Abstract Purpose: In the United States, significant disparities exist in gastric cancer incidence and mortality between Black Americans and non-Hispanic White Americans. Helicobacter pylori (HP) infection is the most important risk factor for developing non-cardia gastric adenocarcinoma (GAC), the most common type of gastric cancer. Non-cardia GAC is thought to occur via progression from HP-induced atrophic gastritis to gastric intestinal metaplasia (GIM), dysplasia, and cancer. The purpose of this study was to enroll racially diverse (~50% self-identified as Black) patients from the endoscopy suite across the spectrum of HP-associated disease (gastritis, GIM, GAC) in a prospective observational cohort to biobank blood and tissue samples with patient-reported survey data and clinical history from the electronic health record. Methods: In an ongoing prospective study funded by an NIH P20 disparities project, we enrolled a diverse, racially balanced cohort of research participants undergoing upper endoscopy. We tracked and optimized screening, enrollment, and collection of blood, tissue, and survey data. Organoids were generated from fresh and/or cryopreserved biopsies of normal gastric tissue, gastritis, GIM, or GAC. Gene expression, immunohistochemistry and DNA mutational profiling were performed in a subset of organoids. Results: To date, 563 patients were identified in screening (47% Black patients) and 250 were successfully included with a 44% enrollment rate (46% Black participants). Rates of successful sample and data collection were: 86% blood, 87% tissue collection, and 89% survey collection. The proportion of Black participants was greater in the HP-positive group (65%) vs. known HP-negative (46%) (p=0.03). Gastric organoids were generated with >90% success from 49 patients, including paired organoid lines from incomplete, complete, and/or extensive GIM, matched tumor and non-tumor and gastric antrum and body samples. 20 organoid lines were derived from cryopreserved endoscopic biopsies. Ongoing characterization of tumor organoids reflects expected heterogeneity among gastric cancer patients and provides a functional measure of cytokine expression. Conclusions: To address health disparities related to gastric cancer, diverse patient cohorts must be established with successful biobanking from groups most affected by the disease. Patient-derived gastric organoids can be generated from diverse populations and across different clinical conditions as one approach to understanding and addressing gastric cancer health disparities. Citation Format: Priya Alagesan, Paula Scotland, HannahSofia Brown, Shannon J. McCall, Meira Epplein, Katherine S. Garman. Biobanking of gastric organoid models from a racially diverse cohort for gastric cancer interception. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5519.

4 Introduction Our group has developed a human umbilical cord-blood derived, macrophage-like cell therapy product, DUOC-01, to treat demyelinating conditions of the central nervous system. Earlier, we demonstrated that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the brain of immune-incompetent mice exposed to the demyelinating agent, cuprizone. To further elucidate the mechanism of action and investigate whether DUOC-01 will be effective in other experimental models of demyelination, we tested DUOC-01 in experimental autoimmune encephalomyelitis (EAE), an animal model used to study multiple sclerosis (MS). Objective In the event of an inflammatory response, it is possible that patients would receive a hydrocortisone (HC) injection at the time of infusing DUOC-01 cells; therefore, it is critical to assess if HC can negatively influence the efficacy of DUOC-01 cells. Methods We induced EAE in C57BL/6 mice by immunizing with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in Freund’s complete adjuvant. DUOC-01 cells were administered at a dose of 3 × 10 cells/mouse into the cerebrospinal fluid by a single intra-cisterna magna injection at the point when mice started showing early disease symptoms of EAE.

