Primary Mediastinal B-cell Lymphoma Cases Research Articles

Cooperative Somatic Alterations in XPO1 and TNFAIP3 (A20) Promote Immune Modulation and Tissue Infiltration in Primary Mediastinal B Cell Lymphoma

Background: Primary Mediastinal B cell lymphoma (PMBCL) is an aggressive large B cell non-Hodgkin lymphoma typically presenting as a mass in the anterior mediastinum in early adulthood, disproportionately affecting females (2:1). Previous studies have identified constitutive activation of the NF-κB pathway and immune evasion as hallmark features of PMBCL. The master negative regulator of canonical NF-κB, TNFAIP3 (A20), is inactivated in ~25-60% of PMBCL cases via disruptive mutations or deletions. Gains of the 2p16.1-15 locus ( REL, XPO1, BCL11A) are seen in ~40-75% of PMBCL cases. Interestingly, PMBCL cases with 2p16.1-15 gain/amplification display concomitant increases in XPO1 expression but lack correlative increases in REL or BCL11A. In addition, XPO1 mutations are seen in ~15-33% of PMBCL cases, with the most prevalent mutation (E571K) implicated in NF-KB activation. Despite the established occurrence of genetic events impacting XPO1 and A20 in PMBCL, the cooperative potential of these alterations remains unexplored. Consequently, we conducted an extensive review of literature describing the PMBCL genomic landscape and utilized in vivo models to examine the combined potential of XPO1 and A20 alterations to promote lymphomagenesis. Methods: Genomic data was obtained from NGS analyses from a total of 233 PMBCL patients across 6 independent published research studies and analyzed for cooccurrence of A20 & XPO1 alterations (Camus et al. 2022; Chapuy et al. 2019; Lacy et al. 2020; Mareschal et al. 2015; Mottock et al. 2019; Tuveri et al 2022). For in vivo analysis, we developed a murine model of B cell specific overexpression of wildtype (WT) or E571K- XPO1 with CD19.cre driven depletion of A20 (Eµ-XPO1xA20 KO). Whole body histopathologic evaluation of FFPE tissue with H&E staining was performed on 6-month-old, age and sex matched littermate mice. Results: We found co-occurrence of inactivating events in A20 and XPO1 missense mutations impacting 15% of PMBCL evaluated cases, with consistent results seen across all studies (range: 13.8-20%). As expected, A20 mutations/del were more prevalent than XPO1 mutations across all studies (OR 2.46, 95% CI 0.84-7.18, p = 0.02). XPO1 mutated cases trended towards a greater likelihood of having co-occurring A20 mutations (OR 8.51, 95% CI 0.84-86.47, p = 0.08). XPO1 mutations and gain (2p16) were found to be mutually exclusive events with gains co-occurring with A20 mutations/del in ~17% of PMBCL cases evaluated. Analysis of survival of PMBCL patients with cooccurring A20 inactivating alterations and XPO1 mutations is ongoing although preliminarily trend towards reduced mean 2-year survival (p=0.056) compared to all others are observed. Taken together, co-occurring genetic alterations effecting A20 and XPO1 are prevalent in PMBCL the alternative biology stemming from potential cooperative mechanisms may represent an aggressive subgroup of PMBCL. To evaluate concurrent B cell-specific alterations of A20 and XPO1 in vivo we utilized Eµ-XPO1xA20 KO mouse models. Performing histopathology in 6-month-old mice we observed enlarged lymph nodes due to marked increases in lymphocytes within the cortex, paracortex, medulla, and sinuses in mice with loss of functional A20, particularly in Eµ-XPO1xA20 KO mice with overexpression of WT XPO1. Increased lymphocyte populations drove overall enlargement of the node (mandibular, mesenteric, pancreatic, and mediastinal) and distorted normal architecture, featuring small lymphocytes populating sinus regions and larger lymphocytes populating areas resembling follicles. Eµ-XPO1(WT & E571K)xA20 KO mice additionally featured mild mononuclear infiltrates in the lung and salivary glands; all of which were observed with increased severity compared to A20KO, Eµ-XPO1, or non-transgenic littermates. Significance: We identified co-occurring genetic lesions impacting A20 and XPO1 in PMBCL tumors and show that these alterations synergize in vivo leading to nodal/extranodal lymphocyte infiltration. The immunomodulatory effects appear to be driven by the cooperation of XPO1 overexpression, more so than mutation, and A20 inactivation in young mice. Further investigation of these models will allow elucidation of underlying cooperative mechanisms and reveal targetable pathways in PMBCL.

Frequent ZNF217 mutations lead to transcriptional deregulation of interferon signal transduction via altered chromatin accessibility in B cell lymphoma

Recent exome-wide studies discovered frequent somatic mutations in the epigenetic modifier ZNF217 in primary mediastinal B cell lymphoma (PMBCL) and related disorders. As functional consequences of ZNF217 alterations remain unknown, we comprehensively evaluated their impact in PMBCL. Targeted sequencing identified genetic lesions affecting ZNF217 in 33% of 157 PMBCL patients. Subsequent gene expression profiling (n = 120) revealed changes in cytokine and interferon signal transduction in ZNF217-aberrant PMBCL cases. In vitro, knockout of ZNF217 led to changes in chromatin accessibility interfering with binding motifs for crucial lymphoma-associated transcription factors. This led to disturbed expression of interferon-responsive and inflammation-associated genes, altered cell behavior, and aberrant differentiation. Mass spectrometry demonstrates that ZNF217 acts within a histone modifier complex containing LSD1, CoREST and HDAC and interferes with H3K4 methylation and H3K27 acetylation. Concluding, our data suggest non-catalytic activity of ZNF217, which directs histone modifier complex function and controls B cell differentiation-associated patterns of chromatin structure.

