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Background: Formalin-fixed, paraffin-embedded (FFPE) tissues are a valuable resource for clinical and basic science research. Paraffin blocks and the resulting unstained sections (USS) are often st...

Diffuse large B-cell lymphomas (DLBCL) represent a clinically heterogeneous group of lymphomas that are classified together based on similarities in morphology and immunophenotype. Gene expression profiling further classifies DLBCL into distinct molecular subgroups based on cell-of-origin (COO), including Germinal Center B-cell type, Activated B-cell type, and Unclassified type. COO assignment of DLBCL has important biological and prognostic significance, as well as emerging therapeutic implications. Herein, we describe the first clinical validation of a digital gene expression profiling assay (Lymph2Cx) to perform COO assignment in the routine work-up of DLBCL using formalin-fixed paraffin-embedded (FFPE) tissue sections and describe the results of 90 consecutive DLBCL cases analyzed prospectively by a College of American Pathologists/Clinical Laboratory Improvement Amendments (CAP/CLIA)-certified clinical molecular diagnostics laboratory.

Tissue microarrays (TMAs) are important tools to conserve precious tissue resources from increasingly smaller biopsies and to control experimental costs and variation across sample sets. The quality assurance assessment of TMA materials created at centralized biobanks has not been standardized. Herein, we outline 2 processes for the construction of tissue microarrays (“recipient block” and “tape” methods) and the associated pre-construction quality control measures (pathology review, protein and RNA assessment, map creation, and storage conditions) developed by the AIDS Cancer Specimen Resource (ACSR) Network’s Science and Technology Core. These steps provide a suggested framework for quality assessment that allows end-users, receiving materials from tissue banks, confidence in their experimental results.

Diffuse large B-cell lymphoma (DLBCL) is up to 17-fold more likely to occur, follows a more aggressive clinical course and frequently presents at advanced stages in HIV infected (+) individuals compared to HIV negative (-) individuals. However, the molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood. We performed a retrospective study examining the transcriptional, genomic and protein expression differences between HIV(+) and HIV(-) germinal center B-cell (GCB) DLBCL cases using digital gene expression analysis, array comparative genomic hybridization (CGH) and immunohistochemistry (IHC). Genes associated with cell cycle progression (CCNA2, CCNB1, CDC25A, E2F1), DNA replication (MCM2, MCM4, MCM7) and DNA damage repair, including eight Fanconi anemia genes (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT/UBE2T, FANCV/MAD2L2), were significantly increased in HIV(+) GCB-DLBCL tumors compared to HIV(-) tumors. In contrast, genes associated with cell cycle inhibition (CDKN1A, CDKN1B) as well as apoptosis regulating BCL2 family members (BCL2, BAX, BIM, BMF, PUMA) were significantly decreased in the HIV(+) cohort. BCL2 IHC confirmed this expression. Array CGH data revealed that HIV(+) GCB-DLBCL tumors have fewer copy number variations than their HIV(-) counterparts, indicating enhanced genomic stability. Together, the results show that HIV(+) GCB-DLBCL is a distinct molecular malignancy from HIV(-) GCB-DLBCL; with an increased proliferative capacity, confirmed by Ki67 IHC staining, and enhanced genomic stability, the latter of which is likely related to the enhanced expression of DNA repair genes.

Primary mediastinal large B-cell lymphoma (PMBCL) is recognized as a distinct entity in the World Health Organization classification. Currently, the diagnosis relies on consensus of histopathology, clinical variables, and presentation, giving rise to diagnostic inaccuracy in routine practice. Previous studies have demonstrated that PMBCL can be distinguished from subtypes of diffuse large B-cell lymphoma (DLBCL) based on gene expression signatures. However, requirement of fresh-frozen biopsy material has precluded the transfer of gene expression–based assays to the clinic. Here, we developed a robust and accurate molecular classification assay (Lymph3Cx) for the distinction of PMBCL from DLBCL subtypes based on gene expression measurements in formalin-fixed, paraffin-embedded tissue. A probabilistic model accounting for classification error, comprising 58 gene features, was trained on 68 cases of PMBCL and DLBCL. Performance of the model was subsequently evaluated in an independent validation cohort of 158 cases and showed high agreement of the Lymph3Cx molecular classification with the clinicopathological diagnosis of an expert panel (frank misclassification rate, 3.8%). Furthermore, we demonstrate reproducibility of the assay with 100% concordance of subtype assignments at 2 independent laboratories. Future studies will determine Lymph3Cx’s utility for routine diagnostic purposes and therapeutic decision making.

