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The chromatin activation landscape of chronic lymphocytic leukemia (CLL) with stereotyped B-cell receptor immunoglobulin is currently unknown. Here, we report the results of a whole-genome chromatin profiling of histone 3 lysine 27 acetylation of 22 CLLs from major subsets which were compared against non-stereotyped CLLs and normal B cell subpopulations. Although subsets #1, #2, and #4 did not differ much from their non-stereotyped CLL counterparts, subset #8 displayed a remarkably distinct chromatin activation profile. In particular, we identified 209 de novo active regulatory elements in this subset, which showed similar patterns with U-CLLs undergoing Richter transformation. These regions were enriched for binding sites of 9 overexpressed transcription factors. In 78/209 regions, we identified 113 candidate overexpressed target genes, being 11 regions associated with more than two adjacent genes. These included blocks of up to 7 genes, suggesting a local co-upregulation within the same genome compartment. Our findings further underscore the uniqueness of subset #8 CLLs, notable for the highest risk of Richter's transformation amongst all CLL, and provide additional clues to decipher the molecular basis of its clinical behavior.

With the introduction of large-scale molecular profiling methods and high-throughput sequencing technologies, the genomic features of most lymphoid neoplasms have been characterized at an unprecedented scale. Although the principles for the classification and diagnosis of these disorders, founded on a multidimensional definition of disease entities, have been consolidated over the past 25 years, novel genomic data have markedly enhanced our understanding of lymphomagenesis and enriched the description of disease entities at the molecular level. Yet, the current diagnosis of lymphoid tumors is largely based on morphological assessment and immunophenotyping, with only few entities being defined by genomic criteria. This paper, which accompanies the International Consensus Classification of mature lymphoid neoplasms, will address how established assays and newly developed technologies for molecular testing already complement clinical diagnoses and provide a novel lens on disease classification. More specifically, their contributions to diagnosis refinement, risk stratification, and therapy prediction will be considered for the main categories of lymphoid neoplasms. The potential of whole-genome sequencing, circulating tumor DNA analyses, single-cell analyses, and epigenetic profiling will be discussed because these will likely become important future tools for implementing precision medicine approaches in clinical decision making for patients with lymphoid malignancies.

Increasing amount of evidence indicates that the deregulation of non-coding elements is a common feature of cancer and therefore, its investigation may uncover new molecular oncogenic mechanisms. In multiple myeloma (MM), the altered expression of a small number of long non-coding RNAs (lncRNAs) has been associated with progression and decreased survival, suggesting that these elements may play a more important role in this disease than previously expected. Nevertheless, an extensive high-throughput analysis that characterizes the deregulation of lncRNAs in MM has not yet been performed. To characterize the transcriptome, including all genomic types of lncRNAs, of MM we performed a paired end strand-specific RNA sequencing (ssRNA-seq) in 38 purified plasma cell (PC) samples from MM patients, as well as PC samples from tonsils (TPCs, n=5) and bone marrow (BMPCs, n=3) of healthy donors as controls. Principal component analysis (PCA) demonstrated that normal PC samples from tonsil and bone marrow cluster separately, suggesting that in spite of being the same cell type, their coding and non coding transcriptomes are very different. Therefore, we selected BMPCs as the normal counterparts for comparison with BM of MM samples. PCA analysis also demonstrated that the well known heterogeneity of MM patients rely not only on the coding transcriptome but also on the lncRNA expression profile. Comparison of MM to BMPCs samples showed 70 previously annotated lncRNAs that were deregulated in MM patients, with 3 lncRNAs showing higher and 67 lower expression than normal BMPCs. Moreover, we identified 40.552 novel MM-specific lncRNAs that were present in at least 3 of the 38 patients, highlighting the magnitude of the deregulation of these non coding elements in MM. To determine the functional role of altered lncRNAs in the biology of MM plasma cells we focused on the study of LINC-MSL1 (Myeloma-Specific LncRNA 1). Analysis of the expression of this lncRNA at different stages of B-cell differentiation (Naïve, Germinal Center, Memory and PC) indicated that it is not expressed at any stage, except for a modest expression in BMPCs. Interestingly, its overexpression was detected in 40% of MM specimens when compared to normal BMPCs which was validated by qPCR in an independent cohort of MM patients. To determine whether the expression of this lncRNA is regulated by epigenetic mechanisms, we studied the DNA methylation state of this gene. DNA methylation analysis in MM demonstrated that the CpGs located upstream of LINC-MSL1 were differentially methylated in comparison with normal counterpart BMPC. These CpGs showed 70% DNA methylation in control samples, about 40% in MGUS, whereas the average of MM was about 20%, showing a remarkable hypomethylation. We validated these results by pyrosequencing, which showed a significantly lower DNA methylation at the promoter region in comparison with B cell populations from tonsil, normal BMPCs and cell lines that do not overexpress LINC-MSL1. We also have observed a gain of active chromatin states analyzed by ChiP-seq in the promoter region of LINC-MSL1 in MM patient samples. These data suggest that epigenetic mechanisms, namely the progressive hypomethylation and the gain of active histone modifications, are the cause of the overexpression of LINC-MSL1 in MM. To analyze the role of the overexpression of LINC-MSL1 in MM, we engineered two MM cell lines that show high levels of LINC-MSL1, MM.1S and MM.1R, to express shRNAs against this lncRNA. Knockdown of LINC-MSL1 by two different shRNAs resulted in a reduced proliferation of the cell lines over time. This effect was not associated with a cell cycle arrest but with a marked increased in the percentage of Annexin V-positive apoptotic cells, indicating that the overexpression of LINC-MSL1 is necessary for the survival of MM cells. All together, these data demonstrate that the alteration of lncRNAs is an important an unexplored feature that contributes to MM pathogenesis. The overexpression of LINC-MSL1 is essential for MM survival and is very specific of MM BMPCs, suggesting it could be a relevant therapeutic target. Disclosures Paiva: Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding. Melnick:Janssen: Research Funding.

