ALBERT

Description: Introduction The ETV6-RUNX1 fusion gene,the most common subtype of childhood pB-ALL, is acquired in utero, producing a persistent and hidden preleukemic clone. However, the underlying mechanism explaining how the preleukemic clone evolves to pB-ALL remains to be identified. The lack of genetically engineered human-like ETV6-RUNX1 pB-ALL models has hampered our understanding of the pathogenesis of this disease. Methods We have used a novel experimental approach to generate a murine strain that mimics the human ETV6-RUNX1 pB-ALL. We expressed ETV6-RUNX1 specifically in hematopoietic stem cells (HSC) of C57BL/6 x CBA mice by placing ETV6-RUNX1 under the control of the Sca1 promoter. Two founder mice were obtained for the Sca1-ETV6-RUNX1 transgene, which had normal gestation, were viable and developed normally. Sca1-ETV6-RUNX1 transgenic mice were characterized with respect to clinical, immunephenotypic and genetic characteristics. For the detection of shared secondary genomic alterations we analyzed three murine Sca1-ETV6-RUNX1 and 11 ETV6-RUNX1 positive human pB-ALL and corresponding germline by whole-exome (WES) and whole-genome sequencing using a HiSeq 2500 (Illumina) platform. Results In our transgenic murine model Sca1-ETV6-RUNX1 transgene expression was detected in HSCs, while there was no detectable expression in pro B cells or later stages of B-cell development, which mimics human ETV6-RUNX1 preleukemic biology. Sca1-ETV6-RUNX1 mice developed exclusively pB-ALL at a low penetrance (7.5%; 3 out of 40) with a CD19+ B220+ IgM- cell surface phenotype. Overall survival was not significantly reduced compared to wild-type mice (P value = 0.7901). pB-ALL in Sca1-ETV6-RUNX1 mice manifested with splenomegaly, disruption of splenic architecture, and appearance of blast cells in the peripheral blood (PB). All leukemic cells displayed clonal immature BCR rearrangement. Tumor pro B cells grew independent of IL-7 and were able to propagate the disease when transplanted into sub-lethally irradiated syngeneic recipient mice. Whole-exome sequencing of murine pB-ALL revealed in one mouse a deletion of three amino acids in the B-cell differentiation factor EBF1, which is well known in the context of human ETV6-RUNX1 leukemia. Additionally we found mutations in genesimplicated in histone modification, i.e. in KDM5C causing a premature translation stop. We compared the genomic alterations detected in the mouse model to published genomic data of pediatric ETV6 -RUNX1 pB-ALL and identified multiple copy number variations, which are shared between the murine and human ETV6 -RUNX1 pB-ALL. Among them were copy number gains and losses including i.e. the tumorsuppressor locus CDKN2A/B with a well-known role in human and mouse pB-ALL. A high proportion of genes implicated in histone modification was also mutated in published data of human ETV6-RUNX1 positive pB-ALL. We validated this novel finding of recurrent alterations of histone modifying genes in both the murine model and the human disease using an independent human ETV6-RUNX1 cohort of 11 patients. In this cohort were able to reproduce this finding. Similar to the murine model, we also detected a missense mutation in the methyltransferase KDM5C in one patient of our cohort of ETV6-RUNX1 positive patients. Conclusion In summary, we have characterized a new Sca1-ETV6-RUNX1 mouse model and this is, to our knowledge the first model, which represents a phenocopy of the human pB-ALL. Sca1-ETV6-RUNX1 mice develop exclusively pB-ALL at a very low penetrance as it is the case in human ETV6-RUNX1 positive pB-ALL. The acquisition of secondary mutations in pB-ALL with a high proportion in histone modifying genes confers the second hit for the conversion of a preleukemic clone into the clinically overt ETV6-RUNX1 positive pB-ALL disease. These findings are important for encouraging novel interventions that might help to prevent or treat ETV6-RUNX1 positive childhood leukemias. Disclosures No relevant conflicts of interest to declare.

