ALBERT

Description: Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both biologically and clinically. Although a number of genetic alterations have been associated with the pathogenesis of DLBCL, survival association of most of the identified mutations remains to be shown. Here we have used exome and RNA sequencing (RNAseq) data to identify novel genetic events associated with treatment outcome after chemoimmunotherapy. Initial screen was performed with eight primary tumor samples and matched normal DNA using exome sequencing. The patients had clinically high risk DLBCL, and were treated in a Nordic phase II protocol with dose dense chemoimmunotherapy and CNS prophylaxis. After stringent filtering (VarScan2 p 〈 0.05), and removal of known germline variants, 967 somatic point mutations and indels in 561 different genes were identified. Of these, 436 were non-synonymous, and 27 were detected in more than one patient. These included both novel and several previously described DLBCL associated mutations, such as MYD88, CD79B, B2M and PTEN. To correlate the identified mutations with survival, we used RNAseq and survival data of 92 DLBCL samples from the Cancer Genome Characterization Initiative (CGCI) repository. The mutational status of the 436 genes identified in our screen was assessed with Bambino, which confirmed 405 of these genes also to be mutated in the CGCI set. Kaplan-Meier survival estimates per gene with at least one single nucleotide variant (SNV; with criteria of minimum 3 reads and 10% coverage and known polymorphisms filtered out) revealed 17 genes that were associated with overall survival (OS, p

Description: Introduction. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoid malignancy with a diverse genomic appearance. To date, the coding genomes of almost 2000 DLBCLs have been recognized, and the translation of this genetic information into clinical practice is awaited. Here, we have investigated translocations and protein expression of the hallmark DLBCL genes BCL2, BCL6 and MYC in a well-characterized sample series. The findings are correlated with gene expression, copy number alterations (CNAs) and exome-wide mutational annotations, and molecular data associated with clinical characteristics and treatment outcome. Methods. A tissue microarray (TMA) was constructed from formalin fixed paraffin embedded (FFPE) tumor tissue of 152 primary DLBCL patients treated with R-CHOP or R-CHOP-like regimen. Translocation status was determined for BCL2, BCL6 and MYC genes with break-apart fluorescent in situ hybridization (FISH). BCL2, BCL6 and MYC protein expression was determined immunohistochemically. RNA sequencing data of the log2 transformed gene expression values and whole-exome sequencing annotations of the mutations and CNAs were derived for each case from the 1001 DLBCLs study (Reddy et. al. Cell 2017; 171:481-494). Results. Overall, 55% of the samples were BCL2+. Of the BCL2+ cases, 58% had an underlying structural variation in the BCL2 locus. In the whole study cohort, structural variants of the BCL2 gene were detected in 35% of the cases, and they were associated with high gene expression and immunoreactivity (Figure 1A). BCL2 translocations were detected in 16% of the cases, and they were mutually exclusive with amplifications. Translocations and co-occurring coding mutations of the BCL2 gene were enriched in the GCB subtype and amplifications in the ABC/non-GCB DLBCLs. In the ABC/non-GCB DLBCLs, BCL2 copy number aberrations were associated with poor survival independent of IPI (Figure 1B). High MYC immunoreactivity (〉70% cut-off, 12 % of all cases) was evenly distributed between the subtypes. However, MYC translocations (14% of all cases) were associated with the GCB phenotype and elevated gene expression (Figure 1C). Nonsynonymous mutations of the MYC gene were only seen in the translocated cases. Co-occurring MYC and BCL2/BCL6 translocations (Double hit lymphomas, DHL) were recognized in 5.6% cases, most of which were GCB DLBCLs with poor outcome (Figure 1D). Dual protein expression (DPE) of BCL2 and MYC was detected in 9.5% of the cases. DPE lymphomas were evenly distributed between the subtypes, and they had adverse prognostic significance independent of IPI and molecular subtype (Figure 1D). BCL6 translocations (15% of the cases) did not associate with elevated gene expression or coding mutations of the BCL6 gene (Figure 1E). Translocations were more prevalent in the non-GCB than the GCB DLBCLs and mutually exclusive with the MYD88 L265P mutations. Furthermore, BCL6 translocation status highlighted a subgroup of patients with a favorable outcome in the non-GCB DLBCLs independent of IPI (Figure 1F). Conclusions. Our study characterizes the prevalence and clinical impact of hallmark structural variations in DLBCL. The results dignify structural, mutational and expression analysis of BCL2, BCL6 and MYC in the clinical practice, and increase our understanding of different entities within the well-established molecular subtypes. Figure 1. A, C, E) Interplay between structural variants, gene expression, and nonsynonymous SNVs of BCL2, MYC and BCL6, and with immunohistochemical staining scores for BCL2 and MYC proteins are illustrated. B) CNAs of BCL2 and poor survival in patients with non-GCB DLBCL. D) DHLs and DPEs (other than DHL associated) and poor survival. F) BCL6 translocations and favorable outcome in patients with non-GCB DLBCL. Figure 1. Figure 1. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy; Bayer: Research Funding.

