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To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper-N-glycosylated (SAMD14 at ASN339 and neurabin-I at ASN1277), explaining their autoimmunogenicity. SAMD14 and neurabin-I induced BCR pathway activation and proliferation of aggressive lymphoma cell lines transfected with SAMD14- and neurabin-I-reactive BCRs. Moreover, the BCR binding epitope of neurabin-I conjugated to truncated Pseudomonas exotoxin-killed lymphoma cells expressing the respective BCRs. These results support the role of chronic antigenic stimulation by posttranslationally modified central nervous system (CNS) driver autoantigens in the pathogenesis of PCNSL, serve as an explanation for their CNS tropism, and provide the basis for a novel specific treatment approach.

Duodenal-type follicular lymphoma (DTFL) is a rare and highly indolent follicular lymphoma (FL) variant. It is morphologically and immunophenotypically indistinguishable from typical FL, characterized by restricted involvement of intestinal mucosa, and lacks extraintestinal manifestations. The molecular determinants of this distinct clinical behavior are largely unknown. Thirty-eight diagnostic biopsies from patients with DTFL were evaluated. The 10-year overall survival rate was 100% in clinically evaluable patients (n = 19). We compared the targeted mutation profile of DTFL (n = 31), limited-stage typical FL (LSTFL; n = 17), and advanced-stage typical FL (ASTFL; n = 241). The mutation frequencies of recurrently mutated genes, including CREBBP, TNFRSF14/HVEM, and EZH2 were not significantly different. However, KMT2D was less commonly mutated in DTFL (52%) and LSTFL (24%) as compared with ASTFL (79%). In ASTFL, 41% of KMT2D-mutated cases harbored multiple mutations in KMT2D, as compared with only 12% in LSTFL (P = .019) and 0% in DTFL (P < .0001). Whole exome and targeted sequencing of DTFL revealed high mutation frequencies of EEF1A1 (35%) and HVCN1 (22%). We compared the immune microenvironment gene expression signatures of DTFL (n = 8) and LSTFL (n = 7). DTFL clearly separated from LSTFL by unsupervised, hierarchical clustering of 147 chemokines and cytokines and was enriched for a chronic inflammation signature. In conclusion, the mutational landscape of DTFL is highly related to typical FL. The lower frequency of multiple mutations in KMT2D in DTFL and LSTFL indicates an increasing selection pressure for complete KMT2D loss in ASTFL pathogenesis. The highly dissimilar immune microenvironment of DTFL suggests a central role in the biology of this disease.

Introduction Rituximab (R) administration results in significant outcome improvement in B cell precursor acute lymphoblastic leukemia (B-ALL) patients (pts), but is usually restricted to pts with ≥20% CD20+ leukemic blasts. Yet, this arbitrary cut-off is not proven biologically sensible. Moreover, CD20 expression might differ between blood (pb) and bone marrow (bm) and varies under prednisone during early treatment. In the present GMALL08/2013 trial R is administered to all BCR-ABL1-negative B-ALL pts irrespective of the initial leukemic CD20 expression. We assessed the initial and post-prephase CD20 expression in GMALL08/2013 pts and correlated the values with MRD response after Induction I (Ind I) and Consolidation I (Cons I). A historical B-ALL GMALL07/2003 cohort without R treatment and with available CD20 expression and MRD data was used to evaluate for R-unrelated effects. Methods Comparative immunophenotypic quantification of CD20 expression in 207 B-ALL pts was performed at diagnosis (pb d0 and/or bm d0) and/or (a/o) after a 5-day dexamethasone- and cyclophosphamide-containing prephase (pb d6) under EuroFlow standardized procedures. CD20 median fluorescence intensities (CD20-MFI) and percentages of CD20+ B-ALL blasts/all blasts (%CD20+ BL) were assessed. Minimal residual disease (MRD) was determined after Ind I (after 1x R) and Cons I (after 4x R) by quantitative PCR for clone-specific immune gene rearrangements to stratify pts as molecular complete response (MolCR, MRD negativity, assay sensitivity at least 1x10 -4), molecular intermediate response (MolIR, MRD positive non-quantifiable or positive Results bm d0/pb d0. In 91 paired bm d0/pb d0 samples %CD20+ BL as well as the CD20-MFI were significantly higher in pb in common/pre-B ALL (c/pre-B ALL) (n=76: paired t-test: p pb d0/pb d6. CD20 expression of circulating blasts significantly increased after a 5-day prephase in c/pre-B ALL but not in pro-B ALL in 106 paired pb d0/pb d6 samples (paired t-test of CD20-MFI and %CD20+ BL; n=20 pro-B ALL, p=.09 and p=.25; n=86 c/pre-B ALL, p Molecular response under R. The values of %CD20+ BL were correlated with MRD response after Ind I and Cons I (Fig. 3). Since the CD20 expression in the present cohort was shown to be significantly modulated in a drug- and compartment-dependent manner, we used the highest measured value of %CD20+ BL out of the three available values per patient (bm d0, pb d0 a/o pb d6). In the historical cohort (n=145) one value per patient for %CD20+ BL was available. Due to low CD20 expression pro-B ALL did not show any differences in the %CD20+ BL among the risk groups in the present (Ind I n=23, Cons I n=20) and the historical (Ind I n=11; Cons I n=11) cohort. The differences in %CD20+ BL in relation to molecular response were significant in c/pre-B ALL between MolCR and MolFAIL after Ind I (n=127) and Cons I (n=120) (Mann-Whitney test: p=.0002 and p=.0028) and of lower significance between MolIR and MolFAIL after Ind I (p=.013) and between MolCR and MolIR after Cons I (p=.029) in the present cohort. Within the historical cohort (Ind I n=145, Cons I n=143) no significant differences were observed. Conclusions Leukemic CD20 expression was modulated between compartments (bm d0/pb d0) and showed a significant increase in a drug-dependent manner in c/pre-B ALL (pb d0/pb d6) probably in response to dexamethasone. The results might challenge the conventional eligibility criteria for CD20 targeted treatment in c/pre-B ALL. MRD persisters showed lower initial CD20 expression compared to MRD responders in the present cohort consistently receiving R, but not in the historical cohort without R treatment. Accordingly, R seems to improve the early MRD response predominantly in pts with higher CD20 expression. Supported by DJCLS Figure 1 Figure 1. Disclosures Szczepanowski: Amgen: Speakers Bureau. Trautmann: Amgen: Speakers Bureau. Ritgen: Roche: Consultancy, Other: Travel support, Research Funding; Abbvie: Consultancy, Other: Travel support, Research Funding; Chugai: Consultancy; MSD: Consultancy, Other: Travel support; Celgene: Other: Travel support. Nachtkamp: Celgene: Other: Travel Support; bsh medical: Speakers Bureau; Jazz: Speakers Bureau. Viardot: Kite/Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; University Hospital of Ulm: Current Employment. Baldus: Jazz: Honoraria; Celgene/BMS: Honoraria; Amgen: Honoraria; Novartis: Honoraria. Schwartz: Morphosys: Research Funding; Gilead: Other: Travel grants, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Travel grants, Speakers Bureau; Novartis: Speakers Bureau; Jazz Pharmaceuticals: Other: Travel grants, Speakers Bureau; BTG International Inc: Membership on an entity's Board of Directors or advisory committees; MSD Sharp & Dohme: Membership on an entity's Board of Directors or advisory committees; Basilea: Other: Travel grants. Goekbuget: Pfizer: Consultancy, Other: Research funding for institution; Amgen: Consultancy, Other: Invited talks for company sponsored symposia (with honoraria); Research funding for institution; Astra Zeneca: Other: Invited talk for company sponsored symposia (with honor); Gilead/Kite: Consultancy; Novartis: Consultancy, Other: Research funding for Institution; Jazz Pharmaceuticals: Other: Research funding for institution; Incyte: Other: Research funding for Institution; Cellestia: Consultancy; Erytech: Consultancy; Morphosys: Consultancy; Servier: Consultancy, Other; Abbvie: Other. Brüggemann: Amgen: Other: Advisory Board, Travel support, Research Funding, Speakers Bureau; Incyte: Other: Advisory Board; Janssen: Speakers Bureau. OffLabel Disclosure: Rituximab administration to patients with CD20-negative (