Members of the Notch gene family have been shown to mediate cell-fate decisions by multipotent precursors in a number of different systems. To determine whether members of this family might play a similar role in hematopoiesis, we asked if homologues of the Notch gene are expressed in human hematopoietic precursors. Using degenerate oligonucleotides corresponding to conserved amino acid sequences in known Notch homologues as primers for the polymerase chain reaction (PCR), we demonstrated that at least one Notch homologue is expressed in human bone marrow CD34+ cells, a population enriched for hematopoietic precursors. Cloning and sequencing of the PCR products identified this Notch homologue as TAN-1, a member of the Notch family previously cloned from a T-cell leukemia with a translocation involving this gene. Subsequent evaluation of bone marrow hematopoietic cells for TAN-1 expression using a reverse transcription-PCR assay confirmed the expression of TAN-1 in CD34+ hematopoietic precursors, including the immature subset that lacks expression of lineage-associated antigens (CD34+lin-). These findings, together with the known role of Notch homologues in other systems, suggest that members of the Notch family, including TAN-1, may be involved in mediating cell-fate decisions during hematopoiesis.

Retroviral mediated gene transfer into stem cells has been proposed as therapy for many inherited hematopoietic diseases. Deficiency of the enzyme adenosine deaminase (ADA) results in depletion of T lymphocytes, causing severe combined immunodeficiency syndrome (SCIDS). In this report, we describe retroviral mediated gene transfer of a murine ADA cDNA into Rhesus monkey hematopoietic stem cells. Immunoselected CD34+ bone marrow cells were exposed to medium containing the ADA retrovirus during culture on a stromal cell line engineered to express the transmembrane form of stem cell factor. After infusion of autologous, transduced cells into irradiated recipients, gene transfer was observed in all three monkeys. The ADA provirus was detected in 2% of circulating granulocytes and T cells from 100 days post-transplantation to longer than 1 year and in B cells from 250 days post-transplantation and beyond. Mouse ADA activity was detected in peripheral blood cells at approximately 3% the activity of monkey ADA. Thus, we have shown gene transfer into repopulating cells that contribute to all hematopoietic lineages with persistent gene expression. These data provide support for the use of stem cell targeted gene transfer for therapy of ADA deficiency.

Murine embryonic stem cells are able to differentiate into embryoid bodies (EBs) in vitro in the absence of leukemia-inhibitory factor with the formation of different types of hematopoietic precursors within these EBs. With the aim of determining the in vitro requirements for the continued development of hematopoietic colony-forming cells (CFCs) and their progeny from embryonic stem-derived cells, cells from EBs disrupted after 9 days of formation in the absence of leukemia-inhibitor factor were cultured under different conditions. Low numbers of day-9 EB cells (5 x 10(5) or less) cultured in the presence of several growth factors (interleukin-3 [IL-3], IL-1, c-kit ligand, basic fibroblast growth factor, insulin growth factor-1, IL-6, granulocyte colony-stimulating factor, fetal liver kinase-2 ligand) develop few or no CFCs after 1 week of culture. When these cells are plated on irradiated NIH-3T3 with IL-3 or c-kit ligand or combinations containing these and other growth factors, they are able to generate CFCs for at least 3 weeks. These cultures were found to include granulocytic, monocytic, erythrocytic, and megakaryocytic cells. Transwell cultures in which NIH-3T3 cells were separated from the EB cells and cultures in which cells were replaced by NIH-3T3 conditioned medium showed that the interaction between EB-derived cells and NIH-3T3 is via a soluble factor(s). These studies show that maximal generation of hematopoietic CFCs from precursors present in day-9 EBs is stimulated by a combination of known hematopoietic growth factors and a soluble factor(s) produced by NIH-3T3 cells.