Description: Introduction Diffuse large B cell lymphoma (DLBCL), the most common lymphoma world-wide, is strikingly heterogeneous. This heterogeneity creates a daunting challenge for conducting well-powered studies connecting molecular features to clinical outcome. Not only is the association of genetic mutations with clinical outcome in DLBCL mostly unknown, the relative importance of other well-described features, such as MYC and BCL2 translocation/expression and cell of origin based subsets (ABC and GCB DLBCL), is difficult to interpret due to conflicting reports. We sought to comprehensively define the spectrum of genetic mutations and their association with clinical outcome in DLBCL. Our calculations indicated that 500 tumor-normal pairs would provide 95% power to define mutations occurring in at least 5% of patients, and that 800 cases would be required to define the clinical correlations with cross-validation. Methods We enrolled 1001 de novo DLBCL patients, with complete IPI and survival data, who were treated uniformly with standard rituximab and anthracycline containing regimens. All tumors were subjected to whole exome and transcriptome sequencing (RNAseq), as well as SNP arrays to confirm genetic alterations. ABC (38%) and GCB DLBCL (36%) subtypes were defined using microarrays and RNAseq in these patients to examine subgroup-based differences in mutations and outcome. MYC and BCL2 expression were quantified separately. Results Gene discovery analysis of somatic mutations and copy number alterations in exome sequencing data from 502 tumor-normal pairs of DLBCL identified 197 recurrently mutated genes, including 155 genes previously identified to be mutated in DLBCLs. In addition, our study uncovered 42 novel driver genes in DLBCL (e.g. BTK, SPEN, CD70). Exome sequencing results were validated by Sanger sequencing of 1120 variants with over 90% concordance. We also identified copy number alterations in these genes, with strong agreement (90%) of amplifications and/or deletions to those detected on Illumina high resolution SNP microarrays. These 197 genes were found to comprise 15 functionally related subnetworks, including those related to histone modification, NFkB, B cell receptor, PI3K and cell cycle (Figure 1). Within each subnetwork, the vast majority of the gene alterations occurred in a mutually exclusive (P

Description: Introduction: Concomitant deregulation of MYC and BCL2, whether at the genomic or protein level, comprises clinically a significant biological high-risk feature in aggressive B-cell lymphomas. Yet, the molecular background of these lymphomas is not fully understood. We sought to comprehensively characterize BCL2 and MYC aberrations and to identify clinically feasible determinants of the recent genomic subtypes and clinical outcome in diffuse large B-cell lymphoma (DLBCL). Patients and Methods: We analyzed BCL2, BCL6 and MYC rearrangements and corresponding protein expression in the context of somatic exome-wide driver gene alterations, copy number annotations, transcriptome and clinical data in 181 patients with primary DLBCL. Results: We found that germinal center B-cell (GCB) and activated B-cell (ABC)-like DLBCLs utilized different mechanisms to deregulate MYC and BCL2 expression. While the structural variations of BCL2 were subtype-specific and specifically increased antiapoptotic BCL2 expression, MYC deregulation was associated with gene expression signatures including biosynthetic pathways and telomerase activity and depletion of JAK-STAT, cytokine and immune cell signatures. Genomic dissection of MYC translocations and protein overexpression revealed associations with other lymphoma drivers, of which we highlight a novel