To address the role of chronic antigenic stimulation in primary central nervous system lymphoma (PCNSL), we searched for autoantigens and identified sterile α-motif domain containing protein 14 (SAMD14) and neural tissue-specific F-actin binding protein I (neurabin-I) as autoantigenic targets of the B-cell receptors (BCRs) from 8/12 PCNSLs. In the respective cases, SAMD14 and neurabin-I were atypically hyper

The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. Here, we provide biochemical, structural, functional and clinical evidence that aberrant Cathepsin S (CTSS) activity induces a supportive immune microenvironment in FL. By targeted DNA sequencing of 305 diagnostic FL biopsies, we identified somatic mutations of CTSS in 8% of cases (24/305), mostly clustered at Y132 (19/24) converting Y to D (16/19). A subset of CTSS Y132 mutations (N=5) occurred at lower variant allele frequencies (5-10%), indicating subclonality. Another 13% of FL had CTSS amplifications (37/286). CTSS Y132 mutations and CTSS amplifications were mutually exclusive. In a cohort of 51 FL, CTSS amplifications were associated with higher CTSS expression (P=0.05). Of note, a subset of FL without CTSS amplifications also had higher CTSS expression, suggesting additional mechanisms of transcriptional dysregulation. CTSS is a cysteine protease that is highly expressed in endolysosomes of antigen presenting cells and malignant B-cells. CTSS is involved in proteolytical processing of antigenic peptides for presentation on MHC-II to be recognized by antigen specific CD4+ T-cells. CTSS is synthesized as an inactive zymogen, which is converted to its active form by autocatalytic cleavage of the autoinhibitory propeptide (pro-CTSS). We used CRISPR/Cas9 to introduce CTSS Y132D into Karpas422, a B-cell lymphoma cell line that harbors the FL hallmark translocation t(14;18). Single-cell derived Y132D mutant clones showed >3-fold higher ratios of active CTSS to pro-CTSS (N=4, P=0.0003). Immunoprecipitated CTSS Y132D had >3-fold higher in vitro substrate cleavage activity compared to CTSS wild type (WT) (N=6, P=0.001). We purified pro-CTSS WT and Y132D and assayed their in vitro autocatalytic cleavage over time. The time required to convert 50% of pro-CTSS decreased from 17 minutes for WT to 11 minutes for Y132D (N=3, P=0.04). In contrast, purified active CTSS WT and Y132 had similar in vitro cleavage activities. Molecular dynamics simulations showed that the Y132D mutation shortens the distances by ~2Å between the catalytic triad of active CTSS (C139, H278, N298) and a stretch of amino acids from the proform (L80, G81, D82, S94), which may facilitate intramolecular cleavage. By mass spectrometry we could indeed detect novel intermediate-sized CTSS fragments. Thus, Y132D does not increase the activity of the mature enzyme but is a gain-of-function mutation by accelerating the conversion from pro-CTSS to catalytically active CTSS. CD74 (invariant chain) is a physiologic CTSS substrate that plays critical roles in the assembly, trafficking and stabilization of peptide-free MHC-II. CTSS cleaves CD74, thereby allowing binding and presentation of antigens on MHC-II to antigen specific CD4+ T-cells. We could show that CTSS Y132D enhanced CD74 cleavage in Karpas422 cells. We then tested the impact of CTSS on antigen specific CD4+ T-cell activation in co-culture assays. CTSS knock-out lymphoma cells were broadly incapable of activating CD4+ T-cells. Overexpression of CTSS WT activated CD4+ T-cells more efficiently compared to empty vector control. CTSS Y132 had the highest capacity to stimulate antigen specific CD4+ T-cell responses. Furthermore, in primary FL biopsies (N=51) CTSS Y132 mutations had gene expression profiles linked with antigen-processing and chemokine perturbation, including CXCL13, a B-cell chemoattractant produced by activated CD4+ T-follicular helper cells. Lastly, we aimed to correlate CTSS aberrations with clinical outcome in patients who received standard immunochemotherapy (R-CHOP) for advanced FL (N=51 with available CTSS mutation and gene expression data). Compared to all other patients (N=34), patients with CTSS Y132 mutations or CTSS overexpression (N=17) had longer failure free survival (P=0.012) and overall survival (P=0.041). In summary, we propose that aberrant CTSS activity - even if only present in a FL subclone - can elicit a CD4+ T-cell driven tumor-promoting immune response, which could be amplified within the microenvironment via pro-inflammatory and chemotactic cytokines and substantially impact the biology and clinical course of the disease. Thus, aberrant CTSS activity is a promising biomarker and therapeutic target in FL and potentially also other tumors. Disclosures Klapper: Roche, Takeda, Amgen, Regeneron: Honoraria, Research Funding. Hiddemann:Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Gilead: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria; Vector Therapeutics: Consultancy, Honoraria. Steidl:Juno Therapeutics: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Tioma: Research Funding; Seattle Genetics: Consultancy; Bayer: Consultancy. Kridel:Gilead Sciences: Research Funding. Weinstock:Celgene: Research Funding; Verastem Oncology: Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

