Porcher, Julie C.

Introduction: Nitrogen dioxide (NO 2 ) is known to be a trigger for asthma exacerbation. However, little is known about the role of seasonal variation in indoor and outdoor NO 2 levels in childhood asthma in a mixed rural-urban setting of North America. Methods: This prospective cohort study, as a feasibility study, included 62 families with children (5-17 years) that had diagnosed persistent asthma residing in Olmsted County, Minnesota. Indoor and outdoor NO 2 concentrations were measured using passive air samples over 2 weeks in winter and 2 weeks in summer. We assessed seasonal variation in NO 2 levels in urban and rural residential areas and the association with asthma control status collected from participants’ asthma diaries during the study period. Results: Outdoor NO 2 levels were lower (median: 2.4 parts per billion (ppb) in summer, 3.9 ppb in winter) than the Environmental Protection Agency (EPA) annual standard (53 ppb). In winter, a higher level of outdoor NO 2 was significantly associated with urban residential living area ( P = .014) and lower socioeconomic status (SES) ( P = .027). For both seasons, indoor NO 2 was significantly higher ( P < .05) in rural versus urban areas and in homes with gas versus electric stoves ( P < .05). Asthma control status was not associated with level of indoor or outdoor NO 2 in this cohort. Conclusions: NO 2 levels were low in this mixed rural-urban community and not associated with asthma control status in this small feasibility study. Further research with a larger sample size is warranted for defining a lower threshold of NO 2 concentration with health effect on asthma in mixed rural-urban settings.

Abstract Background: For most patients with MDS, the disease-associated molecular abnormalities are unknown, which contributes to diagnostic uncertainty and has limited development of effective therapies. Chromosomal deletions such as del(5q) or monosomy 7 are more common than translocations in MDS, but balanced translocations are more likely to be pathobiologically informative. Here we report cloning of an MDS-associated translocation that revealed a novel recurrent molecular abnormality. Methods: We first studied a 71-year old man who presented with MDS and t(6;9)(p21.3;q34) as an isolated chromosomal abnormality in 18 of 20 metaphases. FISH assays for DEK/CAN and ABL rearrangements were unrevealing. We generated somatic hybrid murine/human cell lines from a buffy coat; microdissected and amplified derivative chromosomes; and hybridized to 6p/9q custom comparative genomic hybridization (CGH) arrays. The CGH results considerably narrowed the 6p and 9q breakpoints, which were then amplified by long-range PCR and sequenced to define the rearrangement. Results: The IER3 gene (Immediate Early Response 3; also known as IEX-1) at 6p21.3 was found to be separated from its upstream regulatory elements in this rearrangement and translocated to a transcript-poor region of 9q. RT-PCR confirmed marked downregulation of IER3 expression in the patient compared to healthy controls. IER3 is a plausible candidate for involvement in MDS because of its known role in regulating death receptor-induced apoptosis, interaction with NF-κb pathways, and its importance in response to genotoxic stresses such as ionizing irradiation. We then identified archival bone marrow cell pellets from 204 additional patients with various clonal hematological disorders and chromosomal rearrangements involving 6p21 or 6p22 (i.e., translocations, inversions, deletions, or additions), and designed a splitsignal FISH probe set to assay IER3. FISH results were abnormal in 8 additional patients (9/205 pts total abnormal, 4.4%): 3 split signals and 6 amplifications (one patient had both abnormalities); all 8 patients had MDS and 6p rearrangements as part of a more complex karyotype. FISH studies in MDS patients without 6p rearrangements are ongoing. RTPCR in MDS patients without 6p rearrangements demonstrated down-regulation of IER3 by &gt;2-fold in 38% of patients (67% lower-risk) and up-regulation in 12% of patients (50% higher-risk) when compared to the mean for healthy controls. Direct sequencing of the IER3 coding region and the promoter region in 30 patients without 6p rearrangements revealed no point mutations, but the prevalence of non-synonymous single nucleotide polymorphism rs3094124 (p.A127P) was higher than expected in patients (variant in 28.3 % of MDS alleles vs. 8.6% of 116 HAP-CEU controls, p=0.0015). IER3−/− mice are known to have hypertension, cardiac hypertrophy, and epithelial proliferation, but are not anemic (Sommer SL et al J Appl Physiol 2006). We performed methylcellulose hematopoietic colony growth assays using bone marrow mononuclear cells from IER3−/− mice and wildtype controls, and noted no differences in BFU-E, CFU-E, and CFU-GM growth under unstressed conditions. Studies of IER3 expression induction under stressed conditions in primary MDS cells are ongoing. Conclusion: IER3 rearrangements represent a novel clonally-restricted recurrent genetic abnormality in MDS. Dysregulation of IER3 expression is common in MDS, even in patients without 6p rearrangements. This is the first time IER3 has been linked to human disease. Clarification of the role of IER3 in MDS is likely to yield new insights into MDS pathobiology.