Background & Aim We have developed an umbilical cord-blood derived, macrophage-like cell therapy, DUOC-01, to treat demyelinating conditions of the central nervous system. Previously, we showed that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the brain of immune-incompetent mice exposed to the demyelinating agent, cuprizone. To further explore the mechanism of action and investigate whether DUOC-01 will be effective in other experimental models of demyelination, we tested DUOC-01 in the experimental autoimmune encephalomyelitis (EAE) model, an animal model used to study multiple sclerosis (MS). Methods, Results & Conclusion EAE was induced in C57BL/6 mice by immunizing with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in complete Freund's adjuvant. We have previously established in a phase I trial of intrathecally administered DUOC-01 cells in patients with leukodystrophies, that DUOC-01 is formulated in hydrocortisone (HC) for infusion in the clinic. Therefore, to mimic the practice in the clinic, DUOC-01 cells were incubated in Ringer's Lactate with 3mg/mL HC at a dilution of 1 × 106 cells/ml for 2 hours at room temperature prior to injection. DUOC-01 cells were then administered at a dose of 3 × 105cells/mouse into the cerebrospinal fluid by a single intra-cisterna magna injection at the point when mice started showing early disease symptoms of EAE. Clinical score was recorded for next several days. Compared to mice injected with Ringer's (n=9), mice injected with DUOC-01 (n=9) had decreased severity of the disease, as indicated by lower clinical scores based on severity of ascending paralysis. The spinal cords of mice in the cohorts were studied histopathologically and those with mice injected with DUOC-01 had reduced inflammation and lower cellular infiltration compared to spinal cords of mice injected with Ringer's. The overall number of CD45+ immune cells were less in spinal cords of mice treated with DUOC-01 cells compared to the Ringer's injected mice. Most strikingly, among the various cell types, the number of neutrophils present in the spinal cords from the DUOC-01 treated group was less than the Ringer's treated samples. Presently we are exploring the mechanism(s) through which DUOC-01 cells promote remyelination and decrease immune cell infiltration. In brief, our data suggest that DUOC-01 cell therapy product could be beneficial in treating MS and other diverse neurological conditions with demyelination. We have developed an umbilical cord-blood derived, macrophage-like cell therapy, DUOC-01, to treat demyelinating conditions of the central nervous system. Previously, we showed that DUOC-01 accelerated remyelination, decreased gliosis, and reduced cellular infiltration in the brain of immune-incompetent mice exposed to the demyelinating agent, cuprizone. To further explore the mechanism of action and investigate whether DUOC-01 will be effective in other experimental models of demyelination, we tested DUOC-01 in the experimental autoimmune encephalomyelitis (EAE) model, an animal model used to study multiple sclerosis (MS). EAE was induced in C57BL/6 mice by immunizing with myelin oligodendrocyte glycoprotein peptide (MOG35-55) in complete Freund's adjuvant. We have previously established in a phase I trial of intrathecally administered DUOC-01 cells in patients with leukodystrophies, that DUOC-01 is formulated in hydrocortisone (HC) for infusion in the clinic. Therefore, to mimic the practice in the clinic, DUOC-01 cells were incubated in Ringer's Lactate with 3mg/mL HC at a dilution of 1 × 106 cells/ml for 2 hours at room temperature prior to injection. DUOC-01 cells were then administered at a dose of 3 × 105cells/mouse into the cerebrospinal fluid by a single intra-cisterna magna injection at the point when mice started showing early disease symptoms of EAE. Clinical score was recorded for next several days. Compared to mice injected with Ringer's (n=9), mice injected with DUOC-01 (n=9) had decreased severity of the disease, as indicated by lower clinical scores based on severity of ascending paralysis. The spinal cords of mice in the cohorts were studied histopathologically and those with mice injected with DUOC-01 had reduced inflammation and lower cellular infiltration compared to spinal cords of mice injected with Ringer's. The overall number of CD45+ immune cells were less in spinal cords of mice treated with DUOC-01 cells compared to the Ringer's injected mice. Most strikingly, among the various cell types, the number of neutrophils present in the spinal cords from the DUOC-01 treated group was less than the Ringer's treated samples. Presently we are exploring the mechanism(s) through which DUOC-01 cells promote remyelination and decrease immune cell infiltration. In brief, our data suggest that DUOC-01 cell therapy product could be beneficial in treating MS and other diverse neurological conditions with demyelination.

20 Introduction DUOC-01 cells are a macrophage-like cell therapy product derived from umbilical cord blood that is currently being evaluated in a phase I clinical trial for patients with congenital lysosomal storage diseases (ClinicalTrials.gov Identifier: NCT02254863.) Manufacturing of DUOC-01 involves isolation of mononuclear cells from previously cryopreserved cord blood, plating on tissue culture flasks, and culturing for 21 days. Because of the strong adherent properties of these cells, scraping in addition to trypsinization is required during harvest, which decreases cell viability and is difficult to perform in a GMP environment. Objective The objective of this study was to find a harvest method that was gentler on the DUOC-01 cells and simplified the manufacturing process while maintaining cell function. Methods DUOC-01 cells were grown in either standard tissue culture flasks or Ultra Low Attachment Flasks (ULA, Corning Inc.) for 3 weeks. On day 21, different harvest methods were tested, including TrypLE, cold, PBS-EDTA, and longer incubation time. Cell surface markers were determined by flow cytometry. Activity was determined by intracranial injection of cells into murine model of cuprizone-mediated demyelination, then measuring remyelination in the corpus callosum 1 week later.