Introduction: Treatment with Rituximab has improved the outcome for patients with B-cell precursor ALL (B-ALL) and is conventionally restricted to patients with CD20 expression on leukemic blasts above a level of 20%. However, it is not known whether this arbitrary cutoff is biologically meaningful, or if also patients with lower CD20 expression levels profit from Rituximab treatment. In addition, CD20 expression levels might differ between different compartments and can change during early treatment. Therefore, in the current German Multicentric Acute Lymphoblastic Leukemia (GMALL) 08/2013 trial Rituximab is applied to all BCR-ABL1-negative B-ALL patients during induction and early consolidation irrespective of the initial blast CD20 expression. In this study, CD20 expression on blasts at diagnosis and after a 5-day dexamethasone prephase is thoroughly characterized and correlated to MRD response after induction I including one dose of Rituximab to evaluate its impact on response to Rituximab. Methods: Comparative quantification of CD20 expression levels was performed at diagnosis (blood and bone marrow (BM)) and at the end of the prephase (blood) and correlated to minimal residual disease (MRD) response after early Rituximab treatment. Samples were subjected to the EuroFlow standardized stain, lyse and wash and instrument setting procedures (van Dongen et al., 2012) and stained with a 3-tube, 8-color panel, containing the markers CD45, CD20, CD10, CD66c, CD3, CD19, CD71, CD9, CD13+33, CD34, CD22, CD11a, and CD38. CD20 median fluorescence intensities (CD20-MFI) as well as percentages of CD20+ B-ALL blasts among all blasts (%CD20+) were measured. MRD was quantified at day 22 after induction I using real-time quantitative PCR of clonal immune gene rearrangements. MRD response after induction was defined as MRD decrease to a level below 10-4, MRD persistence as detection of quantifiable MRD ≥10-4. Results: FCM results of a total of 166 samples of 84 B-ALL patients were evaluated. CD20 expression of circulating blasts significantly increased after a 5-day prephase in common/pre-B-ALL (c/pre-B) but not in pro-B-ALL in a cohort of 36 paired peripheral blood samples (Fig. 1 A-B, n=8 pro-B-ALL, paired t-test of CD20-MFI and %CD20: p=.1016 and p=.1660, Fig. 2 A-B, n=28 c/pre-B-ALL, p=.0029 and p=0.0060). Interestingly, the increase of CD20 expression on B-ALL blasts contrasted that on mature normal B-cells, where a decrease occurred in pro-B-ALL and c/pre-B-ALL samples at day 6 (paired t-test: p=.0485 and p In 24 paired blood/BM pre-treatment samples the percentage of CD20+ blasts was significantly higher in blood in c/pre-B-ALL (n=19: paired t-test: p=.0009), but not in pro-B-ALL (n=5: paired t-test: p=.095) (Fig. 1C, 2C). The significance levels were not consistently reached for the comparison of CD20-MFI. Nevertheless, we show that in 3/24 (12.5%) patients the percentage of CD20+ B-ALL blasts in BM did not reach the arbitrary cutoff of 20% whereas the matching diagnostic blood did. Finally, CD20 expression levels from 25 BM and 30 pre-treatment blood samples and 38 blood samples after dexamethasone prephase from 54 patients were assessed (in total 93 samples). In further calculations we correlated MRD response values after one dose of Rituximab with the CD20 expression levels on blasts. In detail, the highest measured percentage value of %CD20 per patient in the group of MRD responders was compared to the highest measured %CD20 per patient in the group of MRD persisters. CD20 expression levels were significantly lower in 33 MRD persisters compared to 21 MRD responders (Fig 3, p=.0336). The difference was mainly attributable to a high frequency of MRD persisters in pro-B-ALL compared to c/pre-B-ALL (MRD persistence in pro-B-ALL 8/9 (89%) compared to 25/45 (56%) in c/pre-B-ALL). Conclusion: We measured significant differences in CD20 expression levels on leukemic blasts (i) between pre-treatment blood and BM and (ii) between pre-treatment blood and blood after prephase in patients with c/pre-B-ALL challenging the conventional eligibility criteria for the CD20 targeted treatment. MRD responders tended to have higher CD20 expression levels compared to MRD persisters. Extension of the patient cohort is ongoing to confirm this observation. Furthermore, we will analyze the impact of 3 rituximab applications after induction phase II. Disclosures Ritgen: abbvie: Research Funding; Roche: Honoraria, Research Funding. Viardot:Gilead Kite: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Amgen: Consultancy. Kneba:Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Goekbuget:Kite / Gilead: Consultancy; Celgene: Consultancy; Novartis: Consultancy, Other: Travel support, Research Funding; Pfizer: Consultancy, Other: Travel support, Research Funding; Amgen: Consultancy, Other: Travel support, Research Funding. Brüggemann:Incyte: Consultancy; Amgen: Consultancy, Research Funding, Speakers Bureau; PRMA: Consultancy; Affimed: Research Funding; Regeneron: Research Funding; Pfizer: Speakers Bureau; Roche: Speakers Bureau.

Diffuse large B-cell-lymphoma (DLBCL) is a heterogeneous disease in its sites of origin, genetic alterations, and clinical behavior. Gene expression profiling (GEP) has led to the identification of two molecular subtypes, GCB-like and ABC-like DLBCL, that follow different molecular circuits and hence, likely represent different diseases. Next to the ABC-like GEP, the rearrangement and/or expression of MYC and TP53 mutations in tumors characterize patient subsets with inferior prognosis. However, the clinical impact of cell of origin (COO) subtyping and the identification of prognostic biomarkers differ between studies. In many clinical trials, patients identified "at risk" experience cure and long-time survival. Important factors modulating the impact of tumor cell-specific factors may be conferred by the host microenvironment, and stromal signatures have been shown to be correlated to survival in DLBCL. The robust analysis of stromal signatures is hampered by the lack of assays applicable to routinely used formalin-fixed paraffin-embedded (FFPE) materials. We have constructed a molecular signature applicable to FFPE, interrogating the quantitative and qualitative composition of the microenvironment in DLBCL. The signature was trained using an algorithm that extracts prognostic information out of the ratios of pairs of genes. The genes that drive prognosis are expressed in T-cells and macrophages and have function in the communication between both cell types. The model was validated using the NanoString assay in a cohort of 466 DLBCL patients enrolled in 7 prospective clinical trials (MInT, MegaCHOEP phase III and observation, RICOVER-60, RICOVER-noRTh, DENSER, SMARTER) of the German High grade non-Hodgkin's lymphoma study group (DSHNHL). Grouping of the patients into quartiles according to the expression of the continuous stromal signature score (ranging from -1.880to 4.441) resulted in three quartiles (Q1-3) with comparable clinical behavior (stromal signature low). Patients from quartile 4 (Q4), however, characterized by high expression of the signature (stromal signature high), showed a clearly inferior outcome in EFS, PFS and OS (Figure 1 a-c). This result could be independently reproduced in the seven clinical trials named above, thus clearly depicting the robustness of the signature. Multivariate analysis revealed that high expression of the stromal signature is a prognostic risk factor independent of the IPI factors in EFS (HR 1.7, 95 % CI 1.2-2.4, p-value =0.001), PFS (HR 1.8, 95 % CI 1.2-2.5, p-value =0.001) and OS (HR1.8, 95 % CI 1.3-2.7, p-value =0.001). Combining stromal signature count with IPI-score led to the identification of a high-risk cohort (Fig. 1 d-f). Of importance, additional multivariate analyses adjusted for the IPI factors performed within selected trials showed that the stromal signature provides prognostic information independent of the COO status, MYC and dual MYC/BCL2 rearrangements, TP53 mutations and the MYC/BCL2 double expresser status. In summary, our data from prospectively randomized trials of the DSHNHL underline the importance of the microenvironment in the prognostic stratification of DLBCL patients and suggest that the composition and quality of the tumor stroma is an independent risk factor in DLBCL. Analysis of stromal features, therefore, may provide a rationale for targeted treatment approaches, e.g. with immunomodulatory substances (IMIDs), in patients at risk. Figure 1. Figure 1. Disclosures Klapper: Regeneron: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Takeda: Honoraria, Research Funding. Richter:HTG Molecular Diagnostics, Inc.: Research Funding. Poeschel:Roche: Other: Travel grants; Amgen: Other: Travel grants. Held:BMS: Consultancy, Other: Travel grants, Research Funding; Spectrum: Research Funding; Roche: Consultancy, Other: Travel grants, Research Funding; Amgen: Research Funding; MSD: Consultancy.