The assignment of diffuse large B-cell lymphoma into cell-of-origin (COO) groups is becoming increasingly important with the emergence of novel therapies that have selective biological activity in germinal center B cell–like or activated B cell–like groups. The Lymphoma/Leukemia Molecular Profiling Project's Lymph2Cx assay is a parsimonious digital gene expression (NanoString)–based test for COO assignment in formalin-fixed paraffin-embedded tissue (FFPET). The 20-gene assay was trained using 51 FFPET biopsies; the locked assay was then validated using an independent cohort of 68 FFPET biopsies. Comparisons were made with COO assignment using the original COO model on matched frozen tissue. In the validation cohort, the assay was accurate, with only 1 case with definitive COO being incorrectly assigned, and robust, with >95% concordance of COO assignment between 2 independent laboratories. These qualities, along with the rapid turnaround time, make Lymph2Cx attractive for implementation in clinical trials and, ultimately, patient management.

Introduction: HIV infected individuals are 17x more likely to receive a diagnosis of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Previously, we demonstrated that HIV-associated [HIV(+)] germinal center B-cell (GCB) DLBCL tumors are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes compared to their HIV-not associated [HIV(-)] counterparts (Maguire et al, Int J Cancer,2019). Given the immunocompromised nature of these patients, herein we assessed whether specific immunological signaling pathways are also altered in HIV(+) GCB DLBCL tumors, that may work in conjunction with, or independently of, increased DNA repair, to drive the enhanced aggressive nature of HIV related DLBCL. Methods: We used our previously studied cohort of patient samples, which included a total of 39 molecularly defined GCB DLBCL cases. Of these, 18 were HIV(+) cases from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 21 were HIV(-) institutional cases. Samples underwent hematopathologist review for diagnosis confirmation and tumor content assessment. Samples with Results: A total of 110 genes were differentially expressed between the HIV(+) and HIV(-) cohorts at p Conclusions: As expected, the results demonstrate a loss in both adaptive and innate immune signaling in the HIV(+) cohort compared to the HIV negative cohort as well as alterations in receptor signaling. In line with our previous observation that HIV(+) GCB DLBCL tumors have higher expression of proliferation genes and the Ki67 antigen than their HIV(-) counterparts, the results of this study also reveal overexpression of TRFC/CD71 mRNA in HIV(+) GCB DLBCL tumors compared to the HIV(-) cases. TFRC/CD71 is well known as a mediator of iron uptake in erythroid cells. Iron uptake through TFRC/CD71 is also necessary for, and positively regulates T and B cell proliferation; and is commonly expressed on aggressive B cell lymphomas. We suggest that TFRC/CD71 may play a role in the enhanced proliferative capabilities of HIV(+) GCB DLBCL. These effects may be mediated through the HIV protein Nef, which has known effects on TFRC/CD71 protein recycling in lymphoid populations (Madrid et al, J Biol Chem 2005), and has been shown to be transferred from HIV infected macrophages to uninfected B-cells via contact-dependent cellular conduits (Xu et al, Nat Immunol 2009). Nef transfer between cells has also been described to occur via microvesicle transfer and trogocytosis (reviewed in Ellwanger et al, Infect Genet Evol 2017). Further analysis of these pathways is ongoing. Disclosures Rimsza: NanoSting: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution].