Potential Utility of Circulating Tumor DNA Monitoring in Primary Mediastinal B-Cell Lymphoma Treated with R-DA-EPOCH

Potential Utility of Circulating Tumor DNA Monitoring in Primary Mediastinal B-Cell Lymphoma Treated with R-DA-EPOCH

STAT6 is a cargo of exportin 1: Biological relevance in primary mediastinal B-cell lymphoma

Primary mediastinal B-cell lymphoma (PMBL) is a distinct B-cell lymphoma subtype with unique clinicopathological and molecular features. PMBL cells are characterised by several genetic abnormalities that conduct to the constitutive activation of the Janus kinase 2/signal transducer and activator of transcription 6 (JAK2/STAT6) signalling pathway. Among recurrent genetic changes in PMBL, we previously reported that the XPO1 gene encoding exportin 1 that controls the nuclear export of cargo proteins and RNAs, is mutated (p.E571K) in about 25% of PMBL cases. We therefore hypothesized that STAT6 could be a cargo of XPO1 and that STAT6 cytoplasm/nucleus shuttle could be altered in a subset of PMBL cells. Using immunocytochemistry techniques as well as the proximity ligation assay, we showed that STAT6 bound XPO1 in PBML cell lines and in HEK-293 cells genetically engineered to produce STAT6. Moreover, XPO1-mediated export of STAT6 occurs in cells expressing either a wild-type or the E571K mutated XPO1 protein.

Primary mediastinal large B-cell lymphoma: a clinicopathologic study of 27 cases

Objective: To study the clinicopathologic characteristics and diagnostic criteria of primary mediastinal B-cell lymphoma (PMBL), and to distinguish PMBL from classic Hodgkin lymphoma(CHL) and systemic diffuse large B-cell lymphoma(DLBCL). Methods: The clinical features, histologic findings, results of immunohistochemical study and prgnosis in 27 PMBL cases were analyzed, with review of literature. Results: The age of patients ranged from 19 to 82 years (median age 34 years). All cases were located in the mediastinum and frequently accompanied by superior vein cava syndrome. Histologically, the tumor cells were pleomorphic and diffusely distributed. Clear cytoplasm and spindle tumor cells were seen in some cases. Varying amount of sclerosing stroma with collagen deposition was seen.Immunohistochemical study showed that the tumor cells were positive for CD20(100%, 27/27), CD30 (64.0%, 16/25), CD23 (77.3%, 17/22) and p63 (16/19). Clonal B cell gene rearrangement was seen. Conclusions: PMBL is a subtype of diffuse large B-cell lymphoma with various histomorphology. Immunohistochemistry can help to confirm the diagnosis, and the prognosis is better than diffuse large B cell lymphoma, not otherwise specified.

Integrated Analysis of IGHV Gene Status, Cell-of-Origin Signature and Genomic Features in Diffuse Large B-Cell Lymphoma

The molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) has been highlighted by gene expression profiling (GEP), dividing DLBCL into the following three main molecular subtypes with different clinical outcomes and responses to immunochemotherapy: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype and the primary mediastinal B-cell lymphoma (PMBL) subtype. Despite frequent translocations involving the IGH (heavy chain) locus, B-cell receptor (BCR) expression is retained in almost all DLBCL, indicating that BCR signaling is crucial for the survival of malignant cells. Furthermore, DLBCL are characterized by recurrent somatic mutations that are supposed to be oncogenic drivers. Here, with the aim to more accurately define the DLBCL pathogenic subgroups, we report a detailed immunogenetic analysis of IGH rearrangements in a well-defined GEP DLBCL cohort and correlate these features with the somatic mutation status of recurrently mutated genes involved in lymphomagenesis. Methods: 204 DLBCL enrolled in the prospective LYSA LNH03 trial program were classified into molecular subtypes by GEP using Affymetrix arrays [82 ABC (40.2%), 77 GCB (37.7%), 29 (14.2%) unclassified and 16 PMBL (7.8%)]. The amplification of complete VDJ rearrangements was realized using BIOMED-2 protocols. Somatic hypermutation (SHM) characteristics were studied (mutation rate, glycosylation N-X-S/T sites, RGYW hotspots, composition of CDR3) and correlated with the molecular subtypes. To obtain a more comprehensive molecular portrait of the DLBCL cases, GEP and IGH VDJ analyses were correlated with the mutational status of a panel of 34 recurrently mutated genes in DLBCL such as MYD88, EZH2, CD79B, TNFAIP3, CARD11 and GNA13 (Dubois et al. Clinical Cancer Research 2016). Results: A total of 153/204 (75%) clonal VDJ rearrangements were successfully determined. Failures to detect clonotypic sequences were predominantly seen in PMBL (56%) and unclassified (31%) cases. The ABC subtype display a significantly lower SHM rate than the 3 other subtypes, especially in contrast to the PMBL subgroup that was characterized by a higher SHM rate (p<0.0001 for each). While IGHV gene usage generally reflected that of normal B cells, a marked increase in IGHV4-34 in ABC cases (30%) was confirmed and those cases tended to have less SHM. Acquisition of new N-glycosylation (N-Gly) sites, a hallmark of follicular lymphoma, was more prevalent in the GCB cases (48%) than in ABC (22%) (p=0.012) but was also observed in 5/7 (71%) PMBL cases with functional IGH rearrangement. This acquisition is correlated with a more frequent t(14;18)(q24;q32) in GCB subtype. As reported in FL, this suggests that lectin binding signals via surface Ig that contain N-Gly sites may provide an extrinsic antigen-independent signal in some DLBCL, and more specifically in t(14;18) DLBCL (Linley et al. Blood 2015 ). Finally, we correlated these immunogenetic features with the mutational status of some drivers genes frequently mutated in DLBCL. Within ABC subtype patients, MYD88 or TNFAIP3 mutated cases displayed a higher SHM rate (13.4% vs 8.4%, p <0.0001; 15.44% vs 11.13%, p=0.03), without any IGH family distribution bias. 9/56 (16%) GCB DLBCL harbored an EZH2 mutation. All expressed an IGHV3 rearrangement (100% vs 52% for the GCB-EZH2 wt, p=0.014). By contrast to MYD88 status, a similar IG SHM rate was observed in mutated and wt patients regarding EZH2, CARD11 or GNA13 genes. Conclusion: The IG characterization and the driver gene mutation analysis according to the cell of origin of the tumor allowed us to refine DLBCL subgroups. Our data suggest that there is a strong interaction between extrinsic mechanisms, including lectin-mediated antigen-independent activation or, in the IGHV4-34 cases, via N-acetyl lactosamine ligands, and intrinsic mechanisms to activate the BCR/NFKB pathway. These data could help us classify the different subgroups of DLBCL more accurately and, in particular, to identify new molecular features for BCR target therapy. DisclosuresHaioun:Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Salles:Mundipharma: Honoraria; Janssen: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