Abstract Background: Many patients (pts) with anemia due to impaired erythropoiesis fail to respond to currently available erythropoiesis-stimulating agents or do not wish to receive these agents due to safety concerns. For these pts, novel approaches are needed. DYRK3, an evolutionarily conserved member of an emerging family of serine-threonine kinases, is expressed at high levels in erythropoietic progenitors; murine models suggest that DYRK3 selectively inhibits red cell production during stress erythropoiesis. We sought to determine DYRK3 expression in pts with anemia, and whether in vitro inhibition of DYRK3 augments erythropoiesis. Methods: We performed quantitative RT-PCR for DYRK3 and 4 control genes using whole peripheral blood (WPB) from 14 healthy persons, 5 pts with anemia due to multiple myeloma (MM), and 3 pts with anemia of chronic disease (ACD); in addition, peripheral blood mononuclear cells (PBMCs) were assayed in the 3 ACD and 5 MM pts and bone marrow mononuclear cells (BMMCs) in the MM pts. Erythrocyte subpopulations (CD36+, CD71+, and dual CD36+/CD71+) were quantified by flow cytometry. CFU-E growth was measured from PBMCs and BMMCs in the presence of varying concentrations of GSK626616, an orally bioavailable first-generation DYRK3 kinase inhibitor. Results: Normalized expression of DYRK3 in WPB was 7.2 fold higher in MM pts (p=0.0001) and 3.4 fold higher in pts with ACD (p=0.026) compared to healthy controls. DYRK3 expression was proportional to the degree of anemia, and WPB and PBMC expression of DYRK3 in MM pts correlated well with BMMC expression. The level of DYRK3 expression was proportional to the population of marrow CD36+/CD71+ erythroid progenitors, and inversely proportional to the size of the more mature CD36−/CD71+ population, suggesting that high DYRK3 expression is associated with maturation arrest in humans at a stage of erythroid differentiation roughly corresponding to pre-Ter119pos/CD71high inhibition observed in murine models. Although incubation of pt-derived BMMC or PBMC with GSK626616 at concentrations up to 30 μM, either in the presence or absence of physiological concentrations of erythropoietin, did not augment in vitro CFU-E formation, CFU-E growth overall was poor in the pt samples studied. Conclusion: DYRK3 is expressed at high levels in pts with anemia due to neoplasia or inflammation, and elevated DYRK3 expression is associated with decreased numbers of CD36−/CD71+ red cells. Further studies of the effects of DYRK3 antagonists on human erythropoiesis in vitro are necessary, and clinical trials in anemic patients will be required to determine if DYRK3 antagonists can reverse DYRK3-associated inhibition.

