The Prolactin Inducible Protein Modulates Antitumor Immune Responses and Metastasis in a Mouse Model of Triple Negative Breast Cancer

The prolactin inducible protein (PIP) is expressed to varying degrees in more than 90% of breast cancers (BCs). Although high levels of PIP expression in BC has been shown to correlate with better prognosis and patient response to chemotherapy, some studies suggest that PIP may also play a role in metastasis. Here, we investigated the role of PIP in BC using the well-established 4T1 and E0771 mouse BC cell lines. Stable expression of PIP in both cell lines did not significantly alter their proliferation, migration, and response to anticancer drugs in vitro compared to empty vector control. To assess the effect of PIP expression on breast tumorigenesis in vivo, the 4T1 syngeneic transplantable mouse model was utilized. In immunocompetent syngeneic BALB/c mice, PIP-expressing 4T1 primary tumors displayed delayed tumor onset and reduced tumor growth, and this was associated with higher percentages of natural killer cells and reduced percentages of type 2 T-helper cells in the tumor environment. The delayed tumor onset and growth were abrogated in immunodeficient mice, suggesting that PIP-mediated modulation of primary tumor growth involves an intact immune system. Paradoxically, we also observed that PIP expression was associated with a higher number of 4T1 colonies in the lungs in both the immunocompetent and immunodeficient mice. Gene expression analysis of PIP-expressing 4T1 cells (4T1-PIP) revealed that genes associated with tumor metastasis such as CCL7, MMP3 and MMP13, were significantly upregulated in 4T1-PIP cells when compared to the empty vector control (4T1-EV) cells. Collectively, these studies strongly suggest that PIP may possess a double-edge sword effect in BC, enhancing both antitumor immunity as well as metastasis.

3123 TGFbeta, Early Cytokine Dysregulation, and Airway Smooth Muscle Dysfunction in Cystic Fibrosis

OBJECTIVES/SPECIFIC AIMS: This study aims to first describe the unique cytokine profile and TGFbeta levels of young children with CF, then understand the pathologic effects of TGFbeta on lung function in a CF animal model. These powerful translational studies linking observations in clinical disease with a transgenic mouse model allow us a unique opportunity to investigate the role of TGFbeta in early CF lung disease. METHODS/STUDY POPULATION: Cytokine levels (TGFbeta, TNFalpha, IL-8, IL-6, HNE, and IL-1beta) in bronchoalveolar lavage fluid (BALF) from CF patients (n = 15) and non-CF control patients (n = 21) under 6 years old were determined by ELISA and Luminex assay. Tracheotomized patients without significant underlying lung disease were chosen as non-CF inflamed control patients, as they had similar levels of neutrophilic inflammation and infection as CF patients. The percentage of BAL neutrophils (% PMNs) in each sample was assessed. The relationships between cytokines were analyzed using linear regression and principal components analysis. In animal studies, CF and non-CF mice (n = 4-5 per group) were treated with intratracheal adenoviral TGFbeta1 vector, an empty vector control, or PBS. After one week, animals were collected; lung function, response to the bronchoconstrictor methacholine, and rescue with albuterol were measured utilizing a FlexiVent machine. Lungs were collected for histology. Immunohistochemistry for alpha-SMA was performed and pictures of all cross-sectional airways were obtained. Burden of ASM was assessed by dividing the square root of alpha-SMA stained airway smooth muscle by the basement membrane perimeter length of each airway. RESULTS/ANTICIPATED RESULTS: Patient characteristics of CF and non-CF inflamed control patients were similar in terms of age (3.6 yrs vs 3.3 yrs respectively, p = 0.49), positive BAL culture (13% vs 14%, p = 0.94), and % PMNs (65% vs 56%, p = 0.64). Despite these similarities, TGFbeta levels were 2-fold higher in CF versus non-CF BAL (p = 0.034). Analysis of BAL cytokines from both patient groups showed that three principal components describe 86% of total variance across the cytokine variables. These components represent different contributions from the cytokines, with TGFbeta, IL6, and % PMNs comprising one component of similarly regulated inflammatory markers. These components can distinguish CF versus non-CF patients with 77% accuracy (area under the curve: 0.77). TGFbeta concentrations were uniquely associated with increased IL-6 in CF samples (r = 0.74; p = 0.0015) but did not demonstrate association with other cytokines. After TGFbeta exposure, CF mice demonstrated greater abnormalities in airway resistance than non-CF mice, with heightened response to methacholine. Importantly, this increase in airway obstruction in CF mice was reversible with albuterol treatment, indicating airway smooth muscle dysfunction as a principal driver of lung function abnormalities. Furthermore, TGFbeta induced an increased ASM burden on lung histology in both CF and non-CF mice (p<0.05). IL-6 levels in the BAL of CF mice showed greater increases after TGFbeta treatment compared to non-CF mice (p<0.05). Empty vector control treatment did not cause lung pathology. DISCUSSION/SIGNIFICANCE OF IMPACT: Young children with CF have a unique pattern of pulmonary inflammation compared to inflamed non-CF control patients. In CF, TGFbeta pulmonary levels are uniquely associated with IL-6, a driver of ASM dysfunction in other pulmonary diseases. We followed up this clinical observation study by investigating the effect of TGFbeta on pulmonary disease in a mouse model. CF mice demonstrate increased pulmonary IL-6, airway obstruction, and ASM dysfunction after TGFbeta exposure. This study provides evidence that TGFbeta is associated with a distinct cytokine pattern that may promote ASM dysfunction in early CF lung disease. Understanding the mechanism of early CF pathophysiology will be critical in developing targeted therapeutics that can prevent early lung damage.

