Burkitt Lymphoma Tumors Research Articles

Inhibition of Hsp90 Suppresses PI3K/AKT/mTOR Signaling and Has Antitumor Activity in Burkitt Lymphoma.

Hsp90 is a molecular chaperone that protects proteins, including oncogenic signaling complexes, from proteolytic degradation. PU-H71 is a next-generation Hsp90 inhibitor that preferentially targets the functionally distinct pool of Hsp90 present in tumor cells. Tumors that are driven by the MYC oncoprotein may be particularly sensitive to PU-H71 due to the essential role of Hsp90 in the epichaperome, which maintains the malignant phenotype in the setting of MYC. Burkitt lymphoma (BL) is an aggressive B-cell lymphoma characterized by MYC dysregulation. In this study, we evaluated Hsp90 as a potential therapeutic target in BL. We found that primary BL tumors overexpress Hsp90 and that Hsp90 inhibition has antitumor activity in vitro and in vivo, including potent activity in a patient-derived xenograft model of BL. To evaluate the targets of PU-H71 in BL, we performed high-affinity capture followed by proteomic analysis using mass spectrometry. We found that Hsp90 inhibition targets multiple components of PI3K/AKT/mTOR signaling, highlighting the importance of this pathway in BL. Finally, we found that the anti-lymphoma activity of PU-H71 is synergistic with dual PI3K/mTOR inhibition in vitro and in vivo Overall, this work provides support for Hsp90 as a therapeutic target in BL and suggests the potential for combination therapy with PU-H71 and inhibitors of PI3K/mTOR. Mol Cancer Ther; 16(9); 1779-90. ©2017 AACR.

Abstract 2656: Polatuzumab vedotin significantly enhances in vitro cell death and overall survival against CD79b+ Burkitt lymphoma (BL)/primary mediastinal large B-cell lymphoma (PMBL) NSG xenograft mice

Abstract Background: Mature B-NHL, including Burkitt lymphoma and primary mediastinal large B cell lymphoma express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood, 2007, Gerrard/Cairo et al. Blood, 2013). However, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease a dismal prognosis (≤ 10% 5 years, EFS) (Cairo et al. JCO, 2012). The antibody drug conjugates (Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et al.Can. Res. 2009). More recently PV has been safe and well tolerated in adult with CD79b refractory CLL (Palanca-Wessels et al. Lancet Oncol, 2014) but its preclinical activity against mature B-NHL (BL/PMBL) is unknown. Objective: To determine the efficacy of the PV against CD79b+ PMBL and rituximab (RTX) sensitive/resistant BL tumor cell lines in-vitro and in-vivo. Design/Methods: Raji/Raji4RH (BL, provided by M. Barth, Roswell Park Cancer Institute) and Karpas1106P and MedB-1(PMBL) were cultured in 10-20% RPMI. Tumor cells were incubated with hu anti-CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and analyzed by flow cytometry, n=3. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), PV (5mg/kg), anti-CD79b mAb (5 mg/kg) and MMAE (5 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji/Raji4RH and Karpas1106P cells as we have previously demonstrated (Awasthi/Cairo et al, BJH, 2015). Mice were treated twice a week for 6 weeks. Tumor burden was monitored by IVIS spectrum system. Results: Anti-CD79b-vc-MMAE compared to anti-CD79b Ab or IgG1 Ab alone (10µg/ml, 24hrs), significantly enhanced cell death in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p=0.0008 and p=0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p=NS and p=0.03), Karpas1106P, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p=0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p=0.002 and 0.0001), respectively. Further, median survival time in mice receiving 5 mg/kg of PV was significantly increased when compared to mice receiving 5 mg/kg of anti-CD79b Ab or isotype control in Raji, Raji4RH and Karpas1106P (35.5 vs.17 vs. 19.5 days, p=0.0001, 0.0003, 50 vs. 18 vs. 18.5 days and 150 vs 89 vs 64 days, p=0.03 ,0.003, respectively) Conclusions: Our preliminary data indicates that PV significantly enhances cell death in RTX sensitive/ resistant BL and PMBL compared to CD79b Ab or isotype control. Furthermore, PV significantly increased survival in BL and PMBL NSG xenografts Citation Format: Aradhana A. Tiwari, Janet Ayello, Christeen Azmy, Carmella van de Ven, Mitchell S. Cairo. Polatuzumab vedotin significantly enhances in vitro cell death and overall survival against CD79b+ Burkitt lymphoma (BL)/primary mediastinal large B-cell lymphoma (PMBL) NSG xenograft mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2656. doi:10.1158/1538-7445.AM2017-2656

Comprehensive Transcriptome and Mutational Profiling of Endemic Burkitt Lymphoma Reveals EBV Type–Specific Differences