Abstract Abstract 88 Introduction: The biology and genetics of anaplastic large T-cell lymphomas (ALCLs) lacking ALK translocations remain poorly defined. We recently identified recurrent translocations involving the DUSP22-IRF4 locus on 6p25.3 in ALK-negative ALCL (Blood 2011;117:915–9). This translocation is present in about 20% of ALK-negative ALCLs and is absent in other T-cell lymphomas; however, the biologic consequences are unknown. In this study, we utilized gene expression profiling (GEP) to compare T-cell lymphomas with and without this translocation. Methods: cDNA was prepared from total RNA extracted from frozen tissue from 25 T-cell lymphoma patients under a Mayo Clinic IRB-approved protocol as follows: 5 ALK-negative ALCLs with 6p25.3 translocations (2 systemic, 3 cutaneous), 12 ALK-negative ALCLs without 6p25.3 translocations (11 systemic, 3 cutaneous), 5 ALK-positive ALCLs, and 3 CD30-positive peripheral T-cell lymphomas (1 transformed mycosis fungoides and 2 not otherwise specified). Partner loci in cases with 6p25.3 translocations were 7p32.3 in 2, 9p24.3 in 1, and unknown in 2. GEP data (Affymetrix U133 plus 2.0) were analyzed by unsupervised clustering; in addition, a GEP signature was developed using genes with raw values >200 in at least 2 cases with at least 2-fold, statistically significantly different expression between 6p25.3-translocated and non-translocated cases (p<0.05 after Benjamini-Hochberg multiple testing correction). Representative overexpressed genes were confirmed by quantitative real-time (qRT-) PCR. Supervised pathway analysis was performed using signatures derived from other studies or curated from public pathway databases. Results: By unsupervised clustering, the 5 cases with 6p25.3 translocations clustered together (281 probe sets). A GEP signature segregating translocated from non-translocated cases included 283 genes (195 up- and 88 down-regulated). Key features of this signature included altered expression of histone-modifying genes (e.g. EZH2, SUZ12, MLLT3, MLL; HDAC4) and up-regulation of cancer-testis antigen (CTA) genes (e.g. SSX4, TEX14, MAGEA12, SSX1, CTAG2). The signature included down-regulation of DUSP22 but not of other genes on 6p25 in the signature. Additional overexpressed genes were validated by qRT-PCR (relative expression: TEX14, 2.9, p=0.01, t test; MAL, 6.5, p=0.003; CCR8, 7.6, p=0.0004). Supervised pathway analysis demonstrated a high proliferation signature in cases with 6p25.3 translocations (p=0.006 vs. untranslocated cases). Conclusions: ALK-negative ALCLs with translocations involving the DUSP22-IRF4 locus on 6p25.3 have a distinct gene expression signature characterized by alterations of histone-modifying genes and CTA genes (which typically are regulated epigenetically, either through histone modifications or changes in methylation). This signature appears independent of anatomic site (systemic vs. cutaneous) and partner locus (7p32.3 vs. other). DUSP22, encoding a dual-specificity phosphatase involved in modulating mitogen-activated protein kinase (MAPK) signaling, is significantly down-regulated in translocated cases; however, pathway analysis did not show significant alterations in MAPK-associated genes. Other known genes in the region of the breakpoint on 6p25 were not affected substantially. These findings suggest the possibility of an unrecognized function of DUSP22. Contributions of the varying partner loci remain unclear. The distinct GEP signature associated with 6p25.3 translocations provides strong support for their biologic relevance. Finally, the genes involved in the histone-modifying signature show overlap with histone-modifying genes mutated in B-cell lymphomas, suggesting potential commonalities in the biology and possibly targeted treatment of B- and T-cell lymphomas. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 633 Background: To date, the only well characterized chromosomal translocations among peripheral T-cell lymphomas (PTCLs) are those involving ALK, which occur in about half of anaplastic large cell lymphomas (ALCLs). ALK-negative ALCLs remain poorly understood, despite morphology and phenotype similar to ALK-positive ALCLs. We recently described translocations near IRF4 on 6p25.3 in cutaneous ALK-negative ALCLs. In the present study, we utilized massively parallel (Next Generation) sequencing to answer three questions about these translocations: (1) Were they recurrent in the more lethal systemic form of ALK-negative ALCL? (2) What was the translocation partner? (3) What were the likely genetic consequences? Methods: We used a mate pair library approach to juxtapose DNA sequences originally lying ~5 kb apart: these paired sequences map to distinct areas of the genome if a translocation breakpoint lies between them. Because only the ends of the original DNA fragments are sequenced, this approach is more resource efficient than whole genome sequencing. Genomic DNA from a systemic ALK-negative ALCL with an uncharacterized 6p25.3 rearrangement was fragmented to 2–5 kb, biotinylated, and separated on an agarose gel. Fragments ~5-5.5 kb were excised, purified, circularized, and further fragmented to ~300-600 bp. Fragments containing biotin (representing the ends of the original 5 kb fragments) were immobilized on streptavidin beads and amplified using the Illumina library protocol. Paired end sequencing was performed on one lane of a flow cell on an Illumina GAIIx. Sequencing data were mapped to the genome using a binary indexing algorithm. Non-mapping and duplicate sequences were discarded. Candidate translocations suggested by >4 unique mate pairs were examined by BLAT to exclude false positives explained by mapping to homologous regions. PCR and Sanger sequencing of tumor DNA was used for validation. Fluorescence in situ hybridization (FISH) was performed on additional PTCLs. mRNA and miRNA expression was quantified using real-time PCR. Results: Of 28.90×106 paired sequences, 8.61×106 were non-duplicate pairs in which both ends mapped to the human genome (bridged coverage, ~7x). Only one candidate translocation involved 6p25.3, represented by 10 unique mate pairs. PCR and Sanger sequencing confirmed a balanced translocation: one breakpoint disrupted the dual-specificity phosphatase gene, DUSP22, telomeric to IRF4 on 6p25.3; the other was telomeric to the FRAH7 fragile site on 7q32.3. Additional ALK-negative ALCLs with known 6p25.3 rearrangements were tested using a 7q32.3 breakapart FISH probe, which showed a 7q32.3 rearrangement in 13/29 (45%; 7 systemic and 6 cutaneous). Dual fusion FISH confirmed t(6;7)(p25.3;q32.3) in 11/11 cases tested. None of 120 PCTLs (including 39 ALCLs) lacking 6p25.3 rearrangements had a 7q32.3 rearrangement. Levels of 5′ and 3′ DUSP22 mRNA in cases with 6p25.3 rearrangements were decreased relative to those in non-rearranged cases (5′: 0.02±0.01 vs 1.00±0.74, p=.002; 3′: 0.09±0.08 vs 1.00±0.55, p=.0002; mean±SD, t test). IRF4 mRNA levels were similar in both groups. Levels of MIR29A and MIR29B1 within FRAH7 in cases with 7q32.3 rearrangements were increased relative to those in non-rearranged cases (MIR29A: 2.44±2.20 vs 1.00±1.34, p=.15; MIR29B: 4.93±4.68 vs 1.00±1.05, p=.007). Conclusions: The t(6;7)(p25.3;q32.3) is the first recurrent translocation characterized in systemic and cutaneous ALK-negative ALCLs. This translocation was entirely specific for ALK-negative ALCLs, and was present in 45% of cases with 6p25.3 rearrangements. The t(6;7)(p25.3;q32.3) was associated with down-regulation of DUSP22 and up-regulation of MIR29, but not dysregulation of IRF4. DUSP22 inhibits mitogen activated protein kinase activity in T cells, and may represent a novel tumor suppressor in ALCLs. Increased MIR29 levels may additionally contribute to tumorigenesis, as recently shown in acute myeloid leukemias. Finally, clinical testing for t(6;7)(p25.3;q32.3) may aid in differentiating ALK-negative ALCL from other PTCLs. Our findings highlight the utility of mate-pair library sequencing to discover novel translocations in lymphoma and other cancers. Disclosures: No relevant conflicts of interest to declare.