Cord blood (CB) mononuclear cells (MNC) are being tested in clinical trials to treat hypoxic-ischemic (HI) brain injuries. Although early results are encouraging, mechanisms underlying potential clinical benefits are not well understood. To explore these mechanisms further, we exposed mouse brain organotypic slice cultures to oxygen and glucose deprivation (OGD) and then treated the brain slices with cells from CB or adult peripheral blood (PB). We found that CB-MNCs protect neurons from OGD-induced death and reduced both microglial and astrocyte activation. PB-MNC failed to affect either outcome. The protective activities were largely mediated by factors secreted by CB-MNC, as direct cell-to-cell contact between the injured brain slices and CB cells was not essential. To determine if a specific subpopulation of CB-MNC are responsible for these protective activities, we depleted CB-MNC of various cell types and found that only removal of CB CD14+ monocytes abolished neuroprotection. We also used positively selected subpopulations of CB-MNC and PB-MNC in this assay and demonstrated that purified CB-CD14+ cells, but not CB-PB CD14+ cells, efficiently protected neuronal cells from death and reduced glial activation following OGD. Gene expression microarray analysis demonstrated that compared to PB-CD14+ monocytes, CB-CD14+ monocytes over-expressed several secreted proteins with potential to protect neurons. Differential expression of five candidate effector molecules, chitinase 3-like protein-1, inhibin-A, interleukin-10, matrix metalloproteinase-9 and thrombospondin-1, were confirmed by western blotting, and immunofluorescence. These findings suggest that CD14+ monocytes are a critical cell-type when treating HI with CB-MNC.

Hypoxic-ischemic (HI) brain injury is a frequent cause of perinatal morbidity and mortality and treatment options are limited. Human umbilical cord blood (CB) mononuclear cells (MNC) are being tested in the clinic for treatment of HI brain injuries with encouraging preliminary results. However the molecular mechanism(s) that underlie this clinical effect are not understood. Recently, we have shown that CB-MNC protected brain neurons from Oxygen-Glucose-Deprivation (OGD)-induced death and suppress astrocyte activation much more effectively than MNC from adult peripheral blood (PB) and that CB-CD14+ monocytes mediated these protective activities. Here, to determine molecules that may contribute to the neuroprotective effect of CB-CD14+ cells, we analyzed the transcriptomes of CB and PB-CD14+ monocytes and found that they differed in expression of many transcripts. We identified seven of these transcripts encoded secreted proteins in CB-CD14+ that could play a paracrine role in neuroprotection. We show that supernatants conditioned by CB CD14+ monocytes exposed to factors released from OGD-shocked brain slices were also neuroprotective. Western blot analysis confirmed that CB monocytes over-expressed five of the proteins corresponding to these candidate genes. Two of these, thrombospondin 1 and chitinase 3-like protein 1, were detected in secretory granules of all CB, but not PB, monocytes by immunofluorescence. The matrix metalloproteinase 9, was abundant in a subpopulation of CB monocytes but was rare in PB monocytes. Our data suggest that CB CD14+ monocytes play a central role in the treatment of HI with CB MNC, allude to specific proteins that may participate in neuroprotection, and suggest new, mechanism-based approaches for developing cell therapy products derived from CB units for treatment of HI brain injury.

10 Cord tissue-derived mesenchymal stem cells (CT-MSC) are increasingly being used in clinical studies to address a variety of health issues and diseases due to their tissue-regenerating and immune-modulatory properties. With this promise of therapies comes the need for the manufacturing of a large number of CTMSCs for dosing. At the Marcus Center for Cellular Cures at Duke, we have created a process for the manufacturing of large batches of CT-MSCs. The path from first isolating CT-MSC progenitors from cord tissue to delivery of the appropriate CT-MSC dose at the treatment site comprises many steps that need to be efficiently connected while conforming to current Good Manufacturing Practice standards. The process starts with the isolation of CT-MSC progenitors from donated cord tissue using enzymatic digestion in a tissue dissociator and subsequent culturing in flasks. We have optimized the timing of supplement addition to CT-MSC growth media and cell harvest for obtaining populations of pure CT-MSC. Expansion of those MSCs were initially carried out in cell culture flasks and then broadened to multilayer stacked flask systems in two expansion steps. In order to increase batch size, improve automation, and reduce the number of cell passages, we currently are testing cell expansion in large closed system bioreactors. Parameters tested include loading conditions, media, feed rates, and harvest conditions. Scaling up cell production also has to be matched with up-scaled procedures for cell washing, addition of cryopreservation media, rapid dispensing into suitable cryopreservation containers, and cell cryopreservation. We will briefly review the factors and equipment used at our facility to complete the scaled-up CTMSC process. Supplying the demand of high numbers of quality CT-MSC product requires a manufacturing process that is closedsystem, automated, and efficient in time and cost throughout all steps. We are developing a manufacturing process that will comply with these requirements and that will provide cells for the increasing demand of MSC clinical trials. STEM CELLS TRANSLATIONAL MEDICINE | StemCellsTM.com © AlphaMed Press 2018 PERINATAL TISSUE BANKING AND THERAPIES, INCLUDING MESENCHYMAL STEM CELLS Official journal of the