Introduction Lymph2Cx lymphoma cell-of-origin assay (LM2CX) was developed by the Lymphoma/Leukemia Molecular Profiling Project (LLMPP) to better categorize diffuse large B-cell lymphoma (DLBCL). NanoString has licensed the assay and is currently pursuing FDA approval. In the meantime, the test is offered exclusively as a lab-developed test (LDT) by the Mayo Clinic Molecular Diagnostics–Arizona Lab (MDAZL) to Mayo Clinic Enterprise patients. In order to comply with these restrictions, the normal workflow for Mayo Clinic Rochester has been modified as cases are sent to Arizona. Rochester consultants order LM2CX using their local laboratory information system (LIS). Slides are prepared by Rochester histology and then shipped to Arizona, where they are entered into the Arizona LIS and processed. In July 2018, we discovered numerous cases that were ordered but not shipped. Because this step took place at the LIS transition between Arizona and Rochester, it was not detected immediately. Methods Allied Health Staff (AHS) colleagues in Rochester and Arizona had the unique opportunity to collaborate. After Arizona AHS identified the problem, they reached out to the Rochester pathology reporting specialists (PRSs) and began a joint improvement project. Together, we were able to measure the impact of the problem, with Arizona auditing digitally, while Rochester audited physical cases. We found 29% of the cases were handled improperly over a 6-month time period. In order to eliminate the gap, Rochester implemented several improvements, including training, tagging all LM2CX cases, and huddle discussions, while Arizona AHS monitored the process digitally. Results Since implementation of improvements, we have had zero defects. Modifications to AHS/consultant training in Rochester will ensure continued success. Conclusions This intervention illustrates the importance of strong collaborations in order to quickly respond to testing issues and provide the greatest value to patients.

Diffuse large B cell lymphoma (DLBCL) is an aggressive form of non-Hodgkin lymphoma. While the initial treatment strategy is highly effective, relapse occurs in 40% of cases. Histone deacetylase inhibitors (HDACi) are a promising class of anti-cancer drugs but their single agent efficacy against relapsed DLBCL has been variable, ranging from few complete/partial responses to some stable disease. However, most patients showed no response to HDACi monotherapy for unknown reasons. Here we show that sensitivity and resistance to the hydroxamate HDACi, PXD101, can be modeled in DLBCL cell lines. Sensitivity is characterized by G 2/M arrest and apoptosis and resistance by reversible G 1 growth arrest. These responses to PXD101 are independent of several negative prognostic indicators such as DLBCL subtype, BCL2 and MYC co-expression, and p53 mutation, suggesting that HDACi might be used effectively against highly aggressive DLBCL tumors if they are combined with other therapeutics that overcome HDACi resistance. Our investigation of mechanisms underlying HDACi resistance showed that cyclin-dependent kinase inhibitors (CKIs), p21 and p27, are upregulated by PXD101 in a sustained fashion in resistant cell lines concomitant with decreased activity of the cyclin E/cdk2 complex and decreased Rb phosphorylation. PXD101 treatment results in increased association of CKI with the cyclin E/cdk2 complex in resistant cell lines but not in a sensitive line, indicating that the CKIs play a key role in G 1 arrest. The results suggest several treatment strategies that might increase the efficacy of HDACi against aggressive DLBCL.

Chemoresistance, due to oxidative stress resistance or upregulation of Bcl-2, contributes to poor outcome in the treatment of hematological malignancies. In this study, we utilize the copper chelator drug, ATN-224 (choline tetrathiomolybdate), to induce cell death in oxidative stress resistant cells and cells overexpressing Bcl-2 by modulating the cellular redox environment and causing mitochondrial dysfunction. ATN-224 treatment decreases superoxide dismutase 1 (SOD1) activity, increases intracellular oxidants and induces peroxynitrite-dependent cell death. ATN-224 also targets the mitochondria, decreasing both cytochrome c oxidase (CcOX) activity and mitochondrial membrane potential (ΔΨm). The concentration of ATN-224 required to induce cell death is proportional to SOD1 levels, but independent of Bcl-2 status. In combination with doxorubicin, ATN-224 enhances cell death. In primary B cell acute lymphoblastic leukemia (B-ALL) patient samples, ATN-224 decreases the viable cell number. Our findings suggest that ATN-224’s dual targeting of SOD1 and CcOX is a promising approach for treatment of hematological malignancies either as an adjuvant or as a single agent.