Uncovering Plant Growth-Mediating Allelochemicals Produced by Soil Microorganisms

Coevolving interactions between a plant population and its microbiota can potentially yield a rhizosphere enriched in metagenomes containing the blueprints for a vast array of natural products. We describe a method of isolating those metabolites through activity-based screening of soil metagenomic libraries. The method allows for the isolation of small molecules produced in vector–host expression systems containing large-insert DNA fragments extracted from the target plant rhizospheres. Allelopathic activities derived from selected clones were screened against a series of controls. Nonmetric multidimensional scaling (NMS) showed similar effects of the set of controls on lettuce growth, whereas annual bluegrass had a broader range of growth responses. Methanol extracts from clones indicating activity showed distinct patterns in grass seedling growth from the empty vector control, but the same extracts showed no effect on lettuce. The results indicate that the metagenomics method and bioassay screen of clone extracts are tools that can be used for initial determination of allelopathic activity from noncultured soil microbiota.

The Microanatomy of the Leukemic Stem Cell Niche in Murine Chronic Myelogenous Leukemia

Objectives and background: Constituents of the bone marrow microenvironment (BMM) influence the proliferation, differentiation and location of hematopoietic stem and progenitor cells (HSPC). Dependent on their maturation stage, different subsets of HSPC are localized at distinct sites in the BMM. This location depends on HSPC-intrinsic, as well as HSPC-extrinsic factors. The BMM protects leukemic stem cells (LSC) from treatment with tyrosine kinase inhibitors or chemotherapy. We, therefore, investigated the microanantomy of the LSC niche hypothesizing that it may differ from the normal HSPC niche. Methods: We used a combination of confocal and 2-photon intravital microscopy (IVM) of the murine calvarium and well-described retroviral models of BCR-ABL1+chronic myelogenous leukemia (CML) and B-cell acute lymphoblastic leukemia (B-ALL). Results: We show here that BCR-ABL1+Lin–c-Kit+Sca-1+ (LKS) CD150+CD48– (LKS SLAM) cells, which harbor the LSC fraction in the CML model, homed to locations further away from the endosteum than their normal counterparts. Prior in-vitro treatment of BCR-ABL1+ LKS with imatinib mesylate, considered standard of care in CML, reversed this phenotype and the cells were found closer to the endosteum. Native BCR-ABL1, as well as the imatinib-resistant BCR-ABL1 point mutants BCR-ABL1Y253F, BCR-ABL1E255K, BCR-ABL1T315I and BCR-ABL1M351T had similar intrinsic catalytic activity, but the BCR-ABL1Y253F, BCR-ABL1E255K, and BCR-ABL1T315I mutants increased the IL-3-independent proliferative capacity of 32D cells relative to native BCR-ABL1. BCR-ABL1Y253F and BCR-ABL1M351T caused increased transformation of primary BM B-lymphoid progenitors in vitro and led to accelerated induction of B-ALL in mice. In the CML model, BCR-ABL1Y253F and BCR-ABL1T315Iinduced myeloproliferative neoplasia with shortened survival and features of accelerated phase disease compared to native BCR-ABL1, whereas BCR-ABL1T315I LKS cells homed closer to osteoblastic cells than LKS cells expressing native BCR-ABL1. Sequential in vivo tracking of leukemic progenitor growth by IVM showed a similar nadir in the number of cells per leukemic cell ‘nest’ 11 days after irradiation and IV transplantation in recipients of DsRed+BCR-ABL1+ or empty vector control-transduced bone marrow. However, between days 18-25 after transplantation there was a significant increase in the number of cells per leukemic cell ‘nest’ compared to the empty vector control group. Sequential immunohistochemistry and TUNEL assays of leukemic bone sections in imatinib- or vehicle-treated recipient mice with CML showed that initial BCR-ABL1+ growth tends to occur at locations further away from the endosteum, whereas erythroid islands were found closer to the endosteum and trabeculae. Apoptosis in response to imatinib appeared most prominent in the metaphysis. Lastly, we could demonstrate by IVM in the CML model that treatment of mice with a combination of imatinib plus granulocyte colony-stimulating factor led to ‘emptying’ of the LSC niche and superior eradication of BCR-ABL1+ leukemic cells compared to treatment with imatinib alone. Conclusions: In summary, these data suggest that the microanatomy of the LSC niche in CML differs from the normal hematopoietic niche. BCR-ABL1 mutation status may affect the positioning of CML LSC in the microenvironment, and location in the niche may be altered pharmacologically, suggesting that niche location may influence clinical outcome. Disclosures Krause: Glycomimetics. Inc.: Research Funding.