interplay between MYC deregulation and TP53 alterations. Double protein expression (DPE) arose from different molecular backgrounds in a subtype-dependent manner. In GCB-DLBCL, concurrent alterations of MYC and BCL2 loci gave rise to the majority of DPE DLBCLs. Importantly, we recognized more GCB lymphomas with double hit -like clinical behavior when BCL2 copy number alterations were considered. In the non-GCB DLBCLs in turn, concurrent alterations were rare and clinical behavior of DPE was dependent on the molecular context. Interestingly, BN2/C1 -like non-GCB lymphomas with BCL6 translocations had excellent outcomes regardless of the DPE status. Overall, DPE DLBCL defined a prognostic entity, which was independent of International Prognostic Index (IPI) and cell-of-origin, and together with TP53 alterations had synergistic dismal impact on survival. Conclusion: Our findings elucidate the pathogenesis and modern biological determinants of high-risk DLBCL and reveal subtype-specific and clinically feasible improvements to risk-stratification and predictors of outcome. Disclosures Leppa: Celgene: Consultancy; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Roche: Consultancy, Honoraria, Research Funding; Bayer: Research Funding.

Description: Follicular Lymphoma (FL) is the most common indolent lymphoma derived from light zone germinal center B cells and characterized by a t(14;18) translocation resulting in upregulation of BCL2 in over 80% of cases. This translocation alone is not sufficient for tumorogenesis, and must be combined with additional genetic mutations to transform B cells. FL is incurable and the disease course can be highly varied, with survival ranging from a few months to decades following diagnosis and treatment with standard chemoimmunotherapy. The heterogeneity of FL poses major challenges to identifying the association of genetic alterations and clinical outcome. Current WHO guidelines recommend establishing grade for each FL case with grade 3 thought to be more aggressive than 1 and 2. The genetic basis and clinical implications of grade in FL are unclear. Recent sequencing studies have identified many genes found to be recurrently mutated in FL including KMT2D and CREBBP. However, the degree to which genetic alterations cooperate with each other or contribute to clinical outcome is unclear. Based on the observed mutational rates in follicular lymphoma, we estimated 900 cases were needed to comprehensively delineate the genetic alterations that underlie histologic grade and clinical outcome. Accordingly, we enrolled a cohort of 1042 patients with newly diagnosed FL. All treated patients received rituximab-containing standard regimens. To go beyond the identification of gene-coding events, we developed a very large panel of 110 Mbp covering exonic (~40Mbp) and non-exonic regions (~70Mbp) of interest to enable a wide range of genomic analysis including mutation calling in both coding and non-coding regions, rearrangement detection, viral identification, and copy number analysis. In addition to the whole exome, we extended coverage to include introns, promoters, and untranslated regions of all known driver genes in cancer. We included the entirety of the immunoglobulin loci, T-cell receptor loci and CD3 loci to detect clonotypes and rearrangements. We also included lymphoma-relevant long non-coding RNAs, microRNAs, enhancers, and breakpoint-prone regions. For viral detection, we targeted the genomes of eight cancer-related viruses: Epstein-Barr virus, human papillomavirus, human immunodeficiency virus, hepatitis B, hepatitis C, Kaposi's sarcoma-associated herpesvirus, human T-lymphotropic virus, and Merkel cell polyomavirus. In addition, to enable