ΝFΚΒΙΕ Deletions: A Novel Marker of Clinical Aggressiveness in Primary Mediastinal B-Cell Lymphoma

Deregulated NF-κB signaling is a hallmark of most, if not all, lymphoid malignancies, and recurrent gene mutations in both the canonical and non-canonical NF-κB pathway are known to lead to NF-κB activation. However, the full compendium of NF-κB gene mutations in lymphoid malignancies remains to be elucidated. Recently, we reported a 4-bp truncating mutation in the NFKBIE gene, which encodes IκBε, a negative regulator of NF-κB, in patients with chronic lymphocytic leukemia (CLL). The NFKBIE deletion was enriched in clinically aggressive CLL patients (7-8%) and associated with a worse clinical outcome. At the functional level, NFKBIE-deleted CLL showed reduced IκBε levels and decreased p65 inhibition, along with increased phosphorylation and nuclear translocation of p65, compared to wildtype patients. Preliminary data has indicated an increased frequency of NFKBIE aberrations in other lymphoid malignancies as well. To explore this further, we screened for NFKBIE deletions in a large cohort of patients diagnosed with a range of different lymphoid neoplasms. Overall, NFKBIE deletions were identified in 76 of 1414 patients (5.4%). While NFKBIE deletions were relatively infrequent in patients diagnosed with follicular lymphoma (3/225, 1.3%), splenic marginal zone lymphoma (3/175, 1.7%), and T-cell acute lymphoblastic leukemia (1/94, 1.1%), moderate frequencies were observed among diffuse large B-cell lymphoma (18/521, 3.5%), mantle cell lymphoma (8/189, 4.2%), and primary CNS lymphoma (1/34, 2.9%) patients. In contrast, a remarkably high frequency of NFKBIE deletions (41/176 cases, 23%) was observed among primary mediastinal B-cell lymphoma (PMBL) patients. Noteworthy, the prevalence of NFKBIE-deleted PMBL cases was similar in the different contributing centers. All PMBL patients in the present series received a CHOP based treatment regime; in ~75% of cases rituximab was added and ~25% were treated with dose intensified schemes. For the latter, the vast majority of patients received CHOEP, while individual cases were treated with MegaCHOEP, DA-EPOCH or ACVBP. Regarding clinicobiological associations, there were no significant differences between NFKBIE-deleted and wildtype PMBL patients with respect to age, sex, Ann Arbor stage, IPI risk-groups, extranodal or bone marrow involvement, bulky disease, and LDH elevation. However, NFKBIE-deleted patients were more likely to be refractory to primary chemotherapy (31% vs. 3%, P=.001) and had a shorter overall survival compared to wildtype patients (5-year overall survival: 63% vs 84%, P=.013). In multivariate analysis (including age, gender, Ann Arbor stage, IPI, and NFKBIE mutation status), NFKBIE mutation status (95% CI: 1.23-10.61; HR: 3.61; P=0.020) remained an independent factor for poor prognosis. In summary, we document NFKBIE deletions as a common genetic event across B-cell malignancies, albeit at varying frequencies. The high frequency of NFKBIE deletions in PMBL alludes to the critical role of this aberration in the pathophysiology of the disease. NFKBIE deletions were associated witha worse clinical outcome, hence potentially representing a novel poor-prognostic marker in PMBL. DisclosuresStamatopoulos:Gilead: Consultancy, Honoraria, Research Funding; Abbvie: Honoraria, Other: Travel expenses; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Other: Travel expenses, Research Funding.

Recurrent mutations of the exportin 1 gene (XPO1) and their impact on selective inhibitor of nuclear export compounds sensitivity in primary mediastinal B-cell lymphoma.

Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity, and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-β nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in GEP-defined PMBL and was associated with shorter PFS. Age, International Prognostic Index and bulky mass were similar in XPO1 mutant and wild-type cases. KPT-185 induced a dose-dependent decrease in cell proliferation and increased cell-death in PMBL cell lines harboring wild type or XPO1 E571K mutant alleles. Experiments in transfected U2OS cells further confirmed that the XPO1 E571K mutation does not have a drastic impact on KPT-330 binding. To conclude the XPO1 E571K mutation represents a genetic hallmark of the PMBL subtype and serves as a new relevant PMBL biomarker. SINE compounds appear active for both mutated and wild-type protein. Am. J. Hematol. 91:923-930, 2016. © 2016 Wiley Periodicals, Inc.

Brentuximab Vedotin in Combination with Multi-Agent Chemotherapy Is Well Tolerated and Shows Promising Activity As Frontline Treatment for Primary Mediastinal B-Cell Lymphoma