Loss of major histocompatibility complex class II (MHC II) expression is associated with poor patient outcome in diffuse large B-cell lymphoma (DLBCL). As MHC II molecules are lost with plasmacytic differentiation in normal cells, we asked whether MHC II loss in DLBCL is associated with an altered differentiation state. We used gene expression profiling, quantum dots, and immunohistochemistry to study the relationship between MHC II and plasma cell markers in DLBCL and plasmablastic lymphoma (PBL). Results demonstrate that MHC II(−) DLBCL immunophenotypically overlap with PBL and demonstrate an inverse correlation between MHC II and plasma cell markers MUM1, PRDM1/Blimp1, and XBP1s. In addition, MHC II expression is significantly higher in germinal center-DLBCL than activated B cell-DLBCL. A minor subset of cases with an unusual pattern of mislocalized punctate MHC II staining and intermediate levels of mRNA is also described. Finally, we show that PBL is negative for MHC II. The results imply a spectrum of MHC II expression that is more frequently diminished in tumors derived from B cells at the later stages of differentiation (with complete loss in PBL). Our observations provide a possible unifying concept that may contribute to the poor outcome reported in all MHC II(−) B-cell tumors.

Introduction: HIV infected individuals are 17x more likely to receive a diagnoses of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals, with up to 70% of patients being primary refractory to chemotherapeutic regimens. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Here, we have assessed the genomic and transcriptional differences between HIV(+) and HIV(-) DLBCL in order to identify the mechanisms driving the enhanced aggressive and refractory nature of HIV related DLBCL. Methods: A total of 66 cases, including 27 HIV(+) from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 39 HIV(-) institutional cases were included in this study. Fresh H&Es were reviewed by a hematopathologist to validate diagnosis and determine tumor content. Samples with Results: Both the HIV(+) and HIV(-) cohorts were found to be GCB enriched with GCB-ABC-UNC distributions of 70%(19/27)-11%(3/27)-19%(5/27) and 54%(21/39)-33%(13/39)-13%(5/39) respectively. For both statistical and biological reasons, analysis was restricted to the GCB-DLBCL subtype only. Array CGH revealed the HIV(+) tumors have less DNA aberrations than their HIV(-) counterparts, indicative of enhanced genomic stability. GEP GSEA analysis revealed significant differences in the HIV(+) cohort compared to the HIV(-) cohort; including increases in DNA replication (MCM2, MCM4, MCM7) and Cell Cycle progression (CDC25A, CCNA2, CCNB1, E2F1) related genes as well as decreases in cell cycle negative regulators (CDKN1A, CDKN1B, CDKN2B, RB1), pro-apoptotic BCL2 genes (BAX, BIM, BMF, PUMA, BNIP3) and pro-survival BCL2 genes (BCL2, BCLW). Analyses also revealed significant increases in DNA repair related genes, particularly those with roles in the Fanconi Anemia-Homologous Recombination-Translesion Synthesis axis (FANCA, FANCD1/BRCA2, FANCE, FANCG, FANCR/RAD51, FANCS/BRCA1, FANCT, FANCV/MAD2L2). Conclusions: Our results show, for the first time, that GCB DLBCL arising in HIV infected individuals possesses a distinct molecular pathology to that which arises in uninfected individuals. These results indicate that the aggressive nature of HIV(+) GCB DLBCL is mediated by increased proliferation in conjunction with reduced cell cycle inhibitory capabilities and potentiated by heightened DNA repair that promotes genomic stability. Moreover, it is probable that the bolstered DNA repair capabilities confer an innate ability to repair DNA damage resulting from the administration of genotoxic chemotherapeutic agents and may be the mechanism underlying the high primary refractory rate to chemotherapeutics in HIV related DLBCL. Disclosures Rimsza: NanoString: Other: Inventor on the patent for the Lymph2Cx assay.