The highly variable clinical course of follicular lymphoma (FL) is determined by the molecular heterogeneity of the tumor cells and complex interactions with the microenvironment. The underlying molecular mechanisms and therapeutic vulnerabilities are not well understood. IL-4 producing follicular helper T cells (TFH) have been identified as a key component of the malignant B-cell niche. IL-4 activates paracrine signaling via STAT6. In a cohort of 258 patients with advanced stage FL, we identified STAT6 mutations in 13% of diagnostic biopsies (n=33). All mutations were clustered within the DNA binding domain, mostly at D419, and included a polymorphic variant (rs11172102). Gene set enrichment analysis (GSEA) revealed that STAT6 mutant cases were significantly enriched for two distinct IL-4 gene expression signatures. Gene expression data and immunohistochemistry of primary FL samples showed significant up-regulation of IL-4/STAT6 target genes in STAT6 mutant cases, including FCER2, which encodes for CD23. We stably expressed wild type STAT6 or mutant STAT6 (D419G, N421K, and D519V) in two B-cell lymphoma lines (OCI-Ly1, OCI-Ly8), both harboring the FL hallmark translocation t(14;18). Upon IL-4 stimulation, cells expressing mutant STAT6 had significantly increased FCER2 transcript levels. Similarly, IL-4 induced expression of membrane-bound as well as soluble CD23 was significantly increased in STAT6 mutant cells. Cells expressing mutant STAT6 showed significantly increased nuclear accumulation of pSTAT6 following IL-4 stimulation. Of note, we did not observe any effect of STAT6 mutations in the absence of IL-4. RNA sequencing of IL-4-stimulated lymphoma cell lines (STAT6 mutant versus wild type) identified PARP14 -a known transcriptional co-activator of STAT6- among the top differentially expressed genes. Bioinformatics and functional experiments demonstrated that PARP14 per se is a novel STAT6 target gene. Furthermore, reporter assays showed increased transactivation activity of mutant STAT6 at the PARP14 promotor, suggesting a regulatory feed-forward loop. Pharmacological inhibition of PARP and knock-down of PARP14 completely abrogated the mutant STAT6 gain-of-function phenotype. In summary, our results suggest that PARP14 is a novel target in STAT6 mutant FL. Our data also imply that the biological and clinical impact of STAT6 mutations will heavily depend on the (targetable) upstream activation of the IL-4 signaling cascade, including the abundance of IL-4 / TFH cells in the microenvironment of FL. Disclosures Richter: HTG Molecular Diagnostics, Inc.: Research Funding. Klapper:Amgen: Honoraria, Research Funding; F.Hoffman-La Roche: Honoraria, Research Funding; HTG Molecular Diagnostics, Inc.: Research Funding; Takeda: Honoraria, Research Funding; Regeneron: Honoraria, Research Funding. Hiddemann:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding. Weigert:Novartis: Research Funding; Roche: Research Funding.

Background: High throughput sequencing of immunoglobuline heavy chain gene rearrangements (IGH) recently demonstrated that B-cell precursor ALL (BCP-ALL) is a highly oligoclonal disease in children (Theunissen, Hematologica, 2017), however, little is known about the degree of oligoclonality in adults with BCP-ALL. Furthermore, no data exist on potential changes of the subclonal composition during therapy. Therefore, we aimed to monitor the clonal architecture of the leukemic blasts before and after Rituximab containing induction treatment in the context of the current German Multicentric Acute Lymphoblastic Leukemia (GMALL) 08/2013 trial. Materials & Methods: We identified and studied complete and incomplete immunoglobulin (IG) heavy chain rearrangements in 100ng of bone marrow (BM) DNA at diagnosis (dx) and 500ng BM DNA after Rituximab-containing induction I in 19 patients with BCP-ALL. We employed IGH-VJ FR1 and IGH-DJ NGS assays developed within the EuroClonality-NGS Consortium (www.euroclonalityngs.org), performing next generation sequencing on Illumina MiSeq. We analysed data with the ARResT/Interrogate bioinformatics platform (Bystry, Bioinformatics, 2017), which is also able to isolate and use the DN-J stem of V/DJ junctions to link clonally related rearrangements. Clones with abundance ≥5 %, and all subclones carrying the same DN-J stem as the dominant clones were considered as leukemia-associated and studied further. Employing the sequence of the DN-J stem, we could also link related complete and incomplete rearrangements, even though those are amplified in two separate PCRs. Results: At dx, 18/19 patients carried at least 1 IGH rearrangement with abundance ≥ 5%, 10 patients carried 2 or more. In all 18 patients with dominant markers detected at dx, subclones with abundance Next, we compared the kinetics of all (sub)clones with abundance ≥1% in at least one of the time-points. Of the 18 patients, 6 only had subclones with abundance Conclusions: It has recently been shown that ALL is a highly oligoclonal disease in children (Theunissen, Hematologica, 2017), and our study extends this finding to adults with BCP-ALL. We furthermore demonstrate that the subclonal composition remains stable in the majority of patient during induction chemoimmunotherapy. The fact that the response to treatment is generally consistent among different (sub)clones has important implications for MRD quantification as it reassures the usage of the dominant clonal IG gene rearrangement for MRD monitoring in ALL. However, also significant changes of the clonal composition may occur in BCP-ALL as exemplified in one patient of our cohort. Further investigations are necessary to elucidate factors that influence subclonal heterogeneity in response to treatment. Disclosures Viardot: Pfizer: Consultancy, Honoraria; Gilead Kite: Consultancy, Honoraria; Roche: Consultancy, Honoraria; Amgen: Consultancy; BMS: Consultancy, Honoraria. Kneba:AbbVie: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Goekbuget:Pfizer: Consultancy, Other: Travel support, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Kite / Gilead: Consultancy; Amgen: Consultancy, Other: Travel support, Research Funding; Celgene: Consultancy. Brüggemann:Incyte: Consultancy; PRMA: Consultancy; Regeneron: Research Funding; Affimed: Research Funding; Pfizer: Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Roche: Speakers Bureau.