Description: Despite improvement in the treatment of advanced classical Hodgkin lymphoma, approximately 30% of patients relapse or die as result of the disease. Current predictive systems, determined by clinical and analytical parameters, fail to identify these high-risk patients accurately. We took a multistep approach to design a quantitative reverse-transcription polymerase chain reaction assay to be applied to routine formalin-fixed paraffin-embedded samples, integrating genes expressed by the tumor cells and their microenvironment. The significance of 30 genes chosen on the basis of previously published data was evaluated in 282 samples (divided into estimation and validation sets) to build a molecular risk score to predict failure. Adequate reverse-transcription polymerase chain reaction profiles were obtained from 262 of 282 cases (92.9%). Best predictor genes were integrated into an 11-gene model, including 4 functional pathways (cell cycle, apoptosis, macrophage activation, and interferon regulatory factor 4) able to identify low- and high-risk patients with different rates of 5-year failure-free survival: 74% versus 44.1% in the estimation set (P 〈 .001) and 67.5% versus 45.0% in the validation set (P = .022). This model can be combined with stage IV into a final predictive model able to identify a group of patients with very bad outcome (5-year failure-free survival probability, 25.2%).

Description: Background: Waldenström's macroglobulinemia (WM) is a rare immunoproliferative neoplasia with indolent characteristics that shows important variability, involving three different stages of presentation: IgM Monoclonal Gammopathy of Undetermined Significance (IgM-MGUS), asymptomatic WM (AWM), and Symptomatic WM (SWM). Whole-genome sequencing and some specific approaches have identified MYD88 L265P (90%) and CXCR4 (29%) mutations as the most recurrent somatic mutations in WM. However, other genetic abnormalities under as well as the mechanisms responsible for this clinical heterogeneity still remain to be clarified. Therefore, our aim was to analyze the genomic landscape of WM, distinguishing between the three stages of the disease, by using a targeted next generation sequencing (NGS) strategy. Methods: In this study, we performed a comprehensive mutation analysis of genes previously described as frequently involved in Waldenstrom Macroglobulinemia in a large and well characterized cohort of WM patients with the aim to dissect relationships between genotype and clinical and biological characteristics to integrate somatic mutations into a clinical/molecular prognostic model. Twelve genes of interest (ARID1A, CD79A, CD79B, TP53, MYBBP1A, TRAF2, TRAF3, RAG2, HIST1H1B, HIST1H1C, HIST1H1D, and HIST1H1E) were analyzed by high throughput sequencing (Illumina MiSeq, San Diego, CA) with a novel custom amplicon-based panel in a cohort of 61 patients (pts) diagnosed according to WHO classification as follows: 14 MGUS, 23 AWM and 24 SWM. DNA was extracted from bone marrow separated CD19+ B-cells and sequenced in a MiSeq (Illumina) using 150-bp paired-end reads and a mean depth of 2000X. Bioinformatics analysis was carried out with Illumina VariantStudio 2.2. Results were correlated with biological and clinical data of the patients. MYD88 and CXCR4 mutation status, available in all cases, was assessed by ASO-PCR and Sanger Sequencing, respectively. Results: Apart from MYD88 L265P mutations (present in 90% of cases) and CXCR4WHIM (21% of cases), 23 non-synonymous mutations were found, corresponding to 18/61 (30%) patients. Only one patient with MGUS demonstrated one additional mutation (7%), while seven of the AWM (30%), and 10 of the SWM (42%) demonstrated additional mutations (p