Endemic Burkitt lymphoma (eBL) is the most common pediatric cancer in malaria-endemic equatorial Africa and nearly always contains Epstein–Barr virus (EBV), unlike sporadic Burkitt lymphoma (sBL) that occurs with a lower incidence in developed countries. Given these differences and the variable clinical presentation and outcomes, we sought to further understand pathogenesis by investigating transcriptomes using RNA sequencing (RNAseq) from multiple primary eBL tumors compared with sBL tumors. Within eBL tumors, minimal expression differences were found based on: anatomical presentation site, in-hospital survival rates, and EBV genome type, suggesting that eBL tumors are homogeneous without marked subtypes. The outstanding difference detected using surrogate variable analysis was the significantly decreased expression of key genes in the immunoproteasome complex (PSMB9/β1i, PSMB10/β2i, PSMB8/β5i, and PSME2/PA28β) in eBL tumors carrying type 2 EBV compared with type 1 EBV. Second, in comparison with previously published pediatric sBL specimens, the majority of the expression and pathway differences was related to the PTEN/PI3K/mTOR signaling pathway and was correlated most strongly with EBV status rather than geographic designation. Third, common mutations were observed significantly less frequently in eBL tumors harboring EBV type 1, with mutation frequencies similar between tumors with EBV type 2 and without EBV. In addition to the previously reported genes, a set of new genes mutated in BL, including TFAP4, MSH6, PRRC2C, BCL7A, FOXO1, PLCG2, PRKDC, RAD50, and RPRD2, were identified. Overall, these data establish that EBV, particularly EBV type 1, supports BL oncogenesis, alleviating the need for certain driver mutations in the human genome.Implications: Genomic and mutational analyses of Burkitt lymphoma tumors identify key differences based on viral content and clinical outcomes suggesting new avenues for the development of prognostic molecular biomarkers and therapeutic interventions. Mol Cancer Res; 15(5); 563–76. ©2017 AACR.

High-throughput sequencing of the B-cell receptor in African Burkitt lymphoma reveals clues to pathogenesis.

Burkitt lymphoma (BL), the most common pediatric cancer in sub-Saharan Africa, is a malignancy of antigen-experienced B lymphocytes. High-throughput sequencing (HTS) of the immunoglobulin heavy (IGH) and light chain (IGK/IGL) loci was performed on genomic DNA from 51 primary BL tumors: 19 from Uganda and 32 from Ghana. Reverse transcription polymerase chain reaction analysis and tumor RNA sequencing (RNAseq) was performed on the Ugandan tumors to confirm and extend the findings from the HTS of tumor DNA. Clonal IGH and IGK/IGL rearrangements were identified in 41 and 46 tumors, respectively. Evidence for rearrangement of the second IGH allele was observed in only 6 of 41 tumor samples with a clonal IGH rearrangement, suggesting that the normal process of biallelic IGHD to IGHJ diversity-joining (DJ) rearrangement is often disrupted in BL progenitor cells. Most tumors, including those with a sole dominant, nonexpressed DJ rearrangement, contained many IGH and IGK/IGL sequences that differed from the dominant rearrangement by < 10 nucleotides, suggesting that the target of ongoing mutagenesis of these loci in BL tumor cells is not limited to expressed alleles. IGHV usage in both BL tumor cohorts revealed enrichment for IGHV genes that are infrequently used in memory B cells from healthy subjects. Analysis of publicly available DNA sequencing and RNAseq data revealed that these same IGHV genes were overrepresented in dominant tumor-associated IGH rearrangements in several independent BL tumor cohorts. These data suggest that BL derives from an abnormal B-cell progenitor and that aberrant mutational processes are active on the immunoglobulin loci in BL cells.

Polatuzumab Vedotin; Antibody Drug Conjugates (ADC, anti-CD79b-vc-MMAE, PV) Significantly Enhances in-Vitro Cell Death Against CD79b+ Burkitt Lymphoma (BL) /Primary Mediastinal Large B Cell Lymphoma (PMBL) and Improved Overall Survival Against Rituximab Sensitive/Resistant-BL NSG Xenograft Mice

Background: Pediatric Burkitt Lymphoma (PBL) represents the most common malignancy in childhood and adolescent non-Hodgkin Lymphoma (NHL) but occurs in less than 5% of adult NHL (Hochberg/Cairo et al, BJH 2009; Miles/Cairo, BJH. 2012). Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood. 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al. Blood. 2013). The prognosis of PBL has significantly improved over 30 years through the use of short and intense multi-agent chemotherapy, however, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease and a dismal prognosis (≤ 20% 5 years, EFS,Cairo et al.Blood. 2007; Cairo et al. JCO.2012, Miles/Cairo et al. BJH. 2012). It is therefore critical to investigate and to develop targeted translational strategies in BL/PMBL in order to reduce acute morbidities, decrease late effects, and provide new options for those with recurrent disease.The anti-CD79b-vc-MMAE antibody drug conjugates (ADC, Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res. 2009; Dornan et al. Blood. 2009). More recently PV has been safe and well tolerated in adults with CD79b refractory NHL (Palanca-Wessels et al. Lancet Oncol.2015), However, PV preclinical activity against mature BL/PMBL is unknown.Objective: To determine the efficacy of the ADC (anti-CD79b-vc-MMAE) against CD79b+ PMBL in-vitro and rituximab (RTX) sensitive/resistant BL tumor cell lines in-vitro/ in-vivo.Methods: Raji/Raji4RH (provided by M. Barth, Roswell Park Cancer Institute) and PMBL: Karpas1106P and MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with hu anti- CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and cell proliferation was analyzed by alamar blue by flow-cytometry, n=3. Six to 8 week old female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ), were divided into 5 groups: PBS only (control), isotype control (IgG), PV (5mg/kg), anti-CD79B mAb (5 mg/kg) and MMAE (5 mg/kg). Mice were xenografted with intravenous injections of Luc+ Raji and Raji4RH cells at 5x106 tumor cells/mouse as we have previously demonstrated (Awasthi/Cairo et al, BJH, 2015). Mice were treated twice a week for 6 weeks. Tumor burden was monitored by IVIS spectrum system.Results: Hu- anti -CD79b-vc-MMAE compared to hu anti-CD79b Ab or control hu IgG1 Ab alone (10µg/ml, 24hrs), significantly enhanced cell death ( apoptosis) in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p=0.0008 and p=0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p=NS and p=0.03), Karpas1106P, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p=0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p=0.002 and 0.0001), respectively. Hu anti- CD79b-vc-MMAE also significantly reduced cell proliferation compared to hu anti- CD79b Ab or control hu anti -IgG1 Ab alone (20µg/ml, 24hrs). Raji, 56.1±5.1% vs 38.1±0.7% vs. 14.8±0.4% (p=0.001 and p=0.0008), Raji4RH, 53.4±5.4% vs 23.4±5.51% vs. 11.3±0.8% (p=0.004 and 0.006), Karpas1106P, 46.4±0.3 %vs 29.0±1.5% vs. 15.9±0.6% (p=0.005 and 0.00007) and MedB-1, 47.2±7.5% vs 27.7±8.5% vs. 12.3±0.5% (p=0.01 and p=0.0005), respectively. Further, median survival time in mice receiving 5 mg/kg of PV was significantly increased when compared to mice receiving 5 mg/kg of anti-CD79b antibody or IgG isotype control in Raji and Raji4RH ( 35.5 vs.17 vs. 19.5 days, p=0.0001, 0.0003 and 50 vs. 18 vs. 18.5 days, p=0.0001, 0.0001, respectively, Figure. A and B)Conclusion: Our preliminary data indicates thathu anti- CD79b-vc-MMAE significantly enhances cell death and reduced cell proliferation in RTX sensitive/ and resistant CD79b+ BL and PMBL compared to CD79b Ab or isotype control IgG1 Ab alone. Furthermore, PV significantly increased survival in BL (RTX sensitive/ resistant) NSG xenografts compared to an equal dose of anti-CD79b alone. [Display omitted] DisclosuresCairo:Celgene: Research Funding.