Abstract Background: Peripheral T-cell lymphomas (PTCLs) comprise a heterogeneous group of relatively uncommon and generally aggressive non-Hodgkin lymphomas. Recent studies have begun to reveal their mutational landscape, such as the recurrent mutations affecting RHOA, IDH2, and TET2 in angioimmunoblastic T-cell lymphomas (AITLs) and related PTCLs. Here, we studied the exomes of 63 PTCLs to address several unanswered questions: (1) How frequently do PTCLs have mutations with a strong rationale for being actionable using clinically available targeted therapies? (2) What pathways are enriched in PTCLs that could point to additional targeted therapies? (3) What additional recurrent mutations exist that may provide insight into PTCL pathogenesis and classification? Methods: We performed exome capture and sequencing in 63 frozen PTCL samples (with matched germline controls from 22), including 13 PTCLs, not otherwise specified; 6 AITLs; 37 anaplastic large cell lymphomas (ALCLs: 12 ALK+, 19 ALK-, and 6 cutaneous); 4 extranodal NK/T-cell lymphomas; 2 cutaneous T-cell lymphomas, and 1 enteropathy associated T-cell lymphoma. Following primary variant calling, data were filtered by removing variants that were: outside the capture region, polymorphic (0.1% in ExAC), seen in paired germline or 50 healthy controls, within repetitive elements, or synonymous. Potentially actionable variants required published evidence for altering protein function and demonstrated in vivo or in vitro sensitivity to an available targeted agent (including open clinical trials). Mutated cancer genes (COSMIC) were examined for enrichment of KEGG canonical signaling pathways. A recurrent mutation encoding MSC E116K was validated in tumor DNA and MSC protein was examined by immunohistochemistry. Results: Of 25 unique variants with published evidence suggesting actionability (including ErbB, JAK/STAT, and RAS genes), at least one variant was present in 16/63 patients (25%). Variants in any COSMIC gene were seen in 55/63 samples (87%). Top genes were STAT3, RHOA, ARID1A, MLL2 (KMT2D), PTPRB, TP53, and TET2. The KEGG ErbB pathway was most significantly enriched for mutated genes in our dataset (EGFR, ERBB2, ERBB3, ERBB4, and others; q=3.88x10-3), followed by neurotrophin and mitogen-activated protein kinase signaling pathways. Among 25 recurrent non-synonymous variants, we identified MSC E116K with predicted gain-of-function exclusively in 3 ALK-ALCLs and 1 cutaneous ALCL. All mutated cases strongly expressed MSC protein. Among 96 cases, 13/18 ALK-ALCLs (72%) were positive for MSC, compared to 9/78 (12%) from other PTCL subtypes. Conclusions: Mutations that were potentially actionable based on published variant-specific data were identified in 25% of PTCLs, indicating promise for the role of sequencing in individualizing therapy. Additionally, remaining variants were enriched for several targetable pathways, most significantly ErbB. A novel recurrent mutation, MSC E116K, was seen exclusively in ALK-ALCLs (systemic and cutaneous). MSC protein, a repressor of E2A-mediated transcription of targets such as MYC, was expressed in most ALK- ALCLs and generally was absent in other PTCL subtypes. This finding extends evidence for a genetic relationship between systemic and cutaneous ALCLs and suggests MSC may help distinguish ALK- ALCLs from other PTCLs. Disclosures Link: Genentech: Consultancy, Research Funding; Kite Pharma: Research Funding.