Background: Primary mediastinal B-cell lymphoma (PMBL) is a rare subtype of diffuse large B-cell lymphoma (DLBCL) with distinctive clinical and immunophenotypic features. Most PMBL patients (pts) present at young age and with bulky mediastinal mass. Over 80% of PMBL cases are CD30 positive by immunohistochemistry (IHC). Standard frontline systemic therapy for PMBL remains controversial and the use of intensive regimens may be associated with increased toxicity. Brentuximab vedotin (BV) is a CD30-directed immunoconjugate with established efficacy in relapsed Hodgkin lymphoma and several other CD30 positive lymphoid malignancies, but this targeted agent has not been utilized in frontline combinations for PMBL.Methods: We designed an open label phase I/II trial using BV administered concurrently with rituximab, cyclophosphamide, doxorubicin, and prednisone (standard R-CHOP with the omission of vincristine to minimize risk of neuropathy) as frontline treatment for PMBL, grey zone lymphoma (GZL), and other CD30-positive DLBCLs. Pts with any Ann Arbor stage and at least equivocal CD30 expression on IHC were eligible. Consolidative radiation was allowed after completion of therapy at discretion of the treating physician. Phase I utilized a 3+3 de-escalation design with starting dose of BV 1.8 mg/kg on Day 1 in combination with rituximab 375 mg/m2 on Day 1, cyclophosphamide 750 mg/m2 on Day 1, doxorubicin 50 mg/m2 on Day 1, and prednisone 100 mg daily on Days 1-5 every 3 weeks for 6 cycles. The primary objective of phase I was assessment of safety and dose limiting toxicity (DLT) defined as any grade 3/4 non-hematologic toxicity observed in Cycle 1 requiring dose delay >14 days from the planned Day 1 of Cycle 2. Secondary end points included overall response rate (ORR), progression free survival (PFS), and overall survival (OS). The primary phase II objective was ORR with secondary endpoints of PFS, OS, safety, and correlation with CD30 expression. Revised Response Criteria for Malignant Lymphoma was used for response assessment (Cheson, 2007). Enrollment began in January 2014 and is reported through July 27, 2015.Results: Twelve pts including 9 PMBL (75%), 1 GZL (8%), and 2 DLBCL (17%) have been enrolled and treated; 11 patients are evaluable after completing protocol defined therapy. Median age was 37 years (range 25 - 58), 5 (45%) were female, 9 (82%) had elevated LDH, and 6 (55%) had stage III-IV disease. No DLT was observed during phase I and no BV dose de-escalation was required. There were no grade 3/4 non-hematologic adverse events (AEs). Hematologic grade 3/4 AEs included febrile neutropenia in 2 pts and afebrile neutropenia in 1 pt. Five pts had grade 2 non-hematologic AEs at least possibly related to BV including mucositis, nausea/vomiting, diarrhea, abdominal pain, anorexia, hypotension, and neuropathy. No study related deaths were observed. The ORR among evaluable pts was 100% (10 pts with CR, 1 pt with GZL with PR). The PFS and OS with median follow-up of 8 months (range: 4 -18 months) is 100%. Eight pts completed consolidative radiation therapy after systemic therapy.Conclusions: We established safety of administrating BV 1.8 mg/kg in combination with rituximab, cyclophosphamide, doxorubicin, and prednisone as frontline therapy for PMBL, GZL and CD30-positive DLBCL. This outpatient regimen was well tolerated. Clinical outcomes observed in this initial cohort are very encouraging and will be validated in the ongoing phase II part of the trial. DisclosuresSvoboda:Celgene: Research Funding; Immunomedics: Research Funding; Seattle Genetics: Research Funding; Celldex: Research Funding. Off Label Use: Brentuximab is not FDA approved for front-line therapy in PMBL and CD30+ DLBCL. Nasta:Seattle Genetics: Research Funding; BMS: Research Funding. Mato:Celgene Corporation: Consultancy, Research Funding; Gilead: Consultancy, Research Funding; TG Therapeutics: Research Funding; Pharmacyclics: Consultancy, Research Funding; Pronai Pharmaceuticals: Research Funding; AbbVie: Consultancy, Research Funding; Genentech: Consultancy. Pro:Seattle Genetics: Consultancy, Other: Travel expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses. Barta:Seattle Genetics: Research Funding. Schuster:Janssen: Research Funding; Celgene: Consultancy, Research Funding; Nordic Nanovector: Membership on an entity's Board of Directors or advisory committees; Genentech: Consultancy; Phamacyclics: Consultancy, Research Funding; Hoffman-LaRoche: Research Funding; Novartis: Research Funding; Gilead: Research Funding.

FOXO1 downregulation contributes to the oncogenic program of primary mediastinal B-cell lymphoma.

Recently we have shown that the transcription factor FOXO1, highly expressed in B cells, is downregulated in classical Hodgkin lymphoma (cHL). As primary mediastinal B cell lymphoma (PMBL) has similarities with the cHL transcription program we investigated FOXO1 expression in this entity. By using immunohistochemistry we found that FOXO1 was absent or expressed at low levels in 19 of 20 primary PMBL cases. PMBL cell lines reproduce the low FOXO1 expression observed in primary cases. By analyzing gene expression profiling data we found that FOXO1 expression inversely correlated with JAK2 in PMBL cases. Targeting JAK2 activity by the small molecular weight inhibitor TG101348 resulted in upregulation of FOXO1 mRNA and protein expression in MedB-1 and U2940 cell lines, and the MYC inhibitor 10058-F4 increased FOXO1 mRNA in MedB-1 cells. Moreover, in MedB-1 cells FOXO1 expression was strongly upregulated by the inhibitor of DNA methylation 5-aza-2-deoxycytidine and by the histone deacetylase inhibitor trichostatin A. Since FOXO1 promoter was unmethylated, this effect is most likely indirect. FOXO1 activation in the FOXO1-negative Med-B1 cell line led to growth arrest and apoptosis, which was accompanied by repression of MYC and BCL2L1/BCLxL. Thus, FOXO1 repression might contribute to the oncogenic program and phenotype of PMBL.

Knock-down of BCL6 / STAT6 sensitizes primary B cell lymphoma cells for treatment with current therapeutic agents.

Primary mediastinal B cell lymphoma (PMBL) is characterized by specific molecular hallmarks including the expression of B Cell Lymphoma factor 6 (BCL6) and the presence of the activated Signal Transducers and Activators of Transcription factor 6 (STAT6). Recently we have shown that combined targeting of BCL6 and activated STAT6 by specific chemical inhibitors in PMBL resulted in additive efficacy regarding their negative effects on cell viability. Given that despite general efficient immuno-chemotherapy in PMBL the delayed treatment-related sequelae remains still a main challenge, we analyzed the role of BCL6 and activated STAT6 in the sensitivity of PMBL cells to the current treatment components. We found that the knock-down of BCL6 / STAT6 by siRNA sensitized the PMBL cells to the effects of R-CHOP components in two of three PMBL cell lines. In one cell line, MedB-1, which is marked by less expression of BCL6 and mutated STAT6, the knock-down of BCL6 / STAT6 did not enhance the efficiency of Doxorubicin, Rituximab, and Vincristin. Thus, the targeting of BCL6 and STAT6 in addition or prior to the treatment with components of the current immuno-chemotherapy may sensitize the PMBL tumor cells for drug effects, at least in parts of PMBL cases.