Comprehensive Genomic Analysis of Adult Burkitt Lymphoma Identifies the B-Cell Receptor Signaling Pathway As a Potential Therapeutic Target

Burkitt lymphoma (BL) is the most common non-Hodgkin lymphoma (NHL) in children. Although it accounts for only 1-5% of NHL in adults, approximately 60% of the BL cases diagnosed each year in western countries occur in patients >40 years of age. Although adult and pediatric BL cases are indistinguishable by molecular classification, pediatric patients have a significantly better outcome than adults. While translocation of MYC to the immunoglobulin heavy or light chain genes is characteristic of pediatric and adult BL, genetic differences may contribute to the superior clinical outcome of childhood cases.Therefore, we aimed to identify the spectrum of additional genetic abnormalities that occur in adult and pediatric BL. Copy number analysis, gene expression profiling (GEP), and targeted sequencing of ~400 genes known to be mutated in NHLs were performed on a cohort of molecularly defined BL samples. Copy number abnormalities (CNAs) were identified by the Affymetrix 250k NspI SNP array in 73 BL tumors (28 adult, 45 pediatric), and sequencing was performed on 52 BLs (21 adult, 31 pediatric). Pediatric cases had fewer CNAs than adults. The most common focal abnormality identified was a gain on 13q31.3 encompassing MIR17HG. It was more frequent in adult compared to pediatric cases (35% vs 16%, p=0.085) and was associated with increased expression of miR-17~92 cluster members; and among adults, patients with this gain trended towards worse overall survival, though the number of cases with available information was small. Gain of 8q was found in ~20% of adult cases, but in no pediatric cases. Surprisingly, cases with 8q gain had significantly lower MYC mRNA expression (p< 0.001) and lower protein expression. In cases with MYC gain 0/4 cases were positive for MYC protein expression by immunohistochemistry; in contrast,6/10 cases with no MYC gain were positive for MYC expression. This suggests that gain of 8q is driven by another gene in the region. Additional genetic alterations included gains of genomic loci encompassing MCL1 and MDM4 (1q21-24) and losses encompassing RB1, p53 and CDKN2A/CDKN2B. Pathway analysis of genes differentially expressed by CN status showed an enrichment of genes involved in cell cycle regulation, the p53 signaling pathway, and the ubiquitin proteasome pathway.The frequencies of mutations in commonly mutated genes including MYC, ID3, TP53, CCND3, DDX3X, ARID1A, and TCF3 were not significantly different in adult and pediatric BL. However, BCL2, (43%, p<0.001), ZFHX3 (24%, p<0.01), SPTBN5 (20%, p=0.02), RB1 (14%, p=0.06), BTG1 (14%, p=0.06), TCF4 (14%, p=0.06), and TNFRSF14 (14%, p=0.06), were exclusively mutated in adult BL. In contrast, mutations in CDH23 (29% vs 5%, p=0.04) and SMARCA4 (35% vs 19%, p=0.05) were more frequent in pediatric BL. When mutations were placed into oncogenic pathways, mutations in genes regulating the PI3K-AKT pathway did not shown significant differences between adult and pediatric cases. Mutations promoting tonic BCR signaling (TCF3 and ID3) by activation of the PI3K pathway had similar frequencies in the two age groups, however, BCR signaling effectors inducing chronic active NF-kB signaling (CD79A, SYK, MYD88, BCL10, CARD11) were significantly associated with adult BL (adult cases with any mutation: 19% vs 7%).Gene expression studies suggested activated BCR signaling in BL cases with CN gain of miR-17~92. In vitro analysis of miR17~92 in BL cell lines (n=4) showed that functional loss of miR-17 ~ 92 expression using a miRNA sponge led to reduced proliferation. Treatment of BL cell lines with anti-IgM induced BCR activation in a time- and dosage-dependent manner, as estimated by increased phosphorylation of downstream mediators (SYK and BLNK). This activation was reduced upon loss of functional miR-17 ~ 92 expression in cell lines. Since miR-17~92 can directly inhibit proximal negative regulators of BCR signaling, treatment with the FDA-approved BTK inhibitor, Ibrutnib, further inhibited proliferation of BL cell lines carrying the miRNA sponge. The BCR signaling pathway is one example of how unique abnormalities in adult BL can provide possible targets for therapeutic intervention. DisclosuresNo relevant conflicts of interest to declare.