Identification of CAR As a Novel Mediator of Erythroid Differentiation and Migration That Is Specifically Downregulated in Erythropoietic Progenitor Cells in Patients with MDS

Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by peripheral cytopenia, an accumulation of dysplastic cells in the bone marrow (BM) and a high risk of transformation to acute myeloid leukemia. Transfusion-dependent anemia develops in most patients suffering from MDS. In patients with refractory anemias the accumulation of dysplastic erythroid BM progenitor cells (EryPC) which paradoxically accompanies peripheral anemia, is a typical finding. Although various hypotheses have been discussed, the biochemical basis of the maturation defect of erythroid cells and their abnormal accumulation in the BM in MDS remains unknown. In order to learn more about abnormally regulated and functionally relevant genes expressed in EryPC, we have recently established a screening program involving mRNA expression profiling studies of EryPC in patients with low-risk MDS (IPSS-R score<7) and control BM samples obtained from patients with other BM neoplasms as well as patients with unexplained cytopenia, normal BM (staging for lymphomas) or reactive/deficiency cytopenias. EryPC were defined as CD45low/CD105+ cells and purified from BM mononuclear cells (MNC) by multicolor flow cytometry (MFC) and cell sorting (purity>95%). mRNA expression profiles were analyzed by Affymetrix array technology (GeneChip U133 Plus 2.0 arrays) and confirmed for a panel of selected mRNA species by qPCR. In mRNA- and MFC-validation experiments, we found that the major Coxsackie-Adenovirus Receptor (CAR) is markedly and specifically down-regulated in CD45low/CD105+ EryPC in patients with MDS when compared to EryPC in normal BM or other control cohorts. In line with this observation, the immature erythroblastic cell lines HEL, K562 and KU812 all stained negative for CAR in flow cytometry analyses. Lentiviral transduction of the full-length CAR gene into these cells resulted in a significantly increased expression of various erythroid differentiation antigens, including CD36, CD71 and CD235a (Glycophorin-A) as determined by flow cytometry and qPCR. As assessed by flow cytometry, the levels of CD235a increased to 158±46% in HEL cells, to 211±23% in K562 cells and to 170±30% in KU812 cells after CAR transduction compared to the empty vector control (p<0.05). In addition, CAR transduction resulted in an increased migration of HEL cells against a serum protein-gradient in a transwell assay (empty vector control: 185±67% vs CAR-transfected cells: 356±74%, p<0.05). Transfection with truncated variants of CAR did not result in an increased expression of erythroid antigens or an increased directed migration. In conclusion, our data show that CAR is a functionally relevant antigen that promotes the expression of early erythroid differentiation antigens on myeloid progenitor cells and their migration against blood serum proteins. In patients with MDS, CAR is specifically downregulated on EryPC, which may have clinical implications both in term of the pathogenesis of the disease and the application of this novel marker in diagnostic MFC algorithms. With regard to functional consequences, we hypothesize that CAR-deficiency is pathogenetically relevant as it may not only contribute to the maturation-defect of erythroid progenitor cells in MDS EryPC but also to the related accumulation of erythroid cells in the BM that is accompanying the peripheral anemia in these patients. Disclosures No relevant conflicts of interest to declare.