high resolution identification of copy number variation (CNV) calls, the entire genome was tiled with probes spaced 10kb apart. DNA and RNA were extracted from all tumors and their paired normal samples, prepared into DNA and RNA sequencing libraries and subjected to sequencing on the Illumina platform to a targeted coverage of 150X. Somatic events were identified and further filtered to identify driver events in both coding and non-coding regions. FLs demonstrated a significant degree of genetic heterogeneity with over 100 genes mutated with a frequency of at least 2%. Nearly 100% of FL cases had a mutation in at least one chromatin-modifying gene. The most frequently mutated genes in follicular lymphoma were KMT2D, BCL2, IGLL5 and CREBBP. In addition, we identified frequent mutations in SPEN, BIRC6 and SETD2. To our knowledge, this is the first description of alterations in these genes in FL. Transcriptome analysis indicated a strong correlation between BIRC6 mutations and the previously described immune response 2 signature that is associated with a poor prognosis. We further performed unbiased clustering of genetic alterations in these FL cases. We identified a cluster that was specifically enriched in BCL6 and TP53 alterations and was strongly associated with grade 3 FLs which are predicted to have poorer outcomes with low intensity therapies. We further examined the genetic profiles of 1001 DLBCLs in comparison to this cohort of FLs. Our data indicate a continuum of highly overlapping genetic alterations with DLBCL displaying more complex patterns that included alterations in MYC, TP53 and CDKN2A (mainly copy number losses), indicating shared pathogenetic mechanisms underlying FL and DLBCL, particularly those germinal center B cell origin. Disclosures Koff: Burroughs Wellcome Fund: Research Funding; V Foundation: Research Funding; Lymphoma Research Foundation: Research Funding; American Association for Cancer Research: Research Funding. Leppä:Roche: Honoraria, Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen-Cilag: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees. Gang:ROCHE: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Hsi:Abbvie: Research Funding; Eli Lilly: Research Funding; Cleveland Clinic&Abbvie Biotherapeutics Inc: Patents & Royalties: US8,603,477 B2; Jazz: Consultancy. Flowers:AbbVie: Consultancy, Research Funding; Denovo Biopharma: Consultancy; BeiGene: Consultancy, Research Funding; Burroughs Wellcome Fund: Research Funding; Eastern Cooperative Oncology Group: Research Funding; National Cancer Institute: Research Funding; V Foundation: Research Funding; Optimum Rx: Consultancy; Millenium/Takeda: Research Funding; TG Therapeutics: Research Funding; Gilead: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Karyopharm: Consultancy; AstraZeneca: Consultancy; Pharmacyclics/Janssen: Consultancy, Research Funding; Spectrum: Consultancy; Bayer: Consultancy; Acerta: Research Funding; Genentech, Inc./F. Hoffmann-La Roche Ltd: Consultancy, Research Funding. Neff:Enzyvant: Consultancy; EUSA Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees. Fedoriw:Alexion Pharmaceuticals: Other: Consultant and Speaker. Reddy:Genentech: Research Funding; BMS: Consultancy, Research Funding; Celgene: Consultancy; KITE Pharma: Consultancy; Abbvie: Consultancy. Mason:Sysmex: Honoraria. Behdad:Loxo-Bayer: Membership on an entity's Board of Directors or advisory committees; Thermo Fisher: Membership on an entity's Board of Directors or advisory committees; Pfizer: Other: Speaker. Burton:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel; Celgene: Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees. Dave:Data Driven Bioscience: Equity Ownership.