Abstract 579: Antibody drug conjugates (anti-CD79b-vc-MMAE, Polatuzumab Vedotin) exhibit enhanced cell death targeted to CD79b+ Burkitt lymphoma (BL) and primary mediastinal large B-cell lymphoma (PMBL)

Abstract Background: Mature B-NHL, including Burkitt lymphoma (BL) and primary mediastinal large B cell lymphoma (PMBL) express CD79b+ and have an excellent prognosis with chemo-immunotherapy (Cairo et al Blood, 2007,Goldman/Cairo et al. Leukemia, 2013, Gerrard/Cairo et al.Blood, 2013). However, a subset of patients with relapsed/refractory mature B-NHL has chemoimmunotherapy resistant disease a dismal prognosis (≤ 10% 5 years, EFS) (Cairo et al. JCO, 2012, Miles/Cairo et al. BJH, 2012). The anti-CD79b-vc-MMAE antibody drug conjugates (Polatuzumab Vedotin, PV) has demonstrated significant preclinical activity against indolent CD79b+NHL (Polson et. al.Can. Res. 2009, Dornan et al. Blood, 2009). More recently PV has been safe and well tolerated in adult with CD79b refractory NHL (Palanca-Wessels et al. Lancet oncol, 2015) but its preclinical activity against mature BL/PMBL is unknown. Objective: To determine the efficacy of ADC (anti-CD79b-vc-MMAE) against RTX sensitive/resistant CD79+ BL and PMBL tumor cell lines in-vitro. Methods: BL: Raji/Raji4RH (provided by M. Barth, Roswell Park Cancer Institute) and PMBL: Karpas1106p and MedB-1 were cultured in RPMI with 10 or 20% FBS. Tumor cells were incubated with hu anti- CD79b-vc-MMAE, and/or anti-CD79b, MMAE or huIgG1 (generously supplied by Genentech Inc.) for 24 hrs. Cell death was evaluated by staining with annexin V/7AAD and cell proliferation was analyzed by alamar blue by flow-cytometry, n = 3. Results: Hu- anti -CD79b-vc-MMAE compared to hu anti-CD79b Ab or control hu IgG1 Ab alone (10μg/ml, 24hrs), significantly enhanced cell death (apoptosis) in Raji, 47.2±1.3% vs 29.1±6.0% vs. 28.2±4.3%, (p = 0.0008 and p = 0.00006), Raji4RH, 29.8±9.1% vs 25.4±3.9% vs. 18.0±8.2% (p = NS and p = 0.03), Karpas1106p, 46.8±5.3% vs 33.8±3.5% vs. 26.2±0.4% (p = 0.02 and 0.006) and MedB-1, 47.4±2.2% vs 27.6±2.4% vs. 23.9±1.7% (p = 0.002 and 0.0001), respectively. Hu anti- CD79b-vc-MMAE also significantly reduced cell proliferation compared to hu anti- CD79b Ab or control hu anti -IgG1 Ab alone (20μg/ml, 24hrs). Raji, 56.1±5.1% vs 38.1±0.7% vs. 14.8±0.4% (p = 0.001 and p = 0.0008), Raji4RH, 53.4±5.4% vs 23.4±5.51% vs. 11.3±0.8% (p = 0.004 and 0.006), Karpas1106p, 46.4±0.3%vs 29.0±1.5% vs. 15.9±0.6% (p = 0.005 and 0.00007) and MedB-1, 47.2±7.5% vs 27.7±8.5% vs. 12.3±0.5% (p = 0.01 and p = 0.0005), respectively. Conclusion: Our preliminary data indicates that hu anti- CD79b-vc-MMAE significantly enhances cell death and reduced cell proliferation in sensitive/ RTX resistant CD79b+ BL and PMBL compared to CD79b Ab or isotype control IgG1 Ab alone. Hu anti- CD79b-vc-MMAE may be a novel agent to investigate as immunotherapeutic agent in patients with relapsed refractory CD79b+ BL and/or PMBL. Citation Format: Aradhana Awasthi Tiwari, Janet Ayello, Carmella van de Ven, Lisa Kurien, Matthew J. Barth, Mitchell S. Cairo. Antibody drug conjugates (anti-CD79b-vc-MMAE, Polatuzumab Vedotin) exhibit enhanced cell death targeted to CD79b+ Burkitt lymphoma (BL) and primary mediastinal large B-cell lymphoma (PMBL). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 579.