The survival of diffuse large B-cell lymphoma patients varies considerably, reflecting the molecular diversity of tumors. In view of the controversy whether cytologic features, immunohistochemical markers or gene expression signatures may capture this molecular diversity, we investigated which features provide prognostic information in a prospective trial in the R-CHOP treatment era. Within the cohort of DLBCLs patients treated in the RICOVER-60 trial of the German High-Grade Lymphoma Study Group (DSHNHL), we tested the prognostic impact of IB morphology in 949 patients. The expression of immunohistochemical markers CD5, CD10, BCL2, BCL6, human leukocyte antigen (HLA)–DR, interferon regulatory factor-4/multiple myeloma-1 (IRF4/MUM1), and Ki-67 was assessed in 506 patients. Expression of the immunohistochemical markers tested was of modest, if any, prognostic relevance. Moreover, the Hans algorithm using the expression patterns of CD10, BCL6, and interferon regulatory factor-4/multiple myeloma-1 failed to show prognostic significance in the entire cohort as well as in patient subgroups. IB morphology, however, emerged as a robust, significantly adverse prognostic factor in multivariate analysis, and its diagnosis showed a good reproducibility among expert hematopathologists. We conclude, therefore, that IB morphology in DLBCL is likely to capture some of the adverse molecular alterations that are currently not detectable in a routine diagnostic setting, and that its recognition has significant prognostic power.

The spectrum of entities, the therapeutic strategy, and the outcome of mature aggressive B-cell non-Hodgkin lymphomas (maB-NHLs) differs between children and adolescents on the one hand and adult patients on the other. Whereas adult maB-NHLs have been studied in detail, data on molecular profiling of pediatric maB-NHLs are hitherto lacking. We analyzed 65 cases of maB-NHL from patients up to 18 years of age by gene expression profiling, matrix comparative genomic hybridization (CGH), fluorescent in situ hybridization (FISH), and immunohistochemistry. The majority of the analyzed pediatric patients were treated within prospective trials (n = 49). We compared this group to a series of 182 previously published cases of adult maB-NHL. Gene expression profiling reclassified 31% of morphologically defined diffuse large B-cell lymphomas as molecular Burkitt lymphoma (mBL). The subgroups obtained by molecular reclassification did not show any difference in outcome in children treated with the NHL-Berlin-Frankfurt-Muenster (BFM) protocols. No differences were detectable between pediatric and adult mBL with regard to gene expression or chromosomal imbalances. This is the first report on molecular profiling of pediatric B-NHL showing mBL to be much more prominent in children than suggested by morphologic assessment. Based on molecular profiling mBL is a molecularly homogeneous disease across children and adults.

The genetic hallmark of Burkitt lymphoma (BL) is the t(8;14)(q24;q32) and its variants leading to activation of the MYC oncogene. It is a matter of debate whether true BL without MYC translocation exists. Here, we identified 59 lymphomas concordantly called BL by 2 gene expression classifiers among 753 B-cell lymphomas. Only 2 (3%) of these 59 molecular BL lacked a MYC translocation, which both shared a peculiar pattern of chromosome 11q aberration characterized by interstitial gains including 11q23.2-q23.3 and telomeric losses of 11q24.1-qter. We extended our analysis to 17 MYC-negative high-grade B-cell lymphomas with a similar 11q aberration and showed this aberration to be recurrently associated with morphologic and clinical features of BL. The minimal region of gain was defined by high-level amplifications in 11q23.3 and associated with overexpression of genes including PAFAH1B2 on a transcriptional and protein level. The recurrent region of loss contained a focal homozygous deletion in 11q24.2-q24.3 including the ETS1 gene, which was shown to be mutated in 4 of 16 investigated cases. These findings indicate the existence of a molecularly distinct subset of B-cell lymphomas reminiscent of BL, which is characterized by deregulation of genes in 11q.

Diffuse large B-cell lymphoma is the most frequent type of B-cell lymphoma in adult patients but also occurs in children. Patients are currently assigned to therapy regimens based on arbitrarily chosen age limits only (eg, 18 or 60 years) and not biologically justified limits. A total of 364 diffuse large B-cell lymphomas and related mature aggressive B-cell lymphomas other than Burkitt lymphoma from all age groups were analyzed by comprehensive molecular profiling. The probability of several biologic features previously reported to be associated with poor prognosis in diffuse large B-cell lymphoma, such as ABC subtype, BCL2 expression, or cytogenetic complexity, increases with age at diagnosis. Similarly, various genetic features, such as IRF4 translocations, gains in 1q21, 18q21, 7p22, and 7q21, as well as changes in 3q27, including gains and translocations affecting the BCL6 locus, are significantly associated with patient age, but no cut-offs between age groups could be defined. If age was incorporated in multivariate analyses, genetic complexity lost its prognostic significance, whereas the prognostic impact of ABC subtype and age were additive. Our data indicate that aging is a major determinant of lymphoma biology. They challenge current concepts regarding both prognostic biomarkers and treatment stratification based on strict age cut-offs.

Sequence variants at the TERT-CLPTM1L locus in chromosome 5p have been recently associated with disposition for various cancers. Here we show that this locus including the gene encoding the telomerase reverse-transcriptase TERT at 5p13.33 is rarely but recurrently targeted by somatic chromosomal translocations to IGH and non-IG loci in B-cell neoplasms, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, mantle cell lymphoma and splenic marginal zone lymphoma. In addition, cases with genomic amplification of TERT locus were identified. Tumors bearing chromosomal aberrations involving TERT showed higher TERT transcriptional expression and increased telomerase activity. These data suggest that deregulation of TERT gene by chromosomal abnormalities leading to increased telomerase activity might contribute to B-cell lymphomagenesis.