Abstract 4465 Introduction: Solid organ transplant recipients are profoundly immune suppressed and are at risk for post-transplant lymphoproliferative disorder (PTLD). PTLD is associated with substantial morbidity and mortality. With limited treatment options, earlier detection of disease and earlier initiation of therapy may be essential for optimizing therapeutic outcomes. Thus, identifying at risk groups is critical. Methods: We conducted an IRB-approved retrospective study of 1,496 solid organ transplant recipients treated at our institution to identify risk factors for the development of PTLD. We identified 104 cases of PTLD going back over the past 20 years, through 1991. A cross-sectional sample of solid organ transplants evaluated at our institution was used as controls (N = 1,392). Charts were reviewed for relevant demographic variables, reason for transplantation, Epstein-Barr virus (EBV) serostatus pre-transplant, and Human Leukocyte Antigen (HLA)-type. Information on outcomes, such as organ rejection over follow up and death was also recorded. In individuals with PTLD, date of diagnosis, tumor characteristics (i.e., EBV status), tumor pathology, and PTLD treatment was recorded. Survival analysis (Kaplan-Meier plots and Cox proportional hazards regression) was conducted on collected data, to determine risk factors for the development of PTLD. Results: In the cohort as a whole, 86/1,496 (5.8%) of individuals were EBV seronegative at baseline, and 20.9% (312/1496) did not have an available documented baseline EBV serology. EBV serostatus was the major risk factor for EBV-associated PTLD (N=74, or 71% of PTLD cases): EBV-seronegative individuals had a Hazard Ratio (HR) =7.70 [95% CI = 3.8 – 15.5, p < 0.001] for EBV-associated PTLD compared to seropositive individuals. Thus, further analyses were stratified by EBV serostatus, to examine the effect of HLA-type on risk of development of PTLD. In an exploratory data analysis, we found that HLA-B40 was a major risk factor for the development of EBV-associated PTLD in EBV-seronegative individuals, while HLA-B8 was a risk factor for the development of EBV-associated PTLD in EBV-seropositive individuals. In baseline seronegative individuals HLA-B40 was associated with a HR=5.99 [95% CI = 1.95 – 18.4, p=0.002] for PTLD (see figure below for Kaplan Meier curves). In baseline seropositive individuals HLA-B8 was associated with a HR = 3.65 [95% CI = 1.52 – 8.76, p= 0.004] for PTLD. In EBV seropositive individuals, receipt of either a liver, cardiac, lung or pancreas transplant (compared to a kidney transplant alone) was associated with an increased risk of PTLD as well, HR = 6.72 [95% CI = 2.78 – 16.24, p < 0.001. Conclusions: While the relation of HLA-type and EBV-associated PTLD seen in the study are novel, we recommend future studies to test these observations. These results demonstrate that risk factors for EBV-associated PTLD may verify substantially with baseline EBV-serostatus. We recommend future studies consider stratifying analyses by baseline EBV-serostatus, as EBV-associated PTLD resulting from primary EBV-infection may represent a distinct pathology than EBV-associated PTLD arising in individuals who are known to be EBV-seropositive. Disclosures: No relevant conflicts of interest to declare.

T-cell non-Hodgkin lymphomas (NHLs) are uncommon malignancies that represent approximately 15% of aggressive lymphomas in Western countries. Contrasting with B-cell lymphomas, very few recurrent chromosomal abnormalities have been identified. Nevertheless, the TCR loci and preferentially the TCRAD at chromosome band 14q11 could be involved in up to 15% of cases. We recently reported a novel and recurrent t(14;19)(q11;q13) chromosomal translocation in peripheral T-cell lymphoma (PTCL). FISH analysis performed in three cases suggested an implication of the TCRAD locus at 14q11 and of BCL3 at 19q13. We now report the molecular cloning of the genomic junctions for these three patients. Sequence analysis confirmed the involvement of the TCRAD on 14q11 and assessed the breakpoint’s localisation to the TCRA variable region for all cases. In the first, the chromosomal break occurred between the J30 and the J31 segments while in the second it was in the J58 segment. Thus, both translocations probably occurred after an illegitimate V(D)J recombination. Remarkably, for the third patient, junctions with the deltaRec and psi(J)alpha elements were identified on the der(14) and der(19) respectively. Consequently this translocation probably results from an illegitimate deltarec-Psi(J)alpha rearrangement (TCRD deletion) during thymopoiesis before Valpha-Jalpha rearrangements. On chromosome 19, our results indicated a new clustering of breakpoints outside the region documented in t(14;19)(q32;q13) in chronic lymphocytic leukemia. Remarkably, all three breaks occurred ~130kb downstream from BCL3, in the PVRL2 gene. Consequently, the TCRA locus enhancer is juxtaposed to PVRL2 and BCL3 on the derivative chromosome 19. For two patients, mRNA expression levels were investigated by real time RT-PCR experiments, which confirmed a high expression of both PVRL2 and BCL3. In conclusion, we identified PVRL2 as the recurrent translocation partner of the TCRA locus in PTCL. Whether this clustering results from an important instability of the PVRL2 gene during thymopoiesis remains to be investigated. The PVRL2 overexpression itself may be an important step for T-cell lymphomagenesis.