Comparative Analysis of Endemic and Sporadic Burkitt Lymphoma By RNA Sequencing

Introduction: Burkitt lymphoma (BL) is an aggressive B-lineage non-Hodgkin lymphoma that has traditionally been classified into 3 subtypes: "endemic", "sporadic", and HIV-associated. The highest incidence of BL - prompting the "endemic" classification - is observed in children in sub-Saharan Africa, where the distribution of the disease is closely associated with that of P. falciparum malaria. The tumor cells in most cases of BL from sub-Saharan Africa are infected with Epstein Barr virus (EBV). In contrast, a minority of BL tumors from Europe and North America - which are commonly described as "sporadic" - are EBV positive. Although excellent outcomes have been observed for patients with BL treated with multi-agent chemotherapy in Europe and North America, the outcomes of BL patients in sub-Saharan Africa are suboptimal. We hypothesized that improved understanding of the molecular heterogeneity that exists within BL, particularly the molecular features that distinguish cases from sub-Saharan Africa from those from Europe/North America, will enable more effective treatment strategies that can readily be implemented in low-resources settings.Methods: Tumor biopsies were collected from 19 pediatric patients who presented to the Uganda Cancer Institute with maxillofacial tumors histologically confirmed to be BL. Total RNA was extracted and polyA-selected sequencing libraries were prepared. Paired-end, 50-base pair sequencing was performed on the Illumina HiSeq 2500 platform at a depth of 100 million reads per sample. A publicly available RNA sequencing dataset from 28 BL cases from Europe and North America that were previously analyzed by both microarray (NEJM 2006; 354[23]: 2431-2442) and RNA sequencing (Nature 2012; 490[7418]: 116-120) was analyzed in parallel for comparison. RNA sequencing on the 28 sporadic BL tumors was performed on polyA-selected sequencing libraries with paired-end, 108-base pair sequence reads generated on the Illumina HiSeq 2000 platform. The reads from all 47 BL tumors were aligned to the human and EBV (GenBank ID KC207813.1) genomes using the STAR aligner. Tumors were deemed EBV positive if the ratio of mapped viral reads to human reads exceeded 0.001%, since the EBV genome is 0.005% the size of the human genome. Normalization and differential expression analysis were performed on aligned reads using the DESeq2 R package. Somatic variants were identified by the Genome Analysis Tool Kit. Analysis of alterative splicing was performed with the SGSeq R package.Results: All of the Uganda BL cases but only 70% of the previously published European/North American cases were obtained from children less than 18 years; 61% and 87% were from male patients, respectively. One half of patients in both groups presented with Ann Arbor stage I or II disease, while the remainder presented with higher stage disease. The median survival in the 19 Ugandan patients was less than one year. Sequence reads from 15 (79%) of the Ugandan tumors and 4 (15%) of the European/North American tumors aligned to the EBV genome. Although BL is reported to express only latency type I EBV genes, expression of latency type I, II, and III genes was detected. Among the 4 tumors from Uganda that did not contain EBV-derived reads, 1 was from a child who was HIV-seropositive, while the others were from HIV-seronegative patients. Unsupervised hierarchical clustering and principal component analysis of all annotated genes failed to separate EBV positive and EBV negative tumors. Cluster analysis of the Ugandan tumors revealed no association with clinical variables. Ongoing analyses are being performed to identify associations between differentially expressed genes and somatic mutations and alternative splicing that may distinguish the tumor subtypes.Conclusions: While the terms "endemic" and "sporadic" have traditionally been used to classify BL, this nomenclature is archaic and does not accurately capture the underlying biology of the tumor subtypes. We performed a comparative whole transcriptome analysis of BL tumors from patients in Uganda to a dataset of BL tumors from patients in Europe/North America to identify features that distinguish the two cohorts. While cluster analysis failed to clearly separate tumors by their EBV or clinical status, ongoing analyses are being performed to identify alterations that may contribute to their distinct gene expression profiles. DisclosuresNo relevant conflicts of interest to declare.

Epstein-Barr virus from Burkitt Lymphoma biopsies from Africa and South America share novel LMP-1 promoter and gene variations.

Epstein Barr virus (EBV) sequence variation is thought to contribute to Burkitt lymphoma (BL), but lack of data from primary BL tumors hampers efforts to test this hypothesis. We directly sequenced EBV from 12 BL biopsies from Ghana, Brazil, and Argentina, aligned the obtained reads to the wild-type (WT) EBV reference sequence, and compared them with 100 published EBV genomes from normal and diseased people from around the world. The 12 BL EBVs were Type 1. Eleven clustered close to each other and to EBV from Raji BL cell line, but away from 12 EBVs reported from other BL-derived cell lines and away from EBV from NPC and healthy people from Asia. We discovered 23 shared novel nucleotide-base changes in the latent membrane protein (LMP)-1 promoter and gene (associated with 9 novel amino acid changes in the LMP-1 protein) of the 11 BL EBVs. Alignment of this region for the 112 EBV genomes revealed four distinct patterns, tentatively termed patterns A to D. The distribution of BL EBVs was 48%, 8%, 24% and 20% for patterns A to D, respectively; the NPC EBV’s were Pattern B, and EBV-WT was pattern D. Further work is needed to investigate the association between EBV LMP-1 patterns with BL.

408. CD19-Targeted CAR T Cells “Armored” To Secrete IL-12 Demonstrate Superior Efficacy Against B Cell Acute Lymphoblastic Leukemia and Resist Treg Immunosuppression

For an adoptive T cell immunotherapy to successfully treat cancer, T cells must be able to recognize the tumor antigen, expand and persist in the tumor microenvironment, and deliver its effective function. Treatment of B cell acute lymphoblastic leukemia (B-ALL) using human T cells modified to express a “second generation” CD19-targeted chimeric antigen receptor (CAR), which includes the CD28 co-stimulatory signaling domain (19-28z) along with the CD3 zeta chain signaling domain, has been successfully demonstrated in both the pre-clinical and clinical setting. However, application of CAR therapy for the treatment of B cell chronic lymphocytic leukemia (B-CLL) and solid tumors remains a challenge. Here, we present preclinical studies demonstrating that further modifying 19-28z T cells to additionally secrete a recombinant fusion protein of the proinflammatory interleukin 12 (IL-12) (19-28z/IL-12 T cells) enhances the potency of adoptive T cell immunotherapy. We demonstrate that these 19-28z/IL-12 “armored” CAR T cells display increased proliferation, enhanced persistence, up-regulation of perforin and granzyme B, as well as a central memory-like phenotype when compared to T cells transduced to express the 19-28z CAR alone. In vivo, treatment of systemic NALM-6 B-ALL tumor-bearing SCID-Beige mice with 19-28z/IL-12 CAR T cells significantly improved survival when compared to mice treated with T cells modified to express the 19-28z CAR alone. Furthermore, in contrast to T cells modified to express the 19-28z CAR alone, 19-28z/IL-12 T cells resist CD4+CD25hiFOXP3+ Treg-mediated immunosuppression in vitro and successfully eradicate Raji Burkitt's lymphoma tumor in SCID-Beige tumor-bearing mice previously infused with CD19-targeted CAR modified Tregs. These findings have important implications for future clinical trials with CAR T cells designed to enhance the clinical outcomes of relapsed B-CLL as well as solid tumor malignancies.