High Expression of Mir-9 down-Regulates the Poor Outcome Prognosticator ERG and Associates with Reduced Relapse-Rates in Acute Myeloid Leukemia

In acute myeloid leukemia (AML) high expression of the transcription factor ERG (ETS related gene) is associated with dismal outcome. The mechanisms that regulate differential ERG expression remain to be fully elucidated. MicroRNAs (miRs), small RNAs that are able to regulate gene expression, have emerged as important players in AML. We hypothesized that part of the differential expression of ERG is mediated by miRs. In silico prediction tools identified three putative miR-9 binding sites (BS) in the 3'-untranslated region (UTR) of ERG. First, we determined the expression levels of ERG & miR-9 in eight leukemia cell lines (i.e. KG1a, K562, THP-1, MV4-11, EOL1, NB4, OCI-AML3 & ME1) & found an inverse correlation between ERG & miR-9 expression (rank correlation -0.90). The cell line KG1a had the highest ERG & low miR-9 expression, and was therefore used for miR-9 overexpression experiments. In these cells miR-9 overexpression decreased ERG expression at the mRNA level to 82±7% (P=.079) & at the protein level to 72±14% (P=.005) after 12 hours (h) compared to empty vector control transfected cells. Next, we tested the activity of the three putative miR-9 BS in the 3'-UTR of ERG using luciferase assays. 12 h after cotransfection of HEK-293T cells with a miR-9 overexpression vector & an appropriate luciferase vector containing two of the putative BS (BS1 & BS2) from the 3'-UTR of ERG, we found the luciferase activity reduced to 52±4% (P=.023). Mutation experiments showed BS1, but not BS2 to be essential for this activity. The insertion of BS3 into the luciferase vector had no effect on reporter gene expression. Thus miR-9 most likely regulates expression of the transcription factor ERG by directly binding to BS1 in its 3'-UTR. To test if a differential expression of miR-9 is also of functional significance in AML, we first analyzed its impact on cell proliferation. Overexpression of miR-9 led to decreased proliferation rates in KG1a cells compared to control vector treated cells. After 5 days, the relative cell count was 133±21% vs. 241±67% in the miR-9 overexpressing cells compared to empty vector expressing cells, respectively. Next, we determined if this difference was based on a higher apoptosis rate. An Annexin V staining revealed no significant difference between the apoptotic threshold of miR-9 overexpressing (21%) and empty vector cells (21%) after 24 h. However, a subsequent cell cycle analysis demonstrated a higher percentage of miR-9 overexpressing cells in the G2/M phase, (39±2%) compared to the empty vector control treated cells (31±3%) after 24 h (P=.084), indicating that the cell cycle is slowed or stopped during cell division. Since miR-9 targets the poor prognostic marker ERG & higher miR-9 expression led to decreased proliferation & reduced cycling in AML cells in vitro we speculated that the differential miR-9 expression would also impact the outcome of AML patients (pts). Mature miR-9 is derived from three precursor molecules of which pre-miR-9-1 & pre-miR-9-3 are known to be expressed in hematopoietic cells. We assessed the pre-miR-9-1 expression of bone marrow by real-time PCR in 131 AML pts (median age 64 [range 22 – 75] years) with favorable (n=4, 3%), intermediate (n=90, 69%), adverse (n=33, 25%), or unknown (n=4, 3%) cytogenetic risk (according to the Medical Research Council [MRC] Classification) who received a RIC-HCT. The median follow-up was 4 years. The pre-miR-9-1 expression levels were normalized to ABL to define high & low pre-miR-9-1 expressers by the median expression of all AML pts. At diagnosis, high pre-miR-9-1 expresser status associated with a lower white blood count (P=.065) and lower % of peripheral blasts (P=.108) by trend. Furthermore, pts with high pre-miR-9-1 expression were more likely to be NPM1 wild-type (P=.047) & FLT3-ITD negative (P=.020). Pts with high pre-miR-9-1 had a lower probability of relapse (P=.048). In conclusion, miR-9 targets & regulates expression of the poor outcome predictor ERG. Overexpression of miR-9 led to decreased proliferation and a pause in AML cell cycling. Furthermore, high pre-miR-9-1 expression associated with reduced relapse rates in AML. Thus a pharmacologically induced expression of miR-9 in AML blasts may improve outcomes of AML pts. Disclosures No relevant conflicts of interest to declare.