Follicular Lymphoma (FL) is the second most common non-Hodgkin lymphoma and remains an incurable disease for the majority of patients who present with advanced stage disease. Virtually all patients subsequently acquire treatment resistance and a third of patients ultimately develop histologically transformed disease with aggressive clinical course and poor prognosis. Thus, there is an unmet clinical need to decipher the underlying biology of this disease, and identify dysregulated pathways that may serve as therapeutic targets. In a cohort of 258 FL specimens, we identified 33 cases with STAT6 mutations (12.8%), including 15 mutations at D419. Overall, 34 out of 35 (97.1%) of STAT6 mutations were within the DNA-binding domain. MutSigCV analysis (Lawrence, Nature 2013) indicated that STAT6 was significantly mutated in FL, i.e., more commonly mutated than expected based on background mutation rate. Previously, STAT6 mutations, including D419 hotspot mutations, have been reported in other lymphomas, including primary mediastinal B-cell lymphoma (Ritz et al., Blood 2009) and FL (Yildiz et al., Blood 2015). STAT6 proteins are transcription factors that are mainly activated by interleukin-4 (IL-4). A recent study identified a subset of IL-4 producing follicular helper T cells to be involved in the survival of FL B cells (Ame-Thomas et al., Blood 2015). We compared the expression levels of selected STAT6 target genes in FL patient samples with or without STAT6 mutations. Gene expression data (nCounter, Nanostring) was available for 138 patient samples with known STAT6 mutation status. Thereof, 13 cases had STAT6 mutations, including six at D419. Among the STAT6 target genes were CD23 (and its soluble form, sCD23) and SOCS1. Both showed significantly higher expression (P CD23 immunohistochemistry was performed on 13 patient samples with known STAT6 mutation status. Five patient samples with wild-type STAT6 stained negative for CD23. In contrast, four out of eight cases with STAT6 mutations stained positive for CD23. To test if STAT6 mutations are directly linked to enhanced CD23 expression, we stably expressed wild-type (wt) STAT6, mutant STAT6 (D419G, N421K, and D519V), or empty vector in two t(14;18) positive B-lymphoma cell lines (OCI-Ly1, OCI-Ly8). Incubation of OCI-Ly1 cell lines with IL-4 (10 ng/mL for 24h) resulted in enhanced CD23 surface expression by flow cytometry (3.2-fold for D419G, 3.1-fold for N421K, and 2.3-fold for D519V), compared to STAT6 wt (N=3), whereas no significant difference was seen in the absence of IL-4. Similar results were observed in OCI-Ly8 cells. To investigate if these findings result from enhanced transcription, we performed quantitative PCR (SybrGreen). Briefly, Ct values were normalized to GAPDH reference gene and Ct values from untreated controls were subtracted from IL-4 treated samples (ΔΔCt method). These experiments showed increased CD23 transcriptional levels for OCI-Ly1 expressing mutant STAT6 (5.6-fold for D419G, 5.0-fold for N421K, and 8.4-fold for D519V), compared to STAT6 wt (N=3). For OCI-Ly8 cells, CD23 transcription was increased 4.6-fold for D419G, 5.2-fold for N421K and 3.0-fold for D519V, compared to STAT6 wt (N=3). In addition to membrane-bound CD23, the soluble CD23 (sCD23) in the microenvironment might further contribute to FL biology. To address this, we established a sCD23 ELISA using OCI-Ly1 cells stably expressing wild type or mutant STAT6. Supernatants of IL-4 stimulated cells expressing mutant STAT6 resulted in a 4.1-fold (D419G), 1.5-fold (N421K), and 1.3-fold (D519V) increase of sCD23 levels, as compared to STAT6 wt. We conclude that STAT6 is recurrently and significantly mutated in FL including hotspot mutations at D419. STAT6 mutations are gain-of-function and may promote disease progression by enhancing IL-4 induced expression of surface and soluble CD23. Disclosures No relevant conflicts of interest to declare.

Background: Follicular lymphoma (FL) is the second most common nodal lymphoma worldwide and remains incurable for most patients (pts). FL is recognized to be a highly heterogeneous disease and a subset of pts experience remarkable poor outcome. In a recent study, 19-26% of pts receiving first-line R-CHOP experienced progression of disease (POD) within 24 months after diagnosis and had a 5-year (yr) overall survival (OS) of only 34-50%, as compared to a 5-yr OS of 90-94% for pts without POD within 24 months (Casulo et al., J Clin Oncol 2015). Consequently, event-free survival at 12 (EFS12) and 24 months (EFS24) have been suggested as novel surrogate endpoints for poor overall survival in clinical trials and useful for patient counseling (Maurer et al., ASH 2014). We have previously shown that a clinicogenetic risk model (m7-FLIPI) that includes the mutation status of seven genes (EZH2, ARID1A, MEF2B, EP300, FOXO1, CREBBP, and CARD11), the FL International Prognostic Index (FLIPI), and the Eastern Cooperative Oncology Group (ECOG) performance status, improves risk stratification for failure-free survival (FFS) and OS in pts with FL receiving first-line immunochemotherapy (Pastore et al., Lancet Oncology 2015). An online tool is available at: http://www.glsg.de/m7-flipi. It would be advantageous to determine prognosis prior to front-line therapy rather than after POD. Thus, we aimed to investigate the predictive utility of the m7-FLIPI for early POD. Patients and Methods: Clinical and mutation data were available from two independent cohorts: 151 pts who received R-CHOP and IFN maintenance as part of a randomized trial of the German Low-Grade Lymphoma Study Group (GLSG), and 107 pts from a population-based registry of the British Columbia Cancer Agency (BCCA) who received R-CVP. Among the latter, 93 (87%) received R-maintenance by intention to treat. All pts had symptomatic, advanced stage or bulky disease considered ineligible for curative radiotherapy, and a biopsy specimen obtained Results: In the GLSG cohort, median age was 57 yrs (range 27-77), 51% had high-risk FLIPI, and 5-yr failure-free survival (FFS) and OS rates were 66% and 83%, respectively (median follow-up for OS 7.7 yrs). In the BCCA cohort, median age was higher (62 yrs, 37-83), but high-risk FLIPI was similarly frequent (50%); 5-yr FFS and OS rates were 58% and 74%, respectively (median follow-up for OS 6.7 yrs). A total of 43 GLSG (28%) and 24 BCCA pts (22%) were classified as high-risk by m7-FLIPI, with a 5-yr OS of 65% and 42%, respectively. In the GLSG cohort, 8 (5%) and 29 pts (19%) had POD within 12 and 24 months, respectively, 4 and 10 pts were censored before POD12 and POD24 (table). High-risk m7-FLIPI pts were significantly more likely to experience early POD with an Odds Ratio (OR) of 20.80 (95% confidence interval (CI) 3.53-395.96; p=0.0052) for POD12 and 6.33 (95% CI 2.67-15.69; p In the GLSG and BCCA cohorts, the m7-FLIPI had a sensitivity of 88% and 50%, a specificity of 75% and 82%, a positive predictive value (PPV) of 16 and 33%, and a negative predictive value (NPV) of 96% and 90% to predict POD12. For POD24, sensitivity was 62% and 46%, specificity 79% and 86%, PPV 42% and 54%, and NPV 82% and 82%, respectively. Conclusion: We conclude that patients classified as high-risk m7-FLIPI are highly enriched among those with early progression of disease. However, approximately one-half of patients with progression by 24 months after therapy initiation are classified as low-risk m7-FLIPI. Thus, further efforts are needed to refine the m7-FLIPI and thereby capture additional cases that may benefit from innovative first-line approaches. Table. Population m7-FLIPI N POD12 POD24 GLSG Total 151 8/147* 29/141* High-risk 43 7 18 Low-risk 108 1 11 BCCA Total 107 16/107 28/107 High-risk 24 8 13 Low-risk 83 8 15 *Pts censored were removed Disclosures Ansell: Bristol-Myers Squibb: Research Funding; Celldex: Research Funding.