Description: Introduction Relapses of diffuse large B-cell lymphoma typically occur within 2-3 years and only 10% of these patients reach a 3-year progression-free survival compared to 65% at diagnosis. Our ability to distinguish patients at risk for relapse remains based on clinical staging. We hypothesized that identifying genetic alterations in serial tumour biopsies at diagnosis and relapse would improve our ability to identify high-risk patients, make therapeutic selections and reveal molecular markers for chemo-immunotherapy resistant tumours. However, relatively few relapsed/refractory biopsies have been sequenced. A unique, clinically annotated, Nordic DLBCL cohort was used to identify significantly mutated genes, assess potential driver genes, comprehensively examine clonal evolution, and gauge the importance of clinical relapsed sampling. Methods To address the lack of information on the molecular foundations of relapsed/refractory DLBCL, we performed whole exome sequencing (WES) on 42 DLBCL cases, with 34% representing relapsed/refractory biopsies and 13 serially sample cases. Enriched with relapsed/refractory diffuse large B-cell lymphoma cases, we performed multiple computational analyses to identify significantly mutated genes (MutSig2CV), mutational signatures (NMF and DeConstructsSig), driver genes (IntOgen and CADD), clonal evolution architecture (SciClone and ClonEvol), druggable gene analysis (DGIdb), and HLA-inference and mutation calling (Polysolver). Results Clonal evolution analysis of 13 paired diagnostic and relapsed biopsies revealed that relapsed/refractory biopsies have remarkable similarities to diagnostic biopsies and often present with late divergent clonal evolution of the tumor. Mutational analysis of 18 serially sampled tumors determined that in the majority of cases druggable oncogenic variants do arise at relapse. In addition, time to relapse correlated with divergence of mutations from the diagnostic biopsy. In addition to being identified as a significantly mutated gene, mutations in HLA-A had an increased incidence in cases that ultimately relapsed. This result led to an in-depth investigation into the mutational prevalence, timing, impact on prognosis, and loss of heterozygosity in the human leukocyte antigen (HLA) haplotypes of relapsed/refractory DLBCL. HLA-A mutagenesis and loss of heterozygosity was discovered as mechanisms of immune evasion in cases that go on to relapse from R-CHOP like therapies (Figure 1). Conclusions Our results yield insight into the development of chemo-immunotherapy resistant diffuse large B-cell lymphoma, and highlight the clinical importance of sampling relapsed biopsies. Analysis of immune evasion through MHC Class I/II, specifically HLA-A, may provide better characterization of patients for relapse prediction. In the age of personalized medicine it will be instrumental to determine if relapsed biopsies offer additional insight for salvage therapy treatment. Divergence of biopsies, as characterized by shared genomic mutations, increase with time and the majority of cases present with new alterations in druggable genes post-therapy. Disclosures Leppa: Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Celgene: Consultancy; Bayer: Research Funding. Holte:Novartis: Honoraria, Other: Advisory board.

Description: Introduction Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease both clinically and biologically. Based on gene expression profiling, two major molecular subtypes, activated B-cell (ABC)- and germinal center B-cell (GCB)-like DLBCLs, have been recognized. More recently, next generation sequencing has also shed light on the mutational heterogeneity of these lymphomas. While several oncogenes are recurrently mutated and aberrantly expressed in DLBCL, little is known of their functional impact and association with survival. Design and Methods To identify mutated oncogenes in DLBCL, we performed whole-exome and RNA sequencing screen for tumor tissue and matched normal DNA from eight high-risk (aaIPI 〉2) DLBCL patients less than 65 years old. The patients were treated in the Nordic phase II protocol with dose-dense chemoimmunotherapy followed by systemic central nervous system (CNS) prophylaxis. Initial screen was expanded to a Nordic discovery cohort consisting of 38 high-risk DLBCL patients treated in the same protocol. Gene expression was analyzed from the tumor tissue using Affymetrix Human Exon 1.0 ST arrays, quantified with