Regulation of STAT3 activity by DARPP-32 in gastric cancer.

47 Background: Dopamine and cAMP regulated phosphoprotein MW 32 kDa (DARPP-32) and its truncated form (t-DARPP) are overexpressed in two-thirds of gastric adenocarcinomas. STAT3 is a member of STAT family. It mediates the expression of a variety of genes in response to cell stimuli, and thus plays a key role in many cellular processes such as cell growth and apoptosis. Constitutive STAT3 activation is associated with various human cancers and commonly suggests poor prognosis. It has anti-apoptotic as well as proliferative effects. The relationship and interaction between DARPP-32 and STAT3 remain unknown. Methods: DARPP-32 was stable or transient overexpressed in gastric cancer cell lines. STAT3 expression and transcriptional activity were investigated by immunofluorescence and STAT3 luciferase reporter assay. DARPP-32 mRNA level was detected in 63 gastric cancer samples. Results: Using stable and transient expression of DARPP-32 and t-DARPP in AGS gastric cancer cell line, lacking endogenous DARPP-32, led to a significant increase of the phosphorylation of STAT3 as compared to empty vector control. Consistent with these findings, immunofluorescence assay also showed the up-regulation of nuclear p-STAT3 in AGS cells following transient expression of DARPP-32, as compared to empty vector control. Using the STAT3 luciferase reporter assay, AGS cells with stable overexpression of DARPP-32 showed higher luciferase activity than empty vector stable cells with and without IL-6 stimulate (p<0.01). Using qPCR in 63 gastric cancers, we found that DARPP-32 is frequently overexpressed in gastric cancer samples (p=0.018). We have also shown that DARPP-32 has a progressive increase in expression from normal to metaplasia, dysplasia, and adenocarcinoma (p<0.001). The relationship between DARPP-32 and STAT3 in these tissues will be examined. Conclusions: Our results suggest that DARPP-32 overexpression may participate in regulating the STAT3 phosphorylation and activating STAT3 signaling pathway in gastric cancer. The precise signaling mechanisms that mediate this activation and its functional impact on gastric tumorigenesis are under investigation.

Plant-mediated RNA interference of effector gene Mc16D10L confers resistance against Meloidogyne chitwoodi in diverse genetic backgrounds of potato and reduces pathogenicity of nematode offspring

Meloidogyne chitwoodi is a major problem for potato production in the Pacific Northwest of the USA. In spite of long-term breeding efforts no commercial potato cultivars with resistance to M. chitwoodi exist to date. The resistance gene against M. chitwoodi has been introgressed from Solanum bulbocastanum into cultivated potato (S. tuberosum), but M. chitwoodi pathotypes are able to overcome this resistance. In this study, an RNA interference (RNAi) transgene targeting the M. chitwoodi effector gene Mc16D10L was introduced into potato cvs Russet Burbank and Désirée, and the advanced breeding line PA99N82-4, which carries the gene. Stable transgenic lines were generated for glasshouse infection assays. At 35 days after inoculation (DAI) with M. chitwoodi race 1 the number of egg masses (g root)−1 formed on RNAi lines of cvs Russet Burbank and Désirée was reduced significantly by up to 68% compared to empty vector control plants. At 55 DAI, the number of eggs was reduced significantly by up to 65%. In addition, RNAi of Mc16D10L significantly reduced the development of egg masses and eggs formed by the resistance-breaking M. chitwoodi pathotype Roza on PA99N82-4 by up to 47 and 44%, respectively. Importantly, the plant-mediated silencing effect of Mc16D10L was transmitted to M. chitwoodi offspring and significantly reduced pathogenicity in the absence of selection pressure on empty vector control plants. This finding suggests that the RNAi effect is stable and nematode infection decreases regardless of the genotype of the host once the RNAi process has been initiated in the nematode through a transgenic plant. In summary, plant-mediated down-regulation of effector gene Mc16D10L provides a promising new tool for molecular breeding against M. chitwoodi.