Background: Sequence analyses of variable immunoglobulin gene fragments of PCNSL from immunocompetent patients have shown a VH4-34 restriction raising speculations on a selection/stimulation of a functional BCR by an autoantigen in the central nervous system. The present study focused on the search for these hypothetic autoantigens presumably driving the malignant transformation of B-cells into PCNSL cells by chronic BCR stimulation in immunocompetent patients. Methods: BCRs were expressed as recombinant Fabs based on corresponding pairs of functional variable region heavy and light chain genes, which had been amplified from isolated genomic DNA of snap-frozen lymphoma specimens and checked for binding to proteins expressed on macroarrays of human cDNA expression libraries. Results: Recombinant BCR expression was attempted in 21 and successful in twelve PCNSL cases. The VH4-34 family was overrepresented, but was found in less than a quarter of the PCNSL patients (4/21). Screening of the recombinant BCRs on protein arrays revealed that 8 of 12 recombinant PCNSL-BCRs reacted with SAMD14 and the SAM domain of neurabin-1, two proteins with high homology and preferential expression in the CNS. Subsequent proteomic analysis of cryoconserved lymphoma specimens showed that SAMD14 and the SAM domain of neurabin-1 were alternatively N-glycosylated in patients with a PCNSL-BCR specific for SAMD14 and neurabin-1, but not in the remaining PCNSL patients with BCR specificities other than for SAMD14/neurabin-1. Compared to SAMD14 and neurabin-1 from healthy controls, Asn 339 of SAMD14 and Asn 1277 of neurabin-1 were shown to carry additionally glycosylated Asn residues. Of interest, both additional glycosylation sites belonged to atypical, non-canonical Asn-Leu-Glu-Gln (N-L-E-Q) sites instead of the Asn-X-Ser/Thre consensus sequence (N-X-S/T; where X can be any amino acid except proline), which is reported to constitute 97% of N-glycosylation sites under physiologic circumstances. These atypical N-hyperglycosylations were shown for every case with sufficient biopsy material for this proteomic analysis and a PCNSL-BCR specific for SAMD14 and neurabin-1 in their PCNSL and CNS, and to a lesser degree in their peripheral blood mononuclear cells and patient-derived EBV-transformed lymphoblastoid cell lines (LCLs). Of the recombinant BCRs of all cases with sufficient material to test for hyperglycosylation, only the BCRs of the 6 cases with hyperglycosylated SAMD14/neurabin-1 reacted against SAMD14/neurabin-1. Of note, glycosylation status of 2/8 cases with recombinant SAMD14/neurabin-1 reactive BCRs could not be analyzed due to insufficient cryomaterial left after variable region gene PCRs. No hyperglycosylation of SAMD14 and neurabin-1 was found in the peripheral blood of 400 healthy controls, 100 newborns and 50 nursery residents. Moreover, antibodies against SAMD14 and neurabin-1 were detected in the sera and cerebrospinal fluids of an independent second cohort of patients with PCNSL (8/22), but not in sera of patients with secondary CNS manifestations of systemic DLBCL (0/17) or of healthy controls (0/92). Conclusion: Our results strongly suggest that the atypical (NLEQ) glycosylation of the highly homologous SAMD14 and the SAM domain of neurabin-1 maintains a chronic autoimmunogenic stimulation in the CNS, ultimately leading to the malignant transformation of B-cells with a BCR specific for these atypically N-hyperglycosylated proteins into an aggressive B-cell lymphoma in the CNS in a majority of patients with PCNSL. The fact that the VH4-34 family represents only a minority of VH families recognizing SAMD14/neurabin-1 underlines the extraordinary autoimmunogenicity of these posttranslationally modified proteins in a broad range of individuals. Our results provide the first and strongest experimental evidence for the role of chronic autoantigenic stimulation as a first step in the pathogenesis of aggressive B-cell lymphomas. Supported by Deutsche Forschungsgemeinschaft DFG, Deutsche José Carreras Leukämie Stiftung, Wilhelm-Sander-Stiftung, Deutsche Krebshilfe e.V. Disclosures No relevant conflicts of interest to declare.

Introduction Burkitt Lymphoma (BL) is the most common subtype of Non-Hodgkin Lymphoma (NHL) in childhood and adolescence. On the molecular level translocations involving the MYC oncogene are known as the hallmark of BL. However, as this event is not sufficient for BL pathogenesis, the search for additional driver mutations is ongoing. Recently published next-generation sequencing studies introduced various recurrently mutated genes in BL and reported the finding of mutations in Inhibitor of DNA-binding 3 (ID3) (Richter et al., Nat Genet 2012; Schmitz et al., Nature 2012; Love et al., Nat Genet 2012). Within the same pathway also the functional partner of ID3, TCF3, and the downstream target CCND3 were shown to harbor recurrent mutations. Functional analyses of the altered proteins hint at increased cellular growth and proliferation, among others by activating PI3K and CDK4/6. Methods To characterize the frequency and clinical relevance we investigated these candidate genes in a well-defined cohort of 63 pediatric BL that were uniformly diagnosed and treated according to NHL-BFM protocols. Also we screened the second most common pediatric B cell malignancies, precursor B cell leukemias (pB-ALL) and Diffuse Large B-cell Lymphoma (DLBCL), for mutations within this pathway. Initial pretreatment tumor samples from 63 BL (including 14 Burkitt leukemias), 15 DLBCL, 6 cases with features intermediate between BL and DLBCL (B-NHL nfc), 96 pB-ALL and an extended group of another 10 relapsed BL were available for analysis. Tumor cell content for DNA extraction was estimated to be at least 60%. Sanger sequencing was performed for the full coding region of ID3 in all entities. Additionally, TCF3 exon 16 and CCND3 exon 5 were investigated in all BL, DLBCL and B-NHL nfc cases. Clinical data were available from the NHL-BFM and ALL-BFM study center. This study was approved by the Ethical Advisory Board of the University of Giessen (A89/11 Amendment 2013). Results Mutation frequencies for the 60/63 MYC translocation positive BL were 48 (80%) for ID3, TCF3 11 (13%) and CCND3 22 (37%) respectively. Most of the cases presented with more than one ID3 mutation and all affected cases had at least one mutation affecting the functional binding helices of ID3. At least one of the three candidate genes was altered by a potential protein-changing mutation in 53/60 cases (88%). In the group of 15 DLBCL three presented with ID3 mutations. Remarkably, in two of these cases a translocation involving MYC was reported in the study database. There were no ID3 mutations in 96 pB-ALL cases, but two cases presented with non-protein changing variants. With respect to clinical characteristics and patient outcome there were no significant differences with respect to ID3, TCF3 and CCND3 mutation status. To further analyze whether ID3, TCF3 and CCND3 mutations at initial diagnosis have prognostic impact on outcome, ten additional cases of initial tumor samples from patients who suffered relapse were analyzed. However, the ID3, TCF3 and CCND3 mutation status at diagnosis did not differ significantly among the 19 relapsed and the 52 non-relapsed BL patients. Discussion The aim of this study was to determine the relevance of ID3, TCF3 and CCND3 gene mutations in a well-defined cohort of pediatric BL. Most interestingly we found ID3 mutations in BL with a significantly higher frequency than previously published in the index studies including patients of all age groups by Richter et al., Schmitz et al. and Love et al., with frequencies ranging between 34 and 68%. The finding that ID3 mutations were virtually exclusive for MYC translocation positive tumors offers the prospect of the ID3 mutation status as a criterion of interest for analysis of intermediate cases between BL and DLBCL. As expected, no ID3 mutations were found in pB-ALL cases, supporting the assumption of exclusive occurrence within the pathogenesis of mature B cell lymphoma. While there was no evidence for a certain role with respect to clinical manifestations and patient outcome, the high frequency of 88% BL cases with at least one mutation in ID3, TCF3 or CCND3 leads to the proposal that this pathway plays an emerging role especially in pediatric BL and it might represent the second hit in BL pathogenesis. This is of special clinical interest as already available kinase inhibitors might successfully address the downstream targets PI3K and CDK4/6. Disclosures No relevant conflicts of interest to declare.