Objective Vascular endothelial growth factor (VEGF) interacts with two high-affinity receptor tyrosine kinases (RTK) on vascular endothelium to initiate complementary but disparate biologic responses. We previously reported that acute myeloid leukemia (AML) cells express VEGF and one or both VEGF-A receptors, Flt-1 (VEGFR-1) and KDR (VEGFR-2). To evaluate receptor-selective trophic response to VEGF-A in AML cells, we investigated receptor-specific ligand activation responsible for VEGF-initiated clonogenic response. Materials and methods Using KG1 (VEGFR-1 + /VEGFR-2 + ) and HL60 (VEGFR-1 + ) cells with differential VEGF receptor display, we investigated ligand-induced clonogenic response and receptor-initiated signaling after stimulation with VEGF-A, the VEGFR-1 selective ligand placental growth factor (PlGF), or receptor-specific antibody agonists. Results Recombinant human (rhu)-VEGF increased S-phase fraction and stimulated colony formation in both KG1 and HL60 cells. Ligation of VEGFR-1 or VEGFR-2 with receptor-specific antibody agonists triggered equivalent and concentration-dependent stimulation of colony recovery in KG1 cells, whereas clonogenic response in HL60 cells was restricted to VEGFR-1 activation by antibody or PlGF. In serum-deprived KG1 and HL60 cells, rhu-VEGF stimulated rapid and sustained phosphorylation of Akt/PKB that was inhibited by the phosphatidyl inositol 3-kinase (PI3-K) kinase inhibitor wortmannin. Preincubation with wortmannin inhibited VEGF-induced colony formation in a concentration-dependent fashion. rhu-VEGF–induced clonogenic response and Akt phosphorylation was abolished by the VEGF-RTK inhibitor SU-5416 at concentrations greater than 10 μM, whereas MEK inhibition by PD98059 (1 and 10 μM) was ineffective. In vivo suppression of Akt phosphorylation was confirmed in myeloblast lysates from three patients with advanced myeloid malignancies treated with SU5416. Conclusion These data indicate that VEGF interaction with either VEGFR-1 or VEGFR-2 initiates a clonogenic response in AML cells that is PI3-kinase dependent. RTK inhibitors with broad specificity for angiogenic receptors represent novel therapeutics that merit further clinical investigation in AML.

Introduction: Diffuse Large B cell Lymphoma (DLBCL) is up to 80-fold more likely to occur and follows a more aggressive clinical course in HIV positive (+) individuals compared to HIV negative (-) individuals. Moreover, the vast majority frequently present at advanced stages with high tumor burdens with poor prognosis. The molecular pathology underpinning the clinical features of DLBCL in HIV(+) patients relative to the general population is poorly understood; however, it is suspected to depend on decreased host immune surveillance and alternative mechanisms of tumorogenesis. In this study, we sought to identify the differences in gene expression between HIV(+) and HIV(-) DLBCL cohorts that may contribute to the unique features of HIV(+) DLBCL. Methods: We performed a retrospective gene expression profiling (GEP) study using a total of 70 DLBCL cases, 31 HIV(+) obtained from AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 39 HIV(-) institutional cases. The H&Es for all cases were reviewed by a hematopathologist and tumor content determined. Cases that did not meet the minimum tumor content threshold of 70% were macro-dissected. RNA was extracted from 4x 5µm formalin fixed, paraffin embedded (FFPE) sections using the Qiagen AllPrep DNA/RNA FFPE Kit and quantified using a NanoDrop. The Lymph2Cx assay, which accurately subtypes DLBCL in FFPE tissues (Scott et al 2014), was used to determine the cell of origin (COO) subtypes: Germinal Center B cell (GCB), Activated B cell (ABC) and Unclassifiable (UNC). Digital GEP was performed using the NanoString PanCancer Pathways and Immunology panels, targeting a total of over 1300 genes with known roles in cancer or immune signaling. Results: Of the 31 HIV(+) cases, Lymph2Cx subtyping showed 74% (23/31) were GCB, 13% (4/31) were ABC and 13% (4/31) were UNC. In a similar distribution to the HIV(+), and comparable to prior literature, 54% (21/39) of HIV(-) cases were GCB, 31% (12/39) were ABC and 15% (6/39) were UNC. Differential gene expression was observed between HIV(+) and HIV(-) cases, but no association with COO subtype was found. When the data was re-analyzed within the GCB subtype alone, stratification by HIV status was observed. Particularly, reduced expression of genes associated with immune regulation was observed in HIV(+) GCB cases compared to HIV(-) GCB cases. In contrast, expression of genes associated with cell cycle regulation and homologous recombination (the predominate double stranded DNA repair pathway employed during the cell cycle), were higher in HIV(+) cases compared to HIV(-) GCB cases. Conclusions: The results show, that within the GCB subtype, marked differences in gene expression are associated with HIV status. Our results suggest that the aggressive nature of DLBCL in HIV(+) patients may, in part, be mediated by enhanced cell proliferation potentiated by aberrant DNA repair. Disclosures Fogel: Natural Selection Inc: Employment. Liu: Natural Selection Inc: Employment. Lamers: Bioinfoexperts LLC: Employment.