Background aims. DUOC-01, a cell product being developed to treat demyelinating conditions, is composed of macrophages that arise from CD14(+) monocytes in the mononuclear cell (MNC) population of banked cord blood (CB). This article demonstrates that expression of multiple gene products that promote remyelination is rapidly up-regulated during manufacturing of DUOC-01 from either MNC or purified CB CD14(+) monocytes. Methods. Cell cultures were initiated with MNC or with immunoselected CD14(+) monocytes isolated from the same CB unit. Cell products present in these cultures after 2 and 3 weeks were compared by three methods. First, quantitative polymerase chain reaction was used to compare expression of 77 transcripts previously shown to be differentially expressed by freshly isolated, uncultured CB CD14(+) monocytes and DUOC-01. Second, accumulation of 16 soluble proteins in the culture medium was measured by Bioplex methods. Third, whole transcriptomes of the cell products were compared by microarray analysis. Results. Key transcripts in multiple pathways that promote remyelination were up-regulated in DUOC-01, and substantial secretion of proteins corresponding to many of these transcripts was detected. Cell products manufactured from MNC or from CD14(+) monocytes were similar with regard to all metrics. Upregulation of gene products characteristic of DUOC-01 was largely completed within 14 days of culture. Conclusion. We demonstrate that expression of multiple gene products that promote remyelination is up regulated during the first 2 weeks of manufacturing of DUOC-01. Measuring these mechanistically important transcripts and proteins will be useful in monitoring manufacturing, evaluating manufacturing changes, and developing mechanism based product potency assays.

Microglia and monocytes play important roles in regulating brain remyelination. We developed DUOC-01, a cell therapy product intended for treatment of demyelinating diseases, from banked human umbilical cord blood (CB) mononuclear cells. Immunodepletion and selection studies demonstrated that DUOC-01 cells are derived from CB CD14+ monocytes. We compared the ability of freshly isolated CB CD14+ monocytes and DUOC-01 cells to accelerate remyelination of the brains of NOD/SCID/IL2Rγnull mice following cuprizone feeding-mediated demyelination. The corpus callosum of mice intracranially injected with DUOC-01 showed enhanced myelination, a higher proportion of fully myelinated axons, decreased gliosis and cellular infiltration, and more proliferating oligodendrocyte lineage cells than those of mice receiving excipient. Uncultured CB CD14+ monocytes also accelerated remyelination, but to a significantly lesser extent than DUOC-01 cells. Microarray analysis, quantitative PCR studies, Western blotting, and flow cytometry demonstrated that expression of factors that promote remyelination including PDGF-AA, stem cell factor, IGF1, MMP9, MMP12, and triggering receptor expressed on myeloid cells 2 were upregulated in DUOC-01 compared to CB CD14+ monocytes. Collectively, our results show that DUOC-01 accelerates brain remyelination by multiple mechanisms and could be beneficial in treating demyelinating conditions.

G protein signaling modulator-3 (GPSM3) is a GoLoco protein that regulates GPCR signaling and is highly expressed in immune cells. Two single nucleotide polymorphisms within the GPSM3 gene (SNPs rs204989, rs204991) are in perfect linkage disequilibrium, inherited together as a single protective allele, and have been associated with a decreased prevalence of some autoimmune diseases in the general population. Consequently, we hypothesized that possessing the allele may correlate with decreased autoimmunity in CVID. Forty-six Caucasian CVID patients were recruited from UNC and Duke. Blood leukocytes were genotyped, assaying genomic DNA for rs204989 SNP using manufacturer protocols. A retrospective chart review was conducted to determine whether patients had autoimmune disease. Of the 46 CVID patients, 29 (63%) did not have the protective GPSM3 allele (SNPM/M), 13 (28%) possessed one allele (SNPM/m), and 4 (9%) possessed both alleles (SNPm/m). 17 of the 46 CVID patients had autoimmunity (37%), and 2 had lymphoproliferation (4%). Four of 17 autoimmune patients (23%) possessed at least one copy of the protective allele (SNPm/m or SNPM/m) compared to 11 of 27 non-autoimmune patients (41%). Both lymphoproliferative patients were heterozygous (SNPM/m). CVID patients with autoimmunity are less likely to possess the GPSM3 protective allele (SNPm/m or SNPM/m). Interestingly, SNP prevalence in CVID was increased overall compared to healthy Caucasians (9% versus 2% SNPm/m and 28% versus 24% SNPM/m), and both lymphoproliferative patients were SNPM/m heterozygous. This observation suggests that this specific GPSM3 allele may be enriched in CVID compared to the general population and could have functional significance.