Recurrent somatic mutations of PTPN1 in primary mediastinal B cell lymphoma and Hodgkin lymphoma

Classical Hodgkin lymphoma and primary mediastinal B cell lymphoma (PMBCL) are related lymphomas sharing pathological, molecular and clinical characteristics. Here we discovered by whole-genome and whole-transcriptome sequencing recurrent somatic coding-sequence mutations in the PTPN1 gene. Mutations were found in 6 of 30 (20%) Hodgkin lymphoma cases, in 6 of 9 (67%) Hodgkin lymphoma-derived cell lines, in 17 of 77 (22%) PMBCL cases and in 1 of 3 (33%) PMBCL-derived cell lines, consisting of nonsense, missense and frameshift mutations. We demonstrate that PTPN1 mutations lead to reduced phosphatase activity and increased phosphorylation of JAK-STAT pathway members. Moreover, silencing of PTPN1 by RNA interference in Hodgkin lymphoma cell line KM-H2 resulted in hyperphosphorylation and overexpression of downstream oncogenic targets. Our data establish PTPN1 mutations as new drivers in lymphomagenesis.

Concurrent Expression Of MYC/BCL2 Protein In Newly Diagnosed DLBCL Is Not Associated With An Inferior Survival Following EPOCH-R Therapy

▪ BackgroundWhile several studies have now demonstrated that patients with MYC-rearranged DLBCL have a worse outcome following treatment with CHOP -with or without rituximab - recent reports suggest that concurrent expression of MYC and BCL2 protein by immunohistochemistry (IHC), independent of a MYC rearrangement, is associated with an inferior clinical outcome following R-CHOP (Johnson et al. JCO 2012 Oct 1; 30 (28): 3452-9). We previously reported that DLBCL patients with a MYC rearrangement have a similar outcome to MYC negative cases (4 year EFS of 83% versus 76% respectively: p=0.46) following dose-adjusted (DA)-EPOCH-R (Dunleavy et al. Ann Oncol 2011 (22) Suppl 4 # 71). MethodsTo assess the prognostic role of MYC and BCL2 protein expression, we performed immunohistochemistry for MYC and BCL2 in patients with newly diagnosed DLBCL who received DA-EPOCH-R or short-course (SC)-EPOCH-RR (if HIV infected). Primary mediastinal B-cell lymphoma (PMBL) cases were excluded as they have a different biology. Tumors were scored positive for MYC if ≥ 40% of cells stained positive. For BCL2, the staining intensity was compared with that in control T cells present in the tumor samples – tumor cells were considered positive if they stained the same or more intensely than T cells. We used the Hans algorithm, as previously described, to predict cell of origin as germinal center B-cell like (GCB) or non-GCB type and correlated this with outcome. ResultsOf 66 patients enrolled on study, characteristics were median (range) age: 48 (18-76) years; male sex 47 (71%); IPI score low versus intermediate/high 18 (27%) versus 48 (73%); HIV positive 22 (33%). IHC was positive for MYC in 28/48 (58%) cases and positive for BCL2 in 24/51 (47%). 36/51 (71%) and 15/51 (29%) of cases were of GCB and non-GCB origin respectively. At a median follow-up time of greater than 10 years, progression-free survival (PFS) and overall survival (OS) were 67.5% and 75% for all patients. We compared survival in the 4 groups: MYC+/BCL2+, MYC+/BCL2-, MYC-/BCL2+ and MYC-/BCL2-. PFS and OS were not significantly inferior in any group (global p value=0.5 (PFS) and 0.8 (OS)). Cell of origin did however predict outcome and at 10 years follow-up, PFS was 78% (GCB) versus 43% (non-GCB) (p=0.016) and OS 80% (GCB) versus 65% (non-GCB) (p=0.24). There was a significant association between MYC+/BCL2+ cases and non-GCB subtype (p=0.01) but as 42% were of GCB origin, MYC+/BCL2+ status alone did not predict a poor outcome. ConclusionsIn DLBCL patients treated with DA-EPOCH-R and SC-EPOCH-RR, concurrent expression of MYC and BCL2 protein did not correspond with a worse clinical outcome. However, cell of origin (GCB versus non-GCB) was predictive of outcome. MYC+/BCL2+ cases segregate with the non-GCB subtype. The overall survival of PMBL cases was 97% at 5 years follow-up. We are currently prospectively studying the DA-EPOCH-R regimen in MYC-rearranged DLBCL in a multicenter study (NCT01092182). Disclosures:No relevant conflicts of interest to declare.

STAT6-mediated BCL6 repression in primary mediastinal B-cell lymphoma (PMBL)

Primary mediastinal B-cell lymphoma (PMBL) is characterized by aberrant activation of JAK/STAT-signaling resulting in constitutive presence of phosphorylated STAT6 (pSTAT6). In primary PMBL samples pSTAT6 is only expressed in a sub-population of lymphoma cells in a pattern that is reminiscent of that of the BCL6 oncogene. Double-fluorescence staining was carried out to determine the association between these two proteins in ten primary PMBL cases and three available PMBL cell line models. Surprisingly, only a minute fraction of double-positive nuclei was observed, while each sample contained considerable fractions of single-positive pSTAT6 and BCL6 nuclei. The intratumoral coexistence of BCL6+/pSTAT6- and BCL6-/pSTAT6+ subpopulations suggests a negative interaction between these factors. In silico screening of the STAT6 /BCL6 promoters for DNA consensus binding sites identified five STAT-binding-sites in the BCL6 promoter. We confirmed STAT6 binding to the BCL6 promoter in vitro and in vivo by band shift / super shift assays and chromatin immunoprecipitations. Using BCL6 luciferase reporter assays, depletion of STAT6 by siRNA, and ectopic overexpression of a constitutive active STAT6 mutant, we proved that pSTAT6 is sufficient to transcriptionally repress BCL6. Recently developed small molecule inhibitors 79-6 and TG101348 that increases BCL6 target gene expression and decreases pSTAT6 levels, respectively, demonstrate that a combined targeting results in additive efficacy regarding their negative effect on cell viability. The delineated pSTAT6-mediated molecular repression mechanism links JAK/STAT to BCL6-signaling in PMBL and may carry therapeutic potential.