Deregulation of DNMT1, DNMT3B and miR-29s in Burkitt lymphoma suggests novel contribution for disease pathogenesis

Methylation of CpG islands in promoter gene regions is frequently observed in lymphomas. DNA methylation is established by DNA methyltransferases (DNMTs). DNMT1 maintains methylation patterns, while DNMT3A and DNMT3B are critical for de novo DNA methylation. Little is known about the expression of DNMTs in lymphomas. DNMT3A and 3B genes can be regulated post-transcriptionally by miR-29 family. Here, we demonstrated for the first time the overexpression of DNMT1 and DNMT3B in Burkitt lymphoma (BL) tumor samples (69% and 86%, respectively). Specifically, the treatment of two BL cell lines with the DNMT inhibitor 5-aza-dC decreased DNMT1 and DNMT3B protein levels and inhibited cell growth. Additionally, miR-29a, miR-29b and miR-29c levels were significantly decreased in the BL tumor samples. Besides, the ectopic expression of miR-29a, miR-29b and miR-29c reduced the DNMT3B expression and miR-29a and miR-29b lead to increase of p16INK4a mRNA expression. Altogether, our data suggest that deregulation of DNMT1, DNMT3B and miR29 may be involved in BL pathogenesis.

Elevation of c-MYC disrupts HLA class II-mediated immune recognition of human B cell tumors.

Elevated levels of the transcription factor c-myc are strongly associated with various cancers, and in particular B cell lymphomas. Although many of c-MYC's functions have been elucidated, its effect on the presentation of Ag through the HLA class II pathway has not been reported previously. This is an issue of considerable importance, given the low immunogenicity of many c-MYC-positive tumors. We report in this paper that increased c-MYC expression has a negative effect on the ability of B cell lymphomas to functionally present Ags/peptides to CD4(+) T cells. This defect was associated with alterations in the expression of distinct cofactors as well as interactions of antigenic peptides with class II molecules required for the presentation of class II-peptide complexes and T cell engagement. Using early passage Burkitt's lymphoma (BL) tumors and transformed cells, we show that compared with B lymphoblasts, BL cells express decreased levels of the class II editor HLA-DM, lysosomal thiol-reductase GILT, and a 47-kDa enolase-like protein. Functional Ag presentation was partially restored in BL cells treated with a c-MYC inhibitor, demonstrating the impact of this oncogene on Ag recognition. This restoration of HLA class II-mediated Ag presentation in early passage BL tumors/cells was linked to enhanced HLA-DM expression and a concurrent decrease in HLA-DO in BL cells. Taken together, these results reveal c-MYC exerts suppressive effects at several critical checkpoints in Ag presentation, which contribute to the immunoevasive properties of BL tumors.

Burkitt's Lymphoma.

Endemic Burkitt's lymphoma (BL) remains the most prevalent pediatric cancer in sub-Saharan Africa even though it was the first human cancer with a viral etiology described over 50years ago. Epstein-Barr virus (EBV) was discovered in a BL tumor in 1964 and has since been implicated in other malignancies. The etiology of endemic BL has been linked to EBV and Plasmodium falciparum malaria co-infection. While epidemiologic studies have yielded insight into EBV infection and the etiology of endemic BL, the modulation of viral persistence in children by malaria and deficits in EBV immunosurveillance has more recently been reified. Renewed efforts to design prophylactic and therapeutic EBV vaccines provide hope of preventing EBV-associated BL as well as increasing the ability to cure this cancer.

Abstract 4202: Deciphering the effects of GNA13 mutations in B-cell lymphomas

Abstract G-proteins and their cognate G-protein coupled receptors (GPCRs) are critical signal transduction molecules that regulate cell survival, proliferation, motility and differentiation. The aberrant expression or function of these molecules has been linked to growth, progression and metastasis of various cancers. Recent genome-wide sequencing efforts have unveiled numerous previously uncharacterized mutations in G-proteins and GPCRs, which could have significant implications to cancer initiation and progression. Of interest, recent analyses have demonstrated that there are frequent mutations in GNA13 in Burkitt's lymphoma and Diffuse Large B cell lymphomas (DLBCL) that are highly statistically significant over background cancer gene mutation rate, and occur in ∼10-13% of patients with these lymphomas. Mutations in the downstream target of G13, RhoA, are also present in several Burkitt's lymphoma tumors, highlighting that mutations in this G13-RhoA pathway may be key to the oncogenic potential of these B cell lymphomas. Although G13 activity has previously been linked cellular transformation and metastatic potential of epithelial cancers, our data suggests that the mutations in GNA13 in these B cell lymphomas are mostly inhibitory in nature. Thus, we have functionally characterized several mutations in GNA13 and RHOA found in Burkitt's lymphoma and DLBCL. Citation Format: Morgan L. O'Hayre, Irina Kufareva, Jose P. Vaqué, Miguel A. Piris, J. Silvio Gutkind. Deciphering the effects of GNA13 mutations in B-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4202. doi:10.1158/1538-7445.AM2014-4202