Our lab is developing cord blood (CB)-derived cell therapies for neuronal damage resulting from hypoxic-ischemic [HI] insult. We are using mouse brain slice cultures subjected to oxygen-glucose deprivation [OGD] to study how CB cells mediate neuroprotection. We previously reported that CD14+ cells account for most of the neuroprotective activity of CB cells in this model. We used immunohistochemistry to further detail the mechanisms of this neuroprotection. Brain slice cultures established from C57BL/6J mice were subjected to 1h OGD on day 9 treated with cell populations or medium immediately after normal conditions were restored. CB CD14+ and CD14+ depleted cells were immunomagnetically prepared from CB mononuclear cells within 48h of collection. Human adult peripheral blood (PB) CD14+ populations were also tested. After 72h, slice cultures were fixed and stained with antibodies to detect astrocytes (GFAP), neurons (NeuN), oligodendrocytes (olig2), and microglia (Iba1). Glial and neuronal cells were enumerated in contiguous images of the periventricular regions using fluorescence confocal microscopy. We also characterized the effects of cell treatment on primary human astrocytes subjected to OGD stress in a microfluidics chamber. In both culture systems treatment with CB-CD14+ cells resulted in an increase in NeuN+ neurons and a decrease in the number of activated GFAP+ astrocytes following OGD shock. Cultures treated with CB-CD14+ had 2-fold more surviving neurons than those not treated. CD14 depleted cells did not protect cultures. We did not detect changes in microglia or oligodendrocytes following cell treatment. We conclude that CB CD14+ cells demonstrate a greater neuroprotective and anti-neuroinflammatory effect than PB CD14+ cells. CB CD14+ cells could mediate neuroprotection either directly on neurons or indirectly through modulation of astrocyte activation. We confirm the therapeutic potential of CB CD14+ cells in the setting of acquired HI.

While virtually all CLL cases are preceded by an asymptomatic phase of monoclonal B cell lymphocytosis (MBL), only 1-2% of MBL cases develop to CLL requiring chemotherapy annually. Specific determinants that lead to progression are unknown thus far, but are highly relevant, since this might contribute to a better understanding of CLL biology. Recently, the application of whole genome and exome sequencing using next generation sequencing (NGS) in CLL resulted in the discovery of novel genes including mutations in SF3B1 and NOTCH1, that could potentially play important roles in CLL leukemogenesis. Additionally NGS enabled in-depth examination of genetic alterations, and therefore has shed new light on clonal evolution of driver mutations which is considered a hallmark for progression. However, the role of genetic mutations and clonal evolution in the development of MBL and progression of MBL to CLL is still largely unknown and NGS studies in MBL samples and paired longitudinal samples is currently scarce. In this study we investigated the role of genetic mutations in the development of MBL and progression of MBL to CLL.

Key Points We identified novel recurrently mutated genes, including WHSC1, RB1, POT1, and SMARCA4, through exome sequencing of 56 cases of MCL. Genetic mutations defining MCL and Burkitt lymphoma were associated with the epigenetically defined chromatin state of their respective B cells of origin.