MEAP (Multiple Exon Array Preprocessing, probe annotation version 70) algorithm, and expression of the mutated genes identified. Samples were classified into GCB, ABC and nonclassified subgroups using the Lymphochip gene predictor. At the time of the analysis, median follow-up time was 65 months and predicted 5-year OS 76%. Survival association of the identified mutations and gene expression was validated using RNAseq data from 92 (Cancer Genome Characterization Initiative (CGCI) repository), and oligonucleotide-based microarray expression data from 233 (Lymphoma/Leukemia Molecular Profiling Project, LLMPP) DLBCL patients, respectively. Patients were treated with chemoimmunotherapy in both validation cohorts. Results In our initial screen 922 genes were affected by somatic mutations. The genes included both novel and previously identified lymphoma-related oncogenes, such as MYD88, CD79B and DTX1. We highlight mutations affecting GC marker Kelch-like family member 6 (KLHL6), which is a predicted oncogene and involved in B-cell receptor (BCR) signaling in DLBCL. In the CGCI validation cohort 16% of the patients carried non-silent KLHL6 mutations. According to Kaplan Meier analysis, the patients with KLHL6 mutations had significantly worse survival in comparison to the patients without mutations (5-year OS; 53% vs 84%; p = .010). In multivariate analysis, the prognostic impact of KLHL6 mutations on the survival was independent of IPI (OS; RR, 3.060; 95% CI, 1.233-7.598; p = .016). KLHL6 mutations were equally distributed between the molecular subtypes. However, when the molecular subtypes were analyzed separately, KLHL6 mutations remained predictive for poor survival only in the ABC-like DLBCL (OS; RR, 6.727; 95% CI 1.576-28.714; p= .010). KLHL6 expression was further analyzed in the Nordic discovery cohort. Consistent with its role as a GC marker, KLHL6 expression tended to be higher in the GCB- than the ABC-like DLBCLs. Overall, low KLHL6 expression was associated with poor survival (5-year OS; 93% vs 65%; p = .044). The difference in survival was restricted to the ABC subtype. The prognostic impact of KLHL6 expression in the patients with the ABC-like DLBCL was confirmed in independent LLMPP validation cohort. Conclusions The results show that although high KLHL6 expression is characteristic for the GCB DLBCL, mutations and low expression of KLHL6 are clinically more relevant in the ABC subtype pinpointing the patients with the most dismal prognosis. Disclosures Leppä: CTI Life Sciences: Honoraria; Roche: Honoraria, Other: Travel Expenses, Research Funding; Takeda: Honoraria, Other: Travel expenses; Bayer: Honoraria, Research Funding; Mundipharma: Research Funding; Janssen: Research Funding; Amgen: Research Funding.

Description: Background: Aggressive non-Hodgkin lymphoma (NHL) relapsing after standard first line chemotherapy represents an unmet clinical need. Currently, a phase 1/2 study with the combination of pixantrone, etoposide, bendamustine, and in CD20 positive tumors, rituximab, in patients with relapsed aggressive NHL of B- or T- cell phenotype (the PREBEN study) is ongoing. Here our aim was to molecularly characterize samples from the PREBEN trial and find clinical correlates for predicting treatment response. Methods: The profiling cohort consisted of 21 patients with pre-treatment RNA samples and clinical data. Nanostring PanCancer Pathways and PanCancer Immune profiling panels (altogether 1348 genes) were utilized for the gene expression analyses. The findings from gene expression analyses were correlated with clinical parameters. Results: Fourteen patients had diffuse large B-cell lymphoma (DLBCL), whereas seven had peripheral T-cell lymphoma (PTCL). In general, the expression of DNA replication genes distinguished DLBCL from PTCL. Additionally, gene expression analyses identified genes having differential expression based on the response to the treatment. Supervised hierarchical clustering of the ten most differentially expressed genes could separate the responding (n=4) and non-responding (n=10) DLBCL patients into two distinct subgroups (Fig. 1A). Similarly, the responding (n=3) and non-responding (n=4) PTCL patients could be separated into distinct subgroups by supervised clustering with the ten most differentially expressed genes (Fig. 1B). Conclusion: Molecular profiles of aggressive NHL are heterogeneous and may be utilized for predicting the treatment response. More detailed molecular analyses are currently ongoing. Disclosures Jørgensen: Gilead: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees. d'Amore:Servier: Research Funding. Leppa:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Research Funding; Bayer: Research Funding; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Honoraria, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. OffLabel Disclosure: combination of bendamustine and pixantrone for relapsed NHL