Comparative study between primary mediastinal B-cell lymphoma and non-mediastinal diffuse large B-cell lymphoma by immunoglobulin gene rearrangement and Epstein-Barr virus infection detection

To investigate the differences between primary mediastinal B-cell lymphoma (PMBCL) and non-mediastinal conventional diffuse large B-cell common lymphoma (DLBCL) in immunoglobulin gene rearrangement and EB virus infections. Twenty cases of PMBCL and 30 cases of non-mediastinal DLBCL were collected from September, 2000 to May, 2011. Pathological data were retrospectively analysed. Immunoglobulin heavy chain and light chain gene rearrangements and EBER in-situ hybridization were performed. Six of 20 cases of PMBCL showed monoclonal gene rearrangement, all of which were weakly detected. Twenty-seven of 30 cases of ordinary diffuse large B-cell lymphoma showed monoclonal gene rearrangement, which were strongly detected (90.0%). Only 1 of 20 cases PMBCL and 2 of 30 cases of DLBCL were positive for EBER in-situ hybridization. The detection rate of immunoglobulin gene rearrangement is significantly lower in PMBCL than that of non-mediastinal DLBCL. However, EB virus infection rates are very low in both types of lymphomas.

A novel A20 (TNFAIP3) antibody (Ber‐A20) can be used to detect unmutated A20 by immunohistology

A20 (TNFAIP3) is a nuclear factor-κB (NF-κB)-inducible component of tumour necrosis factor and Toll-like receptor intracellular signal transduction. It negatively regulates NF-κB, and has been identified as a tumour suppressor. Several studies have described A20 inactivation by deletion of the A20 locus at 6q23, inactivating mutations, and/or methylation of the A20 promoter in various lymphoma entities. We generated a monoclonal antibody against the C-terminus of A20 (Ber-A20) and investigated full-length A20 expression of normal lymphoid tissue and lymphomas for the first time. We identified loss of A20 expression in tumour cells of 24% of classical Hodgkin lymphoma, 27% of diffuse large B-cell lymphoma, 20% of chronic lymphocytic leukaemia, 19% of follicular lymphoma, 13% of mantle cell lymphoma and 8% of primary mediastinal B-cell lymphoma cases by immunohistology. Loss of A20 expression rarely occurred in T-cell non-Hodgkin lymphoma. Our data are in agreement with cytogenetic and molecular analyses. Among 21 cases of ocular adnexal marginal zone lymphomas with known A20 mutation status, we detected complete absence of A20 expression, whereas cases with wild-type A20 were weakly A20-positive. We demonstrate that A20 loss can be detected by immunohistology with a sensitivity similar to that of complex molecular and genetic methods.

JAK2, As Well As PDL1 and PDL2, are Recurrently Targeted by 9p24 Structural and Numerical Aberrations in Lymphoid Neoplasms of Both B- and T-Cell Origin

Abstract 2460 Introduction:Genetic aberrations of the 9p terminal region are recurrent in hematological malignancies. Many of them involve the JAK2 gene located at 9p24, which is non-randomly targeted by chromosomal translocations, amplifications and mutations in various types of leukemia, lymphoma and myeloproliferative neoplasms. Of interest, recent reports showed that also PDL1 and PDL2, two genes located 350kb centromeric to JAK2, are involved in 9p24 aberrations. These genes, coding for the programmed death ligands, were identified as key targets of 9p24 amplification in primary mediastinal B-cell lymphoma (PMBCL) and classical Hodgkin lymphoma (cHL). Moreover, CIITA-PDL1 and CIITA-PDL2 fusions, leading to overexpression of PDL1 and PDL2, respectively, have been reported in two PMBCL cases. Aims:The objective of this study is to characterize structural and numerical 9p24 aberrations in lymphoid malignancies and to assess the molecular consequences of these abnormalities. Study design:Eighty-four cases of B-cell non-Hodgkin lymphoma (B-NHL) and 4 cases of T-cell NHL with 9p24 aberrations were selected for this study. In addition, we included 130 cases of cHL with unknown 9p24 status. All cases were screened by FISH with a dual color break apart assay for JAK2 containing probes covering PDL1 and PDL2. Cases with rearrangements of these loci were further characterized by FISH with BAC and fosmid probes, and by aCGH. In available cases, qRT-PCR and/or IHC was used to determine the expression level of candidate genes. Results:Structural aberrations of 9p24 were identified in 8 patients presenting with CLL in Richter transformation (1), MALT/MZL (2), FL (1), DLBCL (2) and ALL/LL (2). The 9p24 breakpoints were further mapped to a 200 kb region centromeric to JAK2 and harboring PDL1 and PDL2. These aberrations (translocations, insertion, inversion) involved either immunoglobulin (IG) genes (3 cases), or non-IG sequences at 1p34/PHACTR4, 4p13, 3p25, 6q13/EEF1A1 and 13q11 (5 cases, including one with a three-way translocation also involving IGH). In one case with inv(9)(p24p23), a microdeletion affecting the 3' region of JAK2 and the 5' region of PDL1 was detected by FISH and aCGH. The expression pattern of 5 candidate genes analyzed in 5 available cases showed upregulation of PDL1 and PDL2 in 1 case, overexpression of sole PDL2 in 2 cases and a normal level of analyzed transcripts in 2 cases. In both cases with t(9;14)(p24;q32) translocation of JAK2 to der(14) was demonstrated by FISH. Unfortunately, these cases were not available for qRT-PCR.9p24 amplification hallmarked by homogeneously staining region (hsr) was identified in 10 cases of PMBCL and 4 cases of T-NHL. FISH screening of 130 cases of cHL identified 54 lymphomas with low copy number gains (5–10 copies) and 30 cases with 9p24 amplification, including 6 cases with a predominant amplification of the region covering PDL1 and PDL2. In addition, putative involvement of PDL1/PDL2 in unbalanced translocations associated with loss of JAK2 and telomeric 9p24 sequences was found in 3 cases of cHL.Expression of the JAK2, PDL1 and PDL2 mRNA was analyzed in 7 cases of PMBCL and 2 cases of T-NHL. Among the PMBCL samples, 4 showed overexpression of all 3 transcripts, 2 showed upregulation of PDL1 and PDL2, and one showed overexpression of sole PDL1. JAK2 and PDL1 were found to be highly expressed in one case of T-NHL, while a case of ALK+ ALCL revealed only upregulation of PDL1.IHC analysis of P-JAK2, PDL1 and PDL2 proteins in available cases is ongoing. Conclusions:Our study demonstrates that the 9p24 region is recurrently targeted by structural and numerical aberrations in B- and T-cell leukemia/lymphoma. These abnormalities may represent primary as well as secondary genetic changes acquired during progression of the disease. Structural aberrations predominantly involve PDL1 and/or PDL2 that seem to be deregulated by their juxtaposition with regulatory sequences provided by partner genes. Five postulated PDL1/PDL2 partner genes include IGH/14q32, IGL/22q11, PHACTR4/1p34.3 EEF1A1/6q13 and JAK2/9p24. In the analyzed PMBCL and T-NHL cases, 9p24 amplification constantly targets PDL1 and is randomly associated with an aberrant co-expression of either JAK2 or PDL2. Given the role of PDL1/PDL2 in inhibition of T-cell activation, their impaired expression in B- and T-cell lymphoma may lead to “T-cell exhaustion” and deficient cellular immunity favoring tumor progression. Disclosures:No relevant conflicts of interest to declare.