Abstract Triple negative breast cancer (TNBC) represents a particularly aggressive and difficult to treat form of breast cancer. No specific genetic alterations have been described as characteristic of the disease, with the exception of association with BRCA1/2, EGFR, and KRAS mutations. In this study, we sought to define clinically actionable mutations in untreated metastatic tumors as well as compare the mutational status of metastatic samples with germ-line and primary tumors using whole exome sequencing. We prospectively enrolled 38 patients with newly diagnosed metastatic TNBC and collected matched specimens of germ-line DNA, primary tumor and metastatic tumor. Median DFI from time of initial primary diagnosis to recurrence was 18 months (IQR = 1-24 months) and 9 patients presented with de novo metastatic disease. 34/38 patients went on to receive first-line treatment with nab-paclitaxel, carboplatin, and bevacizumab and ORR/PFS/OS are available. Sites of TNBC metastatic tissue (n = 31) included: liver (10), chest wall (13), non-regional lymph nodes (4), and lung (4). 7 patients had inadequate metastatic tumor for sequencing. We performed whole-exome sequencing for all samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 10 GB of sequencing data using high throughput sequencing on the Illumina platform. We observed striking genetic heterogeneity among the metastatic and primary tumors. There was no single driver mutation that was common to the metastatic tumors indicating the diverse genetic pathways that contribute to metastasis. Early analysis suggests that mutations in APC and MTOR occur more frequently in metastatic tumors than in primary tumors. Nonsense mutations of ER were detected in both primary and metastatic tumors but not in germ-line DNA. EGFR and HER2 mutations were not found in any of the primary or metastatic TNBC samples. This data provides the most comprehensive genetic portrait of metastatic and primary TNBC to date, and represents a significant first step in identifying the genetic causes of the disease, drivers of recurrence, and potential therapeutic targets. Full results, including the primary versus metastatic tumor mutational analysis will be presented. This study was funded by a Susan G. Komen Grant SAC 100001. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr S4-03.

Mantle cell lymphoma is an uncommon form of non Hodgkin lymphoma that is characterized by poor responsiveness to chemotherapy and a high rate of mortality. While translocation of CCND1 is a defining feature of the disease, the role of collaborating somatic mutations that contribute to mantle cell lymphoma remains to be better defined.

The t(10;11) chromosomal translocation gives rise to the CALM-AF10 fusion gene and is found in patients with aggressive and difficult-to-treat hematopoietic malignancies. CALM-AF10-driven leukemias are characterized by HOXA gene up-regulation and a global reduction in H3K79 methylation. DOT1L, the H3K79 methyltransferase, interacts with the octapeptide/leucine zipper domain of AF10, and this region has been shown to be necessary and sufficient for CALM-AF10-mediated transformation. However, the precise role of CALM in leukemogenesis remains unclear. Here, we show that CALM contains a nuclear export signal (NES) that mediates cytoplasmic localization of CALM-AF10 and is necessary for CALM-AF10-dependent transformation. Fusions of the CALM NES (NES(CALM)-AF10) or NES motifs from heterologous proteins (ABL1, Rev, PKIA, APC) in-frame with AF10 are sufficient to immortalize murine hematopoietic progenitors in vitro. The CALM NES is essential for CALM-AF10-dependent Hoxa gene up-regulation and aberrant H3K79 methylation, possibly by mislocalization of DOT1L. Finally, we observed that CALM-AF10 leukemia cells are selectively sensitive to inhibition of nuclear export by Leptomycin B. These findings uncover a novel mechanism of leukemogenesis mediated by the nuclear export pathway and support further investigation of the utility of nuclear export inhibitors as therapeutic agents for patients with CALM-AF10 leukemias.

Background Hepatosplenic T-cell lymphoma (HSTL) is a rare form of lymphoma, comprising less than 1% of the cases. However, HSTL extracts a highly disproportionate toll on patients with a median age of diagnosis of 35 years and an expected median survival of less than two years. The vast majority of HSTL patients eventually succumb to their disease. The genetic basis of the disease is largely unknown. Although abnormalities of chromosome 7, including isochromosome 7q occur commonly in the disease, the role of specific genes and genetic mutations to the disease remains essentially unknown. Methods In this study, we sought to define the genetic features of HSTL through the whole genome sequencing and exome sequencing of 32 HSTL tumors and germline DNA (where available) from the same patients. Exome enrichment of DNA was carried out using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes as well as ∼700 human microRNAs. Both whole genome and exome sequencing were performed using the Illumina platform. Results We identified 28 candidate cancer genes that were recurrently mutated in HSTL. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, KRAS) and chromatin modification (e.g. TET1, SETD2 and MLL3), accounting for 16% and 23% of the total genetic events, respectively. Nearly all of these genes have been implicated in HSTL for the first time and provide new insights into the pathogenesis of the disease and potential targets for therapy. Whole genome sequencing confirmed isochromosome 7q as the most common recurrent chromosomal abnormality in HSTL and additional structural genetic alterations in chromosome 7. Conclusion Our study provides the most comprehensive genetic portrait of HSTL to date, and is a significant step in defining the genetic causes of this disease. Disclosures: No relevant conflicts of interest to declare.