Description: Introduction Sequencing of circulating cell-free tumor DNA (ctDNA) has opened a diagnostic avenue for the patients with B-cell neoplasias, including diffuse large B-cell lymphoma (DLBCL). Treatment refractoriness and disease relapse after first-line therapy are associated with dismal survival. We sought to assess the clinical feasibility and prognostic value of ctDNA detection in prospectively collected samples from young high-risk patients treated uniformly in a Nordic phase II study. Materials and Methods The patients were treated in a phase II trial with dose-dense chemoimmunotherapy (biweekly R-CHOEP) and early systemic CNS prophylaxis with high-dose methotrexate (NLG-LBC-05; Leppä et al., 15-ICML; ClinicalTrials.gov NCT01325194). Longitudinal plasma samples from all trial centers were gathered and the plasma purified for cell-free DNA (cfDNA) in Helsinki, Finland (Figure 1A). We designed a 225 kb custom panel targeting most commonly mutated genomic drivers, regions of aberrant somatic hypermutation and immunoglobulin genes. Next-generation sequencing was performed on longitudinal cfDNA samples and primary tumor samples together with matched germline controls from the first 32 patients in the study. Sequencing was completed on Hiseq2500 platform and depth after duplicate removal with unique molecular identifiers was on average 1,926x (ctDNA), 3,073x (primary tumors) and 2,171x (germline controls). Multi-tumor variant calling was performed by Mutect2. Results cfDNA was successfully extracted from 96 patients (Figure 1B). Among the patients with completed sequencing and bioinformatics pipeline, we identified a median of 87 traceable somatic mutations in 97% of the patients. In plasma samples, ctDNA was detectable in 93% of the patients prior to treatment. Compellingly, we observed that the levels of total cfDNA and ctDNA were higher at baseline in the patients who relapsed. We evaluated the concordance between pretreatment ctDNA and primary tumor biopsy mutations in patients with available material. The percentage of shared somatic mutations ranged between 14 - 100%, suggesting substantial spatial heterogeneity in some cases. While the mutations in known lymphoma genes were mostly shared between the baseline ctDNA and the diagnostic tumor biopsy, some lymphomas displayed vast discordance mostly in the immunoglobulin loci, suggesting active somatic hypermutation processes upon clonal divergence in these lymphomas (Figure 1C). In all longitudinally analyzed plasma samples, the levels of ctDNA dropped after the first three treatment courses. At the end of therapy, the levels of ctDNA were undetectable in most of the patients with durable remissions (Figure 1D #1). However, in the patients with progressive disease, the ctDNA levels increased at the end of the therapy (Figure 1D #2). In the patients with late relapse (〉12 months progression free survival), we observed the appearance of a subset of the mutations that were identified at diagnosis, suggesting clonal selection (Figure 1D #3). Conclusion Our findings elucidate the biology of ctDNA in B-cell lymphomas, and the ctDNA kinetics in homogeneously treated patients with primary high-risk DLBCL. Figure 1. A) Collection of cfDNA samples in the CHIC trial. B) Swimmers plot of the patients with extracted cfDNA in the liquid biopsy cohort. C) Concordance between primary tumor and ctDNA mutations at diagnosis. CHIC_100: high concordance. CHIC_88: low concordance. D) Kinetics of ctDNA in patients with different diseases courses. Figure 1 Disclosures Jørgensen: Roche: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Jerkeman:Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Acerta: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Roche: Honoraria, Research Funding. Holte:Novartis: Honoraria, Other: Advisory board. Leppa:Celgene: Consultancy; Bayer: Research Funding; Roche: Consultancy, Honoraria, Research Funding; Takeda: Consultancy, Research Funding; Janssen: Consultancy, Research Funding.