC translocation, BCL2 gain/amp and BCL2 mutation were associated with an inferior 5y-time to progression (TTP, 53% vs 30%, P =0.019; 60% vs 32%, P =0.009 and 52% vs 17%, P =0.026, respectively) in ABC subtype. In GCB subtype, BCL2 translocation, MYC gain/amp and MYC translocation were associated with an inferior 5y-TTP (85% vs 62%, P =0.001; 80% vs 63%, P =0.011; and 79% vs 61%, P =0.013, respectively). In a multivariate Cox model of TTP including IPI and DPE, BCL2 gain/amp remained prognostic (HR=2.4 [1.3-4.5], P =0.008) independent of IPI and DPE in ABC-DLBCL. In GCB-DLBCL, BCL2 translocation and/or MYC gain/amp showed strong prognostic value (HR=3.0 [1.4-6.4], P =0.006) independent of IPI and DPE. In the IPI high risk group (IPI=3-5), the presence of a BCL2 translocation and/or MYC gain/amp defined a remarkably poor outcome group in GCB-DLBCL (5y-TTP; 29%). Similar poor outcome was observed in ABC-DLBCL cases that harbored BCL2 gain/amp (5y-TTP; 22%).Conclusions: The DHIT genotype was only seen in GCB-DLBCL. High-resolution genomic assays identified extremely poor prognostic groups within each COO subtype on the basis of MYC and BCL2 genetic status in a large uniformly R-CHOP-treated population-based cohort of DLBCL. [Display omitted] DisclosuresSavage:Seattle Genetics: Honoraria, Speakers Bureau; BMS: Honoraria; Infinity: Honoraria; Roche: Other: Institutional research funding. Connors:Roche: Research Funding; Seattle Genetics: Research Funding. Scott:Celgene: Consultancy, Honoraria; NanoString: Patents & Royalties: Inventor on a patent that NanoString has licensed.

Expression of MYC but Not pSTAT3 Is an Adverse Prognostic Factor for Diffuse Large B Cell Lymphoma (DLBCL) Treated with Epratuzumab/R-CHOP

AbstractAbstract 1575We recently demonstrated in a phase II study that the anti-CD22 monoclonal antibody epratuzumab added to RCHOP improved DLBCL survival parameters. Despite these advances, 30–40% of DLBCL patients still relapse and die of disease. Recent advances have led to the identification of a variety of intracellular oncogenic pathways as potential targets for lymphoma therapy. Specifically, many studies have found that activation of the Signal transducer and activator of transcription 3 (STAT3) pathway promotes tumor cell survival in various types of cancers. STAT3 is a transcription factor and exerts its anti-apoptototic effect through several downstream targets such as MYC. MYC protein can be expressed in lymphoma cells with or without the presence of MYC translocation. The frequency of phosphorylated STAT3 (pSTAT3) and MYC expression and their prognostic relevance are unknown within diffuse large B-cell lymphoma (DLBCL), germinal center B-cell (GCB) and non-GCB subtypes. This study studied the tumor cell expression of pSTAT3 and MYC by IHC paired with serum cytokine levels in a DLBCL patient population uniformly treated on N0489.DLBCL tumor samples (n=38) were stained for detection of nuclear pSTAT3 expression. Using a threshold of ≥30% of tumor nuclei staining positive, 35 % (14/40) of tumors were pSTAT3+. An additional 17% (7/40) had between 10–30% pSTAT3+ cells. Non-malignant tonsil tissues (n=10) were positive for tSTAT3 in all cases but all but one case were pSTAT3 negative. Twenty-four of the same DLBCL tumors used for pSTAT3 expression were stained for MYC and 50% (12/24) were MYC positive (all nuclear) as defined by the criteria of ≥30% of cells staining positive. In cases (n=23) where both MYC and pSTAT3 IHC were performed, a positive pSTAT3 was more likely to have MYC expression whereas a positive MYC stain did not inform pSTAT3. By using a break apart probe for the MYC gene, MYC translocations in the major breakpoint regions were found in 10% (3/29) of cases. When MYC FISH was correlated with MYC IHC in the 24 DLBCL cases that had both techniques performed, all 3 MYC translocation cases were GCB by IHC, two were strongly positive for MYC by IHC, and 1 was negative. Among the 21 MYC FISH negative cases, 10 were MYC positive by IHC. These data suggest that MYC expression in lymphoma is not only controlled by genetic events such as translocations but also by other signaling pathways such as STAT3. pSTAT3 expression was correlated with an elevated serum LDH (p=0.0007). Neither MYC or pSTAT3 tumor expression correlated with other clinical or pathological features. Survival analysis revealed a trend toward shorter EFS for DLBCL patients whose tumors expressed MYC protein by IHC (p=0.2) or had a MYC translocation (p=0.09) by FISH; pSTAT3 expression status did not predict EFS (p=0.9). Within the GCB group 10 cases were MYC negative (10/17; 59%) and 7 cases were MYC positive (7/17; 41%). Among the non-GCB group 83% (5/6) were MYC positive. These data clearly suggest a trend of higher MYC positivity in the non-GCB DLBCL group (p=0.07). MYC positive cases had a clear trend of inferior EFS in both GCB (p=0.2) and in non-GCB (p=0.5) groups. The distribution of pSTAT3 (n=38) expression was also evaluated in the GCB (n=26) and non-GCB (n=12) DLBCL. In the GCB group 27% (7/26) were pSTAT3 positive compared to 58% (7/12) in the non-GCB group. Thus, there was a clear trend toward higher pSTAT3 positivity in non-GCB DLBCL tumors (p=0.06); however, pSTAT3 status did not correlate with EFS in either GCB or non-GCB DLBCL patients. In the present study we correlated pretreatment serum cytokines levels of these patients with pSTAT3 and MYC tumor cell expression. Out of the 30 cytokines tested, the only pre-treatment cytokines that were significantly correlated with pSTAT3 expression were IL-10 (p=0.05), G-CSF (p=0.03) and TNFa (p=0.04); none were correlated with MYC expression.Overall, our data provide evidence that over-expression of pSTAT3 and MYC is common in DLBCL. These biomarkers have potential as prognostic factors in the case of MYC and as a tool for selecting therapy for pSTAT3. MYC may be especially useful to further identify an adverse group of DLBCL patients within the otherwise favorable GCB tumor group. The availability of JAK/STAT and STAT-specific inhibitors provides the rationale to incorporate pSTAT3 staining in tumors from patients who are participating in these trials to learn if this biomarker can predict response Disclosures:No relevant conflicts of interest to declare.