Protein Tyrosine Phosphatase Type-1 (PTPN1) Is Frequently Mutated In Primary Mediastinal B Cell Lymphoma and Hodgkin Lymphoma

Introduction Hodgkin Lymphoma (HL) accounts for 11% of all lymphomas and despite being one of the most curable lymphomas, 20% of HL patients still ultimately die of their disease. Similarly, a proportion of cases of primary mediastinal B cell lymphoma (PMBCL) have refractory disease or early relapse and frequently fail second-line therapy. Development of more targeted therapeutic approaches is impeded by the lack of knowledge about the mutational landscape in the cancer genomes of these lymphomas. PTPN1 is a protein tyrosine phosphatase gene that encodes the protein, PTP1B. PTP1B dephosphorylates tyrosine residues on many activated kinases to maintain cellular homeostasis. As overactive receptor kinases are critical oncogenic events in cancer, we hypothesized that constitutively active Janus kinase-Signal transducer and activation of transcription (JAK-STAT) observed in HL and PMBCL are in part due to a mutated PTPN1 gene with an impaired functional ability to dephosphorylate this constitutive signaling pathway. Methods and samples Biopsies at the time of primary diagnosis were obtained for 49 PMBCL and 30 HL patients from the British Columbia Cancer Agency, Arizona Lymphoma Repository and the Hôpital Henri Mondor Pathology Department. DNA from PMBCL samples, microdissected Hodgkin Reed Sternberg (HRS) cells and 12 lymphoma-derived cell lines were extracted for PTPN1 exonic PCR amplification (nested PCR was used for HRS cell DNA) and Sanger sequencing. PTPN1 was silenced in a HL cell line (KMH2) by lentiviral transduction of a vector expressing shRNA and confirmed by quantitative real time (qRT) PCR. Wild type and mutant PTPN1 cDNA were cloned into the mammalian expression vector pcDNA 3.1 and expressed in HEK-293 cells. Protein expression of clinical samples, silenced and expressed cells were analyzed by immunohistochemistry and western blotting. Comparisons between groups were performed using two-sample student t tests. Results After exclusion of reported single nucleotide polymorphisms (SNPs) and silent mutations, 16 PTPN1 coding sequence mutations were found in our PMBCL cohort, corresponding to 14 mutations (29%) in clinical samples and 2 in PMBCL-dervied cell lines. Twelve additional mutations were discovered in our HL cohort, corresponding to 6 mutations (20%) in HRS cell samples and another 6 in HL-derived cell lines. In total, 14 (54%) missense, 4 (15%) frameshift, 3 (12%) single amino acid deletions, 4 (15%) nonsense mutations, and 1 (4%) promoter mutation were observed. Eight of these mutations were confirmed as somatic by sequencing of matched constitutional DNA. Silencing of PTPN1 resulted in hyperphosphorylation of JAK1, JAK2, STAT3, STAT5, STAT6 and up-regulation of the oncogenes, MYC and BCL6. Ectopic expression of nonsense and missense PTPN1 mutants in HEK-293 cells led to sustained phosphorylation of STAT6 in comparison to the empty vector control (densitometric values Q9* 0.5 vs. 1.0, R156* 0.7 vs. 1.0, M74L 0.4 vs. 1.0 and M282L 0.8 vs. 1.0). Furthermore, no phosphatase activity was observed for the nonsense mutants and moderate phosphatase activity for the missense mutants using a tyrosine phosphatase-specific substrate (fold change Q9* 2.0, R156* 1.9, M74L 46.7, M282L 46.0 and WT 58.3, compared to empty vector control). Immunohistochemical analysis showed that PTPN1 mutations correspond to decreased protein expression in PMBCL (p=0.03). Discussion PTPN1 is recurrently mutated in PMBCL and HL contributing to constitutive JAK-STAT signaling and oncogene dysregulation. These data suggest PTPN1 mutations as novel driver alterations in these lymphomas and might provide a novel, rational therapeutic target for treating HL and PMBCL patients. Disclosures: Savage: Eli-Lilly: Consultancy. Connors:F Hoffmann-La Roche: Research Funding; Roche Canada: Research Funding.