Background Follicular lymphoma (FL) is a B-cell lymphoma whose cytogenetic hallmark is the translocation t(14;18)(q32;q21) which juxtaposes the BCL2 oncogene to the immunoglobulin heavy chain locus (IGHV). FLs maintain key features of normal germinal center reactions, such as ongoing somatic hypermutation (SHM) of IGHV genes and selection for a functional B-cell receptor. SHM is mediated by activation-induced cytidine deaminase (AID) leading to single nucleotide exchange in IGHV genes and to a much lesser extent in non-IG genes. It was our objective to investigate the clonal evolution of t(14,18) FL from primary to relapse tumors simultaneously on several genetic and epigenetic levels. Methods We studied paired primary and relapsed tumors from 33 patients with t(14;18)-positive FL (76 samples: 25 pairs; 6 trios and 2 quadruples). In a core set of 19 patients we performed Sanger and next generation sequencing of the clonal VHDHJH rearrangements of the IGHV locus. We performed deep sequencing of 9 genes (BCL2, BCL6, MYC, RHOH, PAX5, IRF4, C2TA, REL and PIM1) targeted by aberrant SHM (aSHM) in lymphoma for 69 FL samples including the complete core set. We furthermore analyzed mutations in the coding regions of 10 candidate driver genes of lymphomagenesis (BCL2, MLL2, CREBBP, TNFRSF14, EZH2, EP300, MEF2B, BCL6, MYC, TP53) by deep sequencing. In addition to genetic analyses, evolutionary patterns of DNA-methylation were addressed by Illumina 27k arrays. Results We found strong evidence for ongoing selection against replacement mutations in the IGHV genes both in complementarity determining regions and framework regions, consistent with ongoing dependence of FL on a functional B-cell receptor and stimulation by antigens. Using mean normalized Hamming distance as a quantitative measure for the evolutionary divergence of paired samples (IGHV-divergence) and analyzing phylogenetic trees we classified the patterns of evolution into 3 categories: “No Evolution” (shared IGHV sequences in primary and relapse tumors), “Sequential Evolution” (relapse sequences emerge out of primary ones), “Divergent Evolution” (sequences of primary and relapse sample appear disjoint). We observed a mutation frequency of 62.0 per 100 kb in aSHM targets. These mutations were strongly enriched at the WRCY/RGYW target motifs characteristic for the SHM/AID machinery (OR=3.4; p We detected 197 single nucleotide variants (SNV) affecting the candidate driver genes (4.4 mutations per 100 kb) of which 145 were protein-changing. The genes most frequently affected were CREBBP (52 SNVs in 43/69 samples) and MLL2 (54 in 39/69). High incidence of CREBBP mutations and the high mutated allele frequencies of these mutations suggest that CREBBP deregulation is an early event in FL lymphomagenesis. We defined the aSHM-divergence as the proportion of observed aSHM mutations not present in both samples and found a significant correlation between the IGHV-divergence and the aSHM-divergence in the non-IG aSHM targets (r=0.724 [0.40-0.88]). We calculated a measure for divergence in DNA-methylation within patients by the proportion of all paired CpGs showing a methylation difference of at least 25%. DNA-methylation-divergence was found to correlate with IGHV-divergence (r=0.516 [0.24-0.72]) indicating that evolutionary divergence also affects the level of DNA-methylation. Conclusion Our observations demonstrate correlated evolutionary changes on all genomic and epigenomic levels in FL. This process most likely originates from an ongoing germinal center reaction with a functional BCR driving FL cells, sustaining a continuously active AID-machinery leading to addition of IGHV-mutations and to aberrant mutations in non-IG aSHM-target sites. The drifts in DNA-methylation patterns might be a consequence of additional mutations found in histone modifying genes such as CREBBP. The individual pattern of tumor evolution is likely to impact prognosis and clinical decision making which needs to be investigated in larger series. (Acknowledgment: BMBF/PTJ 0315452) Disclosures: No relevant conflicts of interest to declare.

Abstract 317 Evidence for Antigen-driven Development of Molecularly Classified Burkitt Lymphomas The presence of somatic mutations and intraclonal diversity (ID) in the variable region of rearranged immunoglobulin (Ig) genes is commonly used as a marker of the cellular passage of B cells through the germinal center (GC) reaction in secondary lymphoid tissues following an in vivo antigen encounter. Such features have also been documented in different subgroups of aggressive B-cell lymphomas, suggesting that antigen selection pressure has taken place during lymphoma development. We undertook a detailed analysis of the Ig repertoire and somatic hypermutation (SHM) status of 54 molecularly classified Burkitt lymphomas (BL) (Hummel et al. 2006). The IGHV gene repertoire was remarkably biased, with only three genes accounting for 40.8% of all cases (IGHV4-34, 18.5%; IGHV4-39, 11.1%; IGHV3-30, 11.1%). Of note, this bias was even more evident when cases were grouped according to age < or > 14 years, with the IGVH4-34 and IGVH4-39 rearrangements occurring almost exclusively in pediatric BL (7/11 and 5/6 cases respectively). Only 2/54 cases (3.7%) carried unmutated IGHV genes (100% germline identity, GI); 12/54 cases (22.2%) carried IGHV genes of borderline/minimally mutated status (98–99.9% GI); finally, the remaining 40/54 cases (74.1%) carried mutated IGHV genes ( To assess the presence of ID through ongoing SHM, we evaluated 411 subcloned sequences from 11 IGVH4-34 and 6 IGVH4-39 cases. Only one case carried identical sets of subcloned sequences; seven cases carried mutations in single subcloned sequences that were considered as unconfirmed; finally, 9 cases carried at least one confirmed mutation among two or more subcloned sequences and, thus, were considered as exhibiting confirmed ID. However, analysis of the distribution of the so called unconfirmed changes provided further evidence for the very precise targeting of mutations introduced as part of the ID process. Thus, certain changes identified in single subcloned sequences from one case were shared by most or all subcloned sequences of another case utilizing the same IGHV gene (“confirmed by another case”). In conclusion, our data demonstrate that BL is characterized by a highly distinctive IG gene repertoire with a precisely targeted SHM process. In addition, they provide strong evidence for a functionally driven SHM, supporting a role for antigen-driven selection of the clonogenic progenitors. Disclosures: No relevant conflicts of interest to declare.