Inhibition of lymphoma vascularization and dissemination by estrogen receptor β agonists

AbstractMost lymphomas show an increased incidence and poorer prognosis in males vs females, suggesting endocrine regulation. We have previously shown that tumor growth in vivo of a murine T-cell–derived lymphoma is repressed following activation of estrogen receptor β (ERβ, ESR2). By using ERβ-deficient mice, we now demonstrate that this inhibition is mediated via a direct effect on the tumor cells and not on the microenvironment. Furthermore, we show that the growth-suppressing effects of ERβ agonist are also valid for human B-cell lymphomas as demonstrated in tumors derived from Granta-519 mantle cell lymphoma (MCL) and Raji Burkitt lymphoma (BL) cells. In Granta-519 MCL tumors, activation of ERβ reduced expression of BAFF and GRB7, 2 important molecules involved in B-cell proliferation and survival. Importantly, activation of ERβ inhibited angiogenesis and lymphangiogenesis, possibly mediated by impaired vascular endothelial growth factor C expression. Furthermore, using disseminating Raji BL cells, we show that ERβ activation reduces dissemination of grafted Raji BL tumors. We also show by immunohistochemistry that ERβ is expressed in primary MCL tissue. These results suggest that targeting ERβ with agonists may be valuable in the treatment of some lymphomas, affecting several aspects of the malignant process, including proliferation, vascularization, and dissemination.

Characterizing The CpG Methylation Of Epstein-Barr Virus DNA In The Plasma Of Patients With Hodgkin Lymphoma and HIV-Associated Burkitt Lymphoma

BackgroundEpstein-Barr virus (EBV) is harbored as latent virus within the tumor cells of a subset of classical Hodgkin lymphoma (HL) and HIV-associated Burkitt lymphoma (BL). In HL, we and others have previously shown that elevated EBV DNA copies in plasma accurately distinguish EBV(+) tumors from EBV(-) tumors. However, EBV DNA may be present in plasma due to apoptosis of latently infected EBV(+) tumor cells (or latent EBV(+) benign lymphocytes), as a result of replicative lytic infection (particularly in immunocompromised hosts), or both. Thus, detecting elevated EBV DNA copies in plasma by does not always signify the presence of an EBV(+) tumor. However, a distinction is that the EBV DNA derived from latently infected cells is replicated by cellular DNA polymerase and is usually CpG methylated, whereas EBV DNA in lytic infection is replicated by the viral DNA polymerase and is never CpG methylated. MethodsTo improve upon plasma EBV DNA as a tumor-specific marker in EBV(+) lymphomas, we assessed the CpG methylation of EBV DNA in a subset of plasma specimens from immunocompetent patients with HL (enrolled in the Eastern Cooperative Oncology Group E2496 trial) and a subset from patients with HIV-associated BL (enrolled in the AIDS Malignancy Consortium 048 trial). The EBV tumor status was determined by in situ hybridization (ISH) on tissue for EBV RNAs (EBER). For the CpG methylation assay, methyl binding domain paramagnetic beads were used to first fractionate CpG methylated DNA from CpG unmethylated DNA in plasma. EBV DNA in each fraction were quantified by real time polymerase chain reaction (qPCR) to determine the percentage of methylated EBV DNA. Specimens with > 100 EBV DNA copies/mL plasma were analyzed. ResultsAmong patients with EBER(+) HL and elevated EBV DNA in plasma prior to therapy (n=8), EBV DNA was largely CpG methylated (median 95%, range 54%-99%). By contrast, in the rare cases where patients with EBER(-) HL tumors had high EBV DNA copy number in plasma prior to therapy (4 of 92, 4%), only small percentages of EBV DNA were methylated in 3 patients (5%, 9%, and 12%). In the fourth patient, the plasma EBV DNA was 99% CpG methylated. We suspect that patient 4 had an EBV-associated HL with either a technical failure of EBER-ISH or an instance of a tumor that harbors viral DNA but does not express EBERs. In patients with HIV-associated BL, plasma EBV DNA copies were elevated in 100% of patients with EBER(+) BL (n=4), but also in 4 of 9 (56%) patients with EBER(-) BL. This propensity to detect EBV DNA in the plasma of patients with HIV but without EBV(+) BL likely reflects replicative lytic infection in immunocompromised hosts, making plasma EBV DNA less reliable as a marker of EBV(+) lymphoma in this setting. Thus, we applied the methylation assay to specimens from patients with HIV and BL. Patients with HIV and EBER(+) BL tumors (n=4), EBV DNA was 28%, 46%, 47%, and 60% methylated, whereas a patient with HIV and EBER(-) BL had lower methylation at 8%. To better elucidate the meaning of plasma EBV DNA elevations during lymphoma treatment, we applied the methylation assay to specimens from patients with EBV(+) HL and elevated EBV DNA in plasma at month 6 of therapy (n=4). Dense CpG methylation (96%) of plasma EBV DNA was detected in 1 patient who went on to relapse at Month 15, while the plasma EBV DNA was largely unmethylated in 3 patients who remain in remission each with over 5 years of follow-up. ConclusionIn settings where EBV DNA is detected in plasma, the CpG methylation assay helps differentiate patients with EBV(+) HL or BL from those with EBV(-) tumors. In patients with HIV where lytic EBV replication can contribute greatly to elevations of EBV DNA in plasma, the fractionated quantification of plasma EBV DNA by the methylation assay aids in distinguishing EBV DNA that is tumor-derived from that which is the result of lytic replication. With growing interest in plasma EBV DNA as a serial marker of disease activity in EBV(+) lymphomas, the methylation assay is promising in its ability to differentiate patients with elevated EBV DNA in plasma but ongoing remission from those in whom plasma EBV DNA is tumor-derived and an early harbinger of relapse. Disclosures:No relevant conflicts of interest to declare.