Whole-Exome Analysis Of DLBCL Tumors Reveals a Unique Genetic Signature Associated With Aggressive Disease

While extensive, the genetic studies of DLBCL tumors to date have primarily focused on somatic mutations that are associated with development of lymphoma, i.e., driver mutations. Identification of key genetic events that impact and/or predict response to therapy and clinical outcome of lymphoma patients has not been accomplished. In an exploratory study, we used whole-exome sequencing to identify novel biomarkers that can predict which patients have an aggressive form of DLBCL, and therefore would likely benefit from a different and more aggressive treatment plan. Using whole-exome sequencing data from 54 newly diagnosed DLBCL patients, we evaluated the association of somatic coding single nucleotide (cSNV) and copy number (CNV) variants with aggressive DLBCL. All patients were treated with R-CHOP or immunochemotherapy, and disease aggressiveness was based on relapse, with patients classified as having aggressive disease (AD) (n=13, defined as treatment failure, relapse, or progression within 24 months of diagnosis) versus non-aggressive disease (n=41, defined as achieving at least 24 months of relapse-free survival). An in-house algorithm, patternCNV was used to detect somatic CNVs. Logistic regression was used to assess statistical significance. In the cSNV analysis, 24 genes were found to be significantly (p ≤ 0.05) associated with AD. Of these genes, CIITA (p=0.01) was previously identified as a mutational target in DLBCL and is a recurrent gene fusion partner in lymphoid cancers. Metacore analyses showed that seven of these genes, including CIITA, can be placed in the same regulatory network centered around CREB1. Interestingly, the CREB binding protein (CREBBP) is a known target of pathogenic mutations in DLBCL. In the CNV analyses, we identified 245 gene amplifications and 209 gene deletions associated with AD (p ≤ 0.05). Deleted genes were localized in three major chromosomal regions, 6p22.3-6p22.1 (26 genes), 6q13-6q16.3 (31 genes), and 6q21-6q24.2 (86 genes). In the CNV amplification analysis, the genes were localized in two major chromosomal regions, 3q12.3-3q29 (90 genes) and 19q13.12-19q13.43 (122 genes). We also quantified the association of each regional deletion/amplification with patient outcome. As expected, each deleted region was univariately associated with time to relapse: 6p22.3-6p22.1 (HR = 7.19, 95% CI: 2.66-19.46, p=1.33x10-5), 6q13-6q16.3 (HR = 4.44, 95% CI: 1.74-11.32, p=6.84x10-4), and 6q21-6q24.2 (HR = 5.42, 95% CI: 2.12-13.87, p=9.23x10-5) as well as the two amplification regions: 3q12.3-3q29 (HR = 4.60, 95% CI: 1.49-14.24, p=4.27x10-3) and 19q13.12 -19q13 (HR = 13.79, 95% CI: 4.39-43.34, p=2.81x10-8). Deletions at 6q21-6q24.2 have been previously reported in DLBCL and two tumor suppressor genes associated with lymphoma pathogenesis are localized in the region, TNFAIP3 (p=0.14) and PRDM1 (p=0.02), the latter of which was significantly associated with AD. However, the genes in the 6p21 locus that were most significant (p=0.002) were SLC22A16, GPR6, SOBP, MATTL24, DDO, and REV3L. SLC22A16, which also had cSNVs in two tumors, is an organic cation transporter that has been shown to transport chemotherapeutic drugs including doxorubicin. Successful drug response has been correlated with the level of activity and expression of this transporter in tumor cells. To determine if expression of SLC22A16 would have an impact on doxorubicin transport, and may therefore impact response to R-CHOP we overexpressed either an empty vector control or SLC22A16 in OCI-Ly7 DLBCL cells, which do not express endogenous SLC22A16 mRNA. We found that OCI-Ly7 cells expressing SLC22A16 have increased 14C-doxorubicin uptake and are more sensitive to doxorubicin-induced cell death when compared to the empty vector control cells. These data suggest that loss of SLC22A16 expression through gene deletion could impact the ability of DLBCL cells to respond to therapy and indicates an aggressive form of the disease. In summary, our data are the first to use whole exome sequencing to identify genetic variants associated with aggressive DLBCL, which will require replication in additional samples. Nevertheless, our data highlight the role for somatic CNVs in patient outcome and we biologically validate the potential impact of deletions in SLC22A16 at 6p21. Disclosures: No relevant conflicts of interest to declare.