Inactivating Gene Alterations of MHC Class II Transactivator CIITA Are Recurrent in Primary Mediastinal B Cell Lymphoma and Hodgkin Lymphoma

Abstract 437 INTRODUCTION:Escape from immune surveillance has been proposed to play an important role in cancer and in particular in the pathogenesis of a subgroup of lymphomas. Using massively parallel sequencing, we have recently identified recurrent rearrangements of the CIITA gene in classical Hodgkin lymphoma (HL) and primary mediastinal B cell lymphoma (PMBCL) (Steidl et al, Nature 2011). Specifically, a gene fusion in the HL cell line KM-H2, involving the genes CIITA and FLJ27352, suppressed expression of CIITA-regulated MHC class II genes in a dominant-negative manner. Therefore, we sought to further investigate if gene alterations targeting the CIITA locus are recurrent somatic events in HL and PMBCL that lead to loss of gene function and thereby reduce immunogenicity of the malignant cells. PATIENTS AND METHODS: To characterize CIITA gene alterations, we comprehensively studied the cell lines DEV (nodular lymphocyte predominance HL-derived) and KARPAS1106P (PMBCL-derived) by whole-transcriptome paired-end sequencing (RNA-seq) and single nucleotide polymorphism arrays (Affymetrix SNP 6.0). To identify single nucleotide mutations in the CIITA coding sequence we studied additional 7 mediastinal biopsy specimens of PMBCL by RNA-seq. Fluorescence in-situ hybridization (FISH) was used to determine and validate structural and copy number alterations of the CIITA locus. 23 samples of PMBCL, 3 PMBCL-derived cell lines and 9 HL cell lines were studied for mutations and small deletions in CIITA intron 1 by long-range PCR and direct sequencing. CIITA and HLA-DR expression levels were determined by quantitative reverse transcriptase PCR. RESULTS: We identified chromosomal rearrangements affecting both alleles of CIITA in DEV cells resulting in expression of CIITA-PDL2, CIITA-SOCS1 and SOCS1-CIITA fusion transcripts. In KARPAS1106P cells chromosome 16 deletions result in the loss of one entire CIITA allele and partial loss of the other allele. As a consequence, in both cell lines wildtype CIITA and HLA-DR expression was undetectable. Genomic breakpoints within the CIITA rearrangements, together with previously identified translocations, defined a 1.6Kb breakpoint cluster region in CIITA Intron 1. Further analysis of this breakpoint cluster region revealed small intronic deletions in 10 of 23 (43%) PMBCL cases while no such deletions were detected in 18 diffuse large B cell lymphoma and 15 reactive lymph node samples. Furthermore, sequencing revealed multiple deletions ranging from 1–1948 bp and a high incidence of single nucleotide mutations in the breakpoint cluster region of the deleted alleles. The base pair changes were enriched for C to T and G to A transitions over other transitions and transversions, indicative of a somatic hypermutation process. We found significantly lower CIITA expression in cases with CIITA rearrangement and/or small intronic deletions compared to cases without CIITA alterations (p=0.044). RNAseq revealed three cases with non-synonymous coding-sequence mutations including a truncating mutation in exon 4. These changes were somatic in 3 cases with available matching normal DNA. DISCUSSION:CIITA is the target of multiple genomic hits including chromosomal translocation, deletion and coding sequence mutations in HL and PMBCL. In two HL and PMBCL cell lines this leads to complete gene inactivation and loss of HLA class II expression. These data characterize CIITA alterations as a recurrent underlying genomic event of the previously described immunophenotype of reduced HLA class II expression in a subset of PMBCL cases. Our data show that an immune escape mechanism utilized by tumor cells can be directly linked to somatically acquired alterations in cancer genomes. Disclosures:Siebert:Abbott/Vysis: .

High Rate of Initial Treatment Failure in Patients with Primary Mediastinal B-Cell Lymphoma Treated with R-CHOP