Description: It is assumed that patients with primary refractory myeloma are the most likely to benefit from early HDT. In four series the CR rate was between 8 and 40% and the overall survival ranged from 4 to 6 years. However‚ the number of patients with progressive disease vs those with “stable disease” was not given in published series. The aim of our study was to investigate the efficacy in terms of response up-grading and survival of early HDT in patients with primary refractory myeloma. From October 1999 to December 2003 patients with MM younger than 70 years were given 6 courses of VBMCP/VBAD chemotherapy. Patients with refractory disease were scheduled to receive a tandem transplant‚ the first procedure intensified with busulphan-12 mg/kg-/melphalan-140 or melphalan-200 and the second with the CVB -cyclophosphamide‚ etoposide and BCNU- or with a dose-reduced intensity “allo” conditioned with fludarabine/melphalan-140‚ depending on sibling donor availability. Response and progression were defined according to the EBMT criteria. Forty-nine patients with primary refractory disease were identified. Twenty patients showed progression after their initial chemotherapy while 29 patients had “non-responding‚ non-progressive disease”. Eighty percent of the patients achieved some degree of response after the first autologous transplant (CR or near-CR: 8%‚ PR: 56%‚ MR: 16%) while 10% did not respond and 10% died from the procedure. Twenty-four patients were given a second transplant (autologous: 17‚ allogeneic: 7). Forty-six percent of the 17 patients who received a second autologous transplant upgraded their response (CR: 6%‚ PR: 17%‚ MR: 23%) while 41% had progressive disease or “no-change” and 13% died from the procedure. Three of the seven patients who underwent a dose-reduced intensity “allo” responded (2 CR‚ 1 PR) while two had progressive disease and two died from transplant-related complications. The median survival of the whole series of 49 patients was 32 months. Patients who had progressive disease after the initial chemotherapy had a significantly shorter survival than those who showed “non-responsive‚ non-progressive disease” (median 21 months vs. not reached‚ p=0.003). Finally‚ patients with “non-responding‚ non-progressive disease” had similar survival than those with chemosensitive disease intensified with HDT in the GEM trial. Conclusions. A high-dose therapy approach in patients with primary refractory myeloma results in a high overall response‚ but the CR rate is low‚ patients with progressive disease to the initial chemotherapy have short survival despite intensive therapy‚ and patients with “non-responding‚ non-progressive disease” have similar survival than chemosensitive patients. Whether the good outcome of the latter patients is mainly due the effect of HDT or to the natural history of a more indolent disease remains to be determined.

Description: Background: Bone marrow (BM) examination is essential in the staging of diffuse large B-cell lymphoma (DLBCL). The assessment of BM involvement includes both histology (gold-standard) and flow cytometry (FC), but few studies have compared BM biopsy (BMB) histologic findings with results of FC analysis of BM aspirate. Discordance between both techniques generates debate about the staging and the prognostic significance in these cases. Methods: We performed a retrospective single-center analysis of patients with DLBCL, not otherwise specified (NOS) diagnosed during a 4-year period (2014-2017). Patients were divided in three groups according to BM findings of BMB and FC at diagnosis. Standard FC was performed by 4-color flow panel until 2016 and by 8-color FC since then. We described main characteristics of each group at diagnosis and analyzed survival outcomes. We applied means of descriptive statistics and Pearson's chi-squared test, and analyzed survival outcomes according to Kaplan-Meier, using Cox regression for comparisons. Results: We analyzed 59 cases, which were divided in three groups: 40 cases (67.8%) presented both negative histology and FC (BMB-/FC-), 10 (16.9%) showed BM involvement using both histology and FC (BMB+/FC+) and 9 cases (15.3%) presented discordant results, all of them with negative histology and positive FC (BMB-/FC+). Clinical and biological characteristics of each group at diagnosis are presented in Table 1. Median infiltration by FC analysis of the BMB-/FC+ group was 0.8% (0.1-2.9) and 3/9 patients presented discordant immunophenotype of lymphoma cells between BM and node biopsy. If we considered BM infiltration as positive in all BMB-/FC+ cases, 4/9 (6.8% of all patients) would be upstaged. First-line treatment was homogeneous in all patients. With a median observation time of 18 months, progression-free survival (PFS) after 2 years was 67%, 22% and 22% with BMB-/FC-, BMB-/FC+ and BMB+/FC+, respectively (Figure 1A), with a multivariate hazard ratio (HR) of 1.9 (95% CI 1.2-2.9, p=0.004) and an univariate HR for FC+ (BMB-/FC+ and BMB+/FC+) vs FC- (BMB-/FC-) of 3.3 (95% CI 1.5-7.3, p=0.003). Two-year overall survival (OS) was 68%, 41% and 33% with BMB-/FC-, BMB-/FC+ and BMB+/FC+, respectively (Figure 1B); multivariate HR was 1.6 (95% CI 1.1-2.6, p=0.042) and univariate HR for FC+ vs FC- was 2.5 (95% CI 1.1-5.9, p=0.035). We found no significant difference between BMB-/FC+ and BMB+/FC+ in survival outcomes. Conclusions: In our series, the group with discordant BM infiltration (BMB-/FC+) presented worsen survival outcomes than BMB-/FC-. Such results should be validated in prospective studies because published series are retrospective and not focused specifically on DLBCL. BM infiltration detected by FC analysis but not by BMB could be considered as extranodal involvement at DLBCL NOS diagnosis. Disclosures García Gutiérrez: Novartis: Consultancy, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Research Funding, Speakers Bureau; Incyte: Consultancy, Research Funding, Speakers Bureau.