Clinical and biological features have recognized prognostic significance in acute myeloid leukemia (AML). To evaluate the interaction of these variables and weighted effect on treatment outcome, prognostic variables from 96 previously untreated patients were analyzed for association with expression of the MDR1 gene product P-glycoprotein (Pgp), and effect on response to induction chemotherapy, progression-free survival and overall survival. Multivariate relationships were analyzed using six prognostic variables, including age, cytogenetic pattern, gender, CD34 + surface phenotype, AML type ( de novo versus secondary) and Pgp. Univariate comparisons indicate that Pgp ( P = 0.0001), cytogenetic pattern ( P = 0.0004) and a CD34 + phenotype ( P = 0.0005) are predictive of primary treatment failure, whereas Pgp ( P = 0.0001) had the greatest predictive value in multivariate analysis. Only cytogenetic pattern retained prognostic significance ( P = 0.0143) for response to induction therapy after adjustment for Pgp. Although all variables except gender were associated with Pgp, specimens harboring the favorable karyotypic abnormalities t(15;17), t(8;21) and inv(16) exclusively lacked Pgp expression. In a multivariate model, both Pgp and cytogenetic pattern predicted response and overall survival, whereas secondary AML and cytogenetic pattern influenced remission duration. These findings indicate that cytogenetic pattern has prognostic relevance that is independent of Pgp, and implies the presence of undefined biological mechanisms affecting chemotherapy resistance.

Cultured human pancreatic cancer cells produce transforming growth factor-alpha (TGF-alpha), a potent mitogenic polypeptide. In the present study, we investigated the regulation of TGF-alpha mRNA expression in T3M4 human pancreatic carcinoma cells. TGF-alpha mRNA levels were quantitated by densitometric analysis of autoradiographs obtained following hybridization of size-fractionated cytoplasmic RNA with 32P-labeled cRNA coding for human TGF-alpha. There was a twofold increase in TGF-alpha mRNA levels at 2 h following addition of either epidermal growth factor (EGF) or TGF-alpha. However, TGF-alpha mRNA levels declined to near basal levels by 10 h. At 2 h, one-half maximal stimulation of TGF-alpha mRNA levels occurred at 1 nM and maximal stimulation at 4 nM of either EGF or TGF-alpha. The transcriptional inhibitor actinomycin D (Act D) and the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), mimicked the actions of EGF and TGF-alpha. These findings indicate that the regulation of TGF-alpha mRNA expression in T3M4 cells is complex, and is mediated, in part, via the EGF receptor.

A 1.96 kbp cDNA encoding a male Beagle dog liver cytochrome P-450 of 503 amino acid residues (Mr 57,636) has been isolated and sequenced. The deduced amino acid sequence is 79.8%, 69.3% and 74.1% identical to the P450IIIA forms human NF25, rat PCN1 and rabbit LM3c, respectively. The amino terminal sequence is identical to the first 28 residues of the dog P450IIIA form PBD-1. Southern blot analysis yields restriction patterns consistent with IIIA gene subfamily multiplicity.

Vascular endothelial growth factor (VEGF) is a potent angiogenic peptide with biologic effects that include regulation of hematopoietic stem cell development, extracellular matrix remodeling, and inflammatory cytokine generation. To delineate the potential role of VEGF in patients with myelodysplastic syndrome (MDS), VEGF protein and receptor expression and its functional significance in MDS bone marrow (BM) were evaluated. In BM clot sections from normal donors, low-intensity cytoplasmic VEGF expression was detected infrequently in isolated myeloid elements. However, monocytoid precursors in chronic myelomonocytic leukemia (CMML) expressed VEGF in an intense cytoplasmic pattern with membranous co-expression