Abstract 1601 Introduction:Primary Mediastinal B-cell Lymphoma (PMBCL) is an uncommon variant of Diffuse Large B-cell Lymphoma (DLBCL). Given the rarity of this disease, data guiding management is extrapolated from DLBCL trials, or from small retrospective analyses limited to PMBCL. Prospective evaluation of R-CHOP in the MiNT trial showed excellent results in PMBCL, but this trial was limited to young low risk patients. We present the largest retrospective series to date of R-CHOP for PMBCL in all risk groups. Methods:We identified cases of PMBCL at our institution using a comprehensive clinicopathologic database derived from tumor registry data. Natural language processing software was used to search pathology reports for terms of “mediastinal lymphoma,” “mediastinal large cell lymphoma,” “mediastinal large B-cell lymphoma,” as well as “lymphoma” in mediastinal biopsy specimens. Cases were included if they met clinicopathologic criteria for PMBCL, defined as a large B-cell lymphoma with typical features for PMBCL presenting with a dominant anterior mediastinal mass. All patients had to have been treated with R-CHOP. Progression-free survival (PFS) and overall survival (OS) are calculated by the Kaplan-Meier method and univariate analysis is performed to assess predictors of outcome. Results:Fifty-eight cases from 2000–2011 met inclusion criteria and are included in the analysis. The median age was 38 years (range 20–82) and 60% were male. Forty-four patients (76%) presented at limited Ann Arbor stage and 12 patients (21%) at advanced stage; presenting stage could not be discerned in 2 patients. Fifty-five percent of patients presented with mediastinal bulk ≥10cm in size; median size was 11cm (range 5–17cm). LDH was elevated at diagnosis in 60% of patients, normal in 21%, and unknown in 19%. By revised IPI score, 19% were low-risk (0 risk factors), 60% were intermediate risk (1–2 risk factors) and 12% were high-risk (≥3 risk factors). R-CHOP was given for a median of 6 cycles (range 1–8); 51 of 58 patients received 6 or 8 cycles. Among patients who achieved initial remission, 78% underwent consolidative radiotherapy and the remainder were observed after chemotherapy alone.The overall response rate was 81% (90%CI [71%–89%]) with 72% complete responses and 9% partial responses. Ten patients (17%) had primary refractory disease defined as progression on treatment or within 3 months of completion of therapy. Among 46 patients who achieved a response, 5 (11%) subsequently relapsed. Two patients, both elderly, died during treatment. Among the 10 patients with primary refractory disease, 6 have died from progressive lymphoma, 2 patients are alive with active disease undergoing salvage therapy, 1 is alive and free of disease greater than 8 years from diagnosis, and 1 was lost to follow-up. Among 5 patients with relapsed disease, 2 are alive without disease at last follow-up, while 3 have died of progressive lymphoma. Median follow-up for the entire series is 58 months. Five-year PFS is 68% (95% CI, 55% to 80%) and 5-year OS is 76% (95% CI, 65% to 88%). On univariate analysis, advanced Ann Arbor stage and high R-IPI score were associated with inferior PFS and OS. (p=0.006 and p<0.001, respectively for PFS, p=0.005 and p<0.001 for OS, log-rank test). Conclusion:PMBCL treated with R-CHOP carries an overall favorable prognosis, though primary refractory disease occurs in a significant number of patients, and is rarely curable with second line therapy. Advanced stage disease and high R-IPI scores are associated with inferior outcome. Novel treatment approaches warrant evaluation in high-risk patients. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Chromosomal Alterations in Gene Expression-Defined Pediatric Aggressive B-Cell Non-Hodgkin Lymphoma (B-NHL).

Abstract 2922Poster Board II-898 Introduction:Lymphomas derived from mature lymphocytes in children and adolescents are predominantly aggressive B-cell non-Hodgkin lymphomas (B-NHL), including Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). Differences in the clinical course between pediatric and adult aggressive B-NHL suggest distinct pathogenetic mechanisms. This study sought to identify both shared and unique genetic alterations between gene expression-defined pediatric and adult cases of BL and DLBCL. Patients and Methods:Gene expression profiling (GEP) was done on 45 BL and 18 DLBCL specimens from patients 18 years of age or younger, and 38 BL and 106 DLBCL from adult patients. Pediatric specimens were collected from the Cooperative Human Tissue Network (CHTN) pediatric NHL repository through the Children's Oncology Group (COG) and adult specimens were collected from the Nebraska Lymphoma Study Group Registry and Tissue Bank through the Lymphoma/Leukemia Molecular Profiling Project (LLMPP). Previously-published gene signatures were used to classify lymphomas molecularly into mBL and mDLBCL groups (Dave et al., NEJM, 2006). The mDLBCL tumors were further classified into activated B-cell-like (ABC), germinal center B-cell-like (GCB), and primary mediastinal B-cell lymphoma (PMBL) subtypes (Rosenwald et al., JEM, 2003). High resolution array comparative genomic hybridization (aCGH) was done on a subset of the pediatric cases using the 250K NspI Human Mapping Array (Affymetrix) to detect DNA copy number alterations (CNA). Results:Molecular classification of the pediatric cases resulted in a 20% reclassification rate for cases with a morphologic diagnosis of BL or DLBCL. Among the 63 pediatric cases, there were 38 mBL (3 of which were DLBCL by morphology), 23 mDLBCL (9 of which were BL by morphology) and two cases which were unclassifiable by the molecular gene signatures. Comparison of the GEP profiles for adult and pediatric mBL failed to identify pathways that differed significantly; however high resolution aCGH analysis revealed a number of abnormalities in the pediatric cases not previously reported in BL, including gains of 3q21, 11q13 and 16p11. A predominance of the GCB to ABC subtype (3:1) was found among pediatric mDLBCL patients. Two cases with mediastinal tumors were classified as PMBL molecularly. Both PMBL cases were female and carried copy number gains of the Rel/BCL11A locus. Comparison of adult and pediatric GCB mDLBCL gene expression revealed enrichment in B-cell surface molecules and markers of antigen-dependent B-cell activation in the adult cases. aCGH analysis identified abnormalities that were both shared (+12q15, +19q13, -6q) between adult and pediatric mDLBCL and unique (-4p14, -19q13.32, +16p11.2) to the pediatric cases. Correlation of DNA copy number and gene expression revealed potential candidate genes for these loci. Conclusions:Pediatric BL and DLBCL classified by morphology were reclassified molecularly in a significant fraction of cases. Although pediatric BL and DLBCL are treated similarly, defining homogeneous molecular entities will be relevant for developing new therapies and future clinical trials. In general, pediatric cases have a more favorable outcome relative to adult patients. However, it is unclear whether this is due to the ability of children to tolerate very intensive therapies or whether distinct pathogenetic mechanisms modulate the disease course. Higher B-cell receptor signaling in adult relative to pediatric GCB DLBCL may be relevant to the outcome. The identification of previously undetected chromosomal alterations unique to the pediatric cases also suggests distinct pathogenetic mechanisms. Elucidation of the underlying genes may provide insight into factors which modulate outcome and could provide novel therapeutic targets with reduced toxicity. Disclosures:Gascoyne:Roche Canada: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.