Epstein–Barr virus patterns in US Burkitt lymphoma tumors from the SEER residual tissue repository during 1979–2009

Burkitt lymphoma (BL) occurs at all ages, but the patterns of Epstein-Barr virus (EBV) positivity in relation to human immunodeficiency virus (HIV), immunoprofiles and age have not been fully explored. BL tissues from residual tissue repositories, and two academic centers in the United States were examined by expert hematopathologists for morphology, immunohistochemistry, MYC rearrangement, EBV-encoded RNA (EBER), and diagnosed according to the 2008 WHO lymphoma classification. Analysis was done using frequency tables, Chi-squared statistics, and Student's t-test. Of 117 cases examined, 91 were confirmed as BL. The age distribution was 26%, 15%, 19%, and 29% for 0-19, 20-34, 35-59, 60+ years, and missing in 11%. MYC rearrangement was found in 89% and EBER positivity in 29% of 82 cases with results. EBER positivity varied with age (from 13% in age group 0-19 to 55% in age group 20-34, and fell to 25% in age group 60+ years, p = 0.08); with race (56% in Blacks/Hispanics vs 21% in Whites/Asians/Pacific Islanders, p = 0.006); and by HIV status (64% in HIV positive vs 22% in HIV negative cases, p = 0.03). EBER positivity was demonstrated in about one-third of tumors and it was strongly associated with race and HIV status, and marginally with age-group.

The genetic landscape of mutations in Burkitt lymphoma

Burkitt lymphoma is characterized by deregulation of MYC, but the contribution of other genetic mutations to the disease is largely unknown. Here, we describe the first completely sequenced genome from a Burkitt lymphoma tumor and germline DNA from the same affected individual. We further sequenced the exomes of 59 Burkitt lymphoma tumors and compared them to sequenced exomes from 94 diffuse large B-cell lymphoma (DLBCL) tumors. We identified 70 genes that were recurrently mutated in Burkitt lymphomas, including ID3, GNA13, RET, PIK3R1 and the SWI/SNF genes ARID1A and SMARCA4. Our data implicate a number of genes in cancer for the first time, including CCT6B, SALL3, FTCD and PC. ID3 mutations occurred in 34% of Burkitt lymphomas and not in DLBCLs. We show experimentally that ID3 mutations promote cell cycle progression and proliferation. Our work thus elucidates commonly occurring gene-coding mutations in Burkitt lymphoma and implicates ID3 as a new tumor suppressor gene.

Burkitt lymphoma pathogenesis and therapeutic targets from structural and functional genomics

Burkitt's lymphoma (BL) can often be cured by intensive chemotherapy, but the toxicity of such therapy precludes its use in the elderly and in patients with endemic BL in developing countries, necessitating new strategies. The normal germinal centre B cell is the presumed cell of origin for both BL and diffuse large B-cell lymphoma (DLBCL), yet gene expression analysis suggests that these malignancies may use different oncogenic pathways. BL is subdivided into a sporadic subtype that is diagnosed in developed countries, the Epstein-Barr-virus-associated endemic subtype, and an HIV-associated subtype, but it is unclear whether these subtypes use similar or divergent oncogenic mechanisms. Here we used high-throughput RNA sequencing and RNA interference screening to discover essential regulatory pathways in BL that cooperate with MYC, the defining oncogene of this cancer. In 70% of sporadic BL cases, mutations affecting the transcription factor TCF3 (E2A) or its negative regulator ID3 fostered TCF3 dependency. TCF3 activated the pro-survival phosphatidylinositol-3-OH kinase pathway in BL, in part by augmenting tonic B-cell receptor signalling. In 38% of sporadic BL cases, oncogenic CCND3 mutations produced highly stable cyclin D3 isoforms that drive cell cycle progression. These findings suggest opportunities to improve therapy for patients with BL.

TLR9 agonists induced cell death in Burkitt's lymphoma cells is variable and influenced by TLR9 polymorphism

Toll-like receptor 9 (TLR9) triggering is a promising novel strategy to combat cancer as it induces innate and adaptive immunity responses. B-cell lymphoma is unique in this context as tumor cells express TLR9 and may harbor latent Epstein-Barr virus (EBV), a gamma-herpesvirus with remarkable oncogenic potential when latent. Latent EBV may be promoted by TLR9 triggering via suppression of lytic EBV. Here, we elaborated an initial assessment of the impact of TLR9 triggering on EBV-positive and EBV-negative B-cell lymphoma using Burkitt's lymphoma (BL) cell lines as an in vitro model. We show that, independent of the presence of EBV, the TLR9 ligand oligodeoxynucleotide (ODN) CpG-2006 may or may not induce caspase-dependent cell death in BL cells. Moreover, ODN CpG-2006-induced cell death responses of BL cells were associated with TLR9 single-nucleotide polymorphisms (SNPs) rs5743836 or rs352140, which we detected in primary BL tumors and in peripheral blood from healthy individuals at similar frequencies. Thus, our findings suggest that the effect of TLR9 agonists on BL cells should be tested in vitro before installment of therapy and TLR9 SNPs in BL patients should be determined as potential biological markers for the therapeutic response to treatment targeting innate immunity.