Description: Key Points Crebbp inactivation perturbs B-cell development, but cooperates with Bcl2 overexpression to promote lymphoma. Transcriptional and epigenetic signatures of Crebbp loss implicate Myc in disease etiology.

Description: Follicular lymphoma (FL) is genetically characterized by translocations of the BCL2 oncogene that are found in ~90% of patients, and mutations of chromatin modifying genes that are found in up to 96% of patients. The latter include inactivating mutations of KMT2D and CREBBP, and activating mutations of EZH2, among others. However, CREBBP has yet to be investigated using this approach. We recently defined the evolutionary hierarchy of somatic mutations in FL and found that CREBBP mutations were most frequently acquired as early events during disease evolution and were maintained throughout disease progression and transformation. Recent studies, using transgenic mouse models, have shown that inactivation of KMT2D and introduction of the activating EZH2 mutation results in perturbed B-cell development and lymphomagenesis. Here, we extended upon these observations by performing targeted next generation sequencing of an additional cohort of tumors allowing the identification of the spectrum of CREBBP mutations across 200 FLs. This identified CREBBP mutations in 55% of tumors, and found that 31% of these mutations reside within the lysine acetyltransferase domain. Furthermore, 30% of mutations altered a single amino acid, arginine 1408, to either a cysteine or histidine residue. We performed a sensitive in vitro acetyltransferase assay for these point mutants and show that they result in 〉90% loss of catalytic activity. As our results show that CREBBP mutations result in a loss of function, we modeled these events in mice by floxing one or both alleles of Crebbp and crossing with the Mb1-cre strain. This yielded mice that deleted Crebbp specifically in B-cells. We additionally crossed these mice with the EµBcl2 strain that over-expresses Bcl2 in B-cells. Inactivation of Crebbp in B-cells was associated with deficits in B-cell development, with significantly reduced numbers of total B-cells that were contributed to by reductions in multiple B-cell subsets. These deficits were partially rescued by the EµBcl2 transgene. After 14-21 months, some mice became ill and necropsy revealed lymphadenopathy and splenomegaly as a result of B-cell lymphoma. We noted increased penetrance and decreased latency of lymphoma with one vs two alleles of Crebbp deleted, and with absence vs presence of the EµBcl2 transgene (Figure 1). We investigated the molecular etiology of these tumors by isolating splenic B-cells from these mice and performing transcriptome profiling and epigenetic profiling for the histone H3 lysine 18 acetylation (H3K18Ac) mark that is catalyzed by Crebbp. Transcriptional profiling identified a signature of 335 genes with increased expression and 370 genes with decreased expression, including an incremental increase in Myc expression when one or both alleles of Crebbp were deleted, respectively. Surprisingly, changes in transcript abundance were not associated with changes in H3K18Ac in the proximal regulatory regions of those genes. Regions of significantly altered H3K18Ac were instead localized primarily to intragenic regions. Analysis of the DNA sequences in these regions identified a significant enrichment of motifs that contained Myc consensus sequences, and these were present in 〉60% of regions with altered H3K18Ac. In addition, ChIP-seq data from the ENCODE database showed a strong level of Myc binding to the center of these regions with altered H3K18Ac. Together, our results demonstrate that inactivating mutations of Crebbp may have a role in altering B-cell development. The significant induction of Myc expression that was associated with Crebbp deletion, and epigenetic changes in regions that are bound by Myc, suggest that Crebbp inactivation may have a role in the induction of Myc expression and activity. This may be important with respect to transformation of FL, which may proceed via induction of MYC. However, our results also demonstrate some important discrepancies between the role of CREBBP mutations in human FL, and the role of Crebbp deletion in murine models. Disclosures Lunning: Celgene: Consultancy; Spectrum: Consultancy; TG Therapeutics: Consultancy; Gilead: Consultancy; Genentech: Consultancy; Juno: Consultancy; Bristol-Myer-Squibb: Consultancy; AbbVie: Consultancy; Pharmacyclics: Consultancy.