MYC Translocation Partner Gene Determines Survival in Large B-Cell Lymphoma with MYC- or Double Hit MYC/BCL2 Translocation

Abstract 542MØP and AOG shared the first authorship. Background:In large B-cell lymphoma (LBCL) chromosomal translocations involving the MYC protooncogene (8q24) with or without concurrent BCL2 translocation (double hit) have been associated with inferior survival. We recently found in a prospective cohort of LBCL patients that double hit MYC/BCL2 translocations had no impact on overall survival (Pedersen et al., Eur.J.Haematol. 2012). However, further stratification of patients with double hit MYC/BCL2 translocation indicated an inferior survival related to immunoglobulin MYC translocation partner gene (MYC-IG). We sought to confirm this in a larger prospective cohort of LBCL patients. Materials and methods:All patients diagnosed with LBCL (diffuse large B-cell lymphoma, DLBCL, or B-cell lymphoma, unclassifiable, with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma, BCLU), at Dept. of Pathology and subsequently treated at Dept. of Hematology, Copenhagen University Hospital in Herlev, were prospectively collected from 2009–2011. Tumors were classified according to morphology and immunophenotype (2008 WHO classification). Chromosomal translocations were examined with FISH, including BCL2, MYC, MYC/IGH, kappa and lambda probes. Cases which were MYC/IGH fusion signal positive or MYC split signal positive + kappa or lambda split signal positive were classified as MYC-IG. Clinical data were collected from patient files. A total of 237 patients (163 primary LBCL, 49 transformed LBCL, 25 relapsed LBCL) were included. Results:MYC translocation was found in 28/225 patients, with translocation partner gene MYC-IG in 12/24 patients and MYC-nonIG in 12/24 patients. Double hit MYC/BCL2 was found in 23/228 patients, with translocation parter gene MYC-IG in 9/19 patients and MYC-nonIG in 10/19 patients. Cox regression models were performed for calculating p-values and survival curves (Fig. 1+2). The presence of MYC translocation or MYC/BCL2 double hit translocation showed no correlation with survival. However, stratification according to MYC translocation partner gene showed an inferior overall survival related to MYC-IG compared to MYC-nonIG (p=0.03), and to MYC translocation negative (Fig. 1). Among patients with double hit MYC/BCL2 translocation, a similar picture evolved where MYC-IG/BCL2 had an inferior overall survival compared to MYC-nonIG/BCL2 (p=0.006) and MYC/BCL2 translocation negative cases (Fig. 2). Most patients were treated with standard Rituximab containing chemotherapy and treatment was comparable between the groups. Conclusion:MYC translocation, with or without concurrent BCL2 translocation, was associated with inferior survival only if MYC had immunoglobulin translocation partner gene, in this prospective cohort of LBCL patients. This suggests that prognostic stratification by MYC and MYC/BCL2 translocations should include examination of MYC translocation partner genes. An overrepresentation of transformed cases which was observed in the MYC-nonIG group could lead to an underestimation of the prognostic effect of MYC-IG. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Concurrent Expression of MYC and BCL2 in Diffuse Large B-Cell Lymphoma Treated With Rituximab Plus Cyclophosphamide, Doxorubicin, Vincristine, and Prednisone

Diffuse large B-cell lymphoma (DLBCL) is curable in 60% of patients treated with rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP). MYC translocations, with or without BCL2 translocations, have been associated with inferior survival in DLBCL. We investigated whether expression of MYC protein, with or without BCL2 protein expression, could risk-stratify patients at diagnosis. We determined the correlation between presence of MYC and BCL2 proteins by immunohistochemistry (IHC) with survival in two independent cohorts of patients with DLBCL treated with R-CHOP. We further determined if MYC protein expression correlated with high MYC mRNA and/or presence of MYC translocation. In the training cohort (n = 167), MYC and BCL2 proteins were detected in 29% and 44% of patients, respectively. Concurrent expression (MYC positive/BCL2 positive) was present in 21% of patients. MYC protein correlated with presence of high MYC mRNA and MYC translocation (both P < .001), but the latter was less frequent (both 11%). MYC protein expression was only associated with inferior overall and progression-free survival when BCL2 protein was coexpressed (P < .001). Importantly, the poor prognostic effect of MYC positive/BCL2 positive was validated in an independent cohort of 140 patients with DLBCL and remained significant (P < .05) after adjusting for presence of high-risk features in a multivariable model that included elevated international prognostic index score, activated B-cell molecular subtype, and presence of concurrent MYC and BCL2 translocations. Assessment of MYC and BCL2 expression by IHC represents a robust, rapid, and inexpensive approach to risk-stratify patients with DLBCL at diagnosis.