Key PointsDUSP22-rearranged ALCLs belong to a distinct subset of ALCLs lacking activated STAT3. DUSP22-rearranged ALCLs have a unique molecular signature characterized by DNA hypomethylation and an immunogenic phenotype.

Abstract Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a “wastebasket” category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

Abstract Interferon regulatory factor-4 (IRF4) is a tightly-regulated transcription factor involved in growth and differentiation of normal lymphocytes. IRF4 is expressed in multiple lymphoid malignancies and its expression drives tumor growth. Thus, characterization of the molecular events regulating IRF4 levels is key to design any targeting strategy. Here, we define a novel regulatory mechanism controlling IRF4 expression in peripheral T-cell lymphoma (PTCL), an aggressive non-Hodgkin lymphoma with poor overall survival rates and high levels of this transcription factor. In normal T cells, IRF4 was induced upon external activation of protein kinase C (PKC) by phorbol myristate acetate and ionomycin, an effect reversed by the PKC inhibitor, enzastaurin. Unlike normal T cells, PTCL cells constitutively expressed IRF4 in the absence of external stimulation, and this expression was resistant to enzastaurin. To identify regulators of IRF4 expression in PTCL, we performed a pharmacological screen using inhibitors that targeted PKC-associated pathways, including mitogen-activated protein kinases, PI3K/AKT/mTOR, and NF-κB. Of the inhibitors tested, only the proteasome inhibitor, bortezomib, showed a decrease in IRF4 expression in a dose- and time-dependent manner. To confirm this result we used a more specific NF-κB inhibitor blocking IκB kinase, BMS-345541. This inhibitor dramatically reduced IRF4 expression by 95.6% (P&lt;0.0001). We used cell-based assays to investigate further the role of NF-κB in the transcriptional regulation of IRF4. A reporter assay for the IRF4 promoter demonstrated reduced promoter activity by siRNA knockdown of RelB and p52 NF-κB subunits to 31.4% and 36.9% of control (P=0.009). IRF4 promoter activity also was inhibited 67.9% by IκBαΔN, a dominant negative inhibitor of NF-κB activity (P=0.002), and was increased 71% by siRNA knockdown of IκBα (P&lt;0.05). Chromatin immunoprecipitation assays confirmed the occupancy of NF-κB subunits in the IRF4 promoter, and this interaction was diminished in the presence of BMS-345541. Finally, knockdown of RelB and p52 subunits had a tendency to decrease IRF4 gene expression by 24.4% and 27.3% (P=0.1). Taken together, these data demonstrate transcriptional regulation of IRF4 by the NF-κB family in PTCL, and suggest the NF-κB pathway may represent a therapeutic target for PTCLs with concurrent IRF4 and NF-κB expression. Citation Format: Rebecca L. Boddicker, N. Sertac Kip, Luciana L. Almada, Julie C. Porcher, Deanna M. Grote, Stephen M. Ansell, Martin E. Fernandez-Zapico, Andrew L. Feldman. Interferon regulatory factor-4 is transcriptionally regulated by NF-κB in peripheral T-cell lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2323. doi:10.1158/1538-7445.AM2014-2323

Key PointsThe NF-κB subunits p52 and RelB increase IRF4 promoter activity and expression in PTCL cells. A positive feedback loop involving CD30, NF-κB, and IRF4 drives PTCL cell proliferation and can be blocked by NF-κB inhibitors.