Description: Hematopoietic stem and progenitor cells (HSPCs) and leukocytes circulate between the bone marrow (BM) and peripheral blood following circadian oscillations. Autonomic sympathetic noradrenergic signals have been shown to regulate HSPC and leukocyte trafficking, but the role of the cholinergic branch has remained unexplored. We have investigated the role of the cholinergic nervous system in the regulation of day/night traffic of HSPCs and leukocytes in mice. We show here that the autonomic cholinergic nervous system (including parasympathetic and sympathetic) dually regulates daily migration of HSPCs and leukocytes. At night, central parasympathetic cholinergic signals dampen sympathetic noradrenergic tone and decrease BM egress of HSPCs and leukocytes. However, during the daytime, derepressed sympathetic noradrenergic activity causes predominant BM egress of HSPCs and leukocytes via β3–adrenergic receptor. This egress is locally supported by light-triggered sympathetic cholinergic activity, which inhibits BM vascular cell adhesion and homing. In summary, central (parasympathetic) and local (sympathetic) cholinergic signals regulate day/night oscillations of circulating HSPCs and leukocytes. This study shows how both branches of the autonomic nervous system cooperate to orchestrate daily traffic of HSPCs and leukocytes.

Description: Abstract 3660 Poster Board III-596 Introduction Despite the major advances in the treatment of classical Hodgkin Lymphoma (cHL) patients, around 30% to 40% of cases in advanced stages may relapse or die as result of the disease. Current predictive systems, based on clinical and analytical parameters, fail to identify accurately this significant fraction of patients with short failure-free survival (FFS). Transcriptional analysis has identified genes and pathways associated with clinical failure, but the biological relevance and clinical applicability of these data await further development. Robust molecular techniques for the identification of biological processes associated with treatment response are necessary for developing new predictive tools. Patients and Methods We used a multistep approach to design a quantitative RT-PCR-based assay to be applied to routine formalin-fixed, paraffin-embedded samples (FFPEs), integrating genes known to be expressed either by the tumor cells and their reactive microenvironment, and related with clinical response to adriamycin-based chemotherapy. First, analysis of 29 patient samples allowed the identification of gene expression signatures related to treatment response and outcome and the design of an initial RT-PCR assay tested in 52 patient samples. This initial model included 60 genes from pathways related to cHL outcome that had been previously identified using Gene Set Enrichment Analysis (GSEA). Second, we selected the best candidate genes from the initial assay based on amplification efficiency, biological significance and treatment response correlation to set up a novel assay of 30 genes that was applied to a large series of 282 samples that were randomly split and assigned to either estimation (194) or validation series (88). The results of this assay were used to design an algorithm, based on the expression levels of the best predictive genes grouped in pathways, and a molecular risk score was calculated for each tumor sample. Results Adequate RT-PCR profiles were obtained in 264 of 282 (93,6%) cases. Normalized expression levels (DCt) of individual genes vary considerably among samples. The strongest predictor genes were selected and included in a multivariate 10-gene model integrating four gene expression pathway signatures, termed CellCycle, Apoptosis, NF-KB and Monocyte, which are able to predict treatment response with an overall accuracy of 68.5% and 73.4% in the estimation and validation sets, respectively. Patients were stratified by their molecular risk score and predicted probabilities identified two distinct risk groups associated with clinical outcome in the estimation (5-year FFS probabilities 75.6% vs. 45.9%, log rank statistic p≈0.000) and validation sets (5-year FFS probabilities 71.4% vs. 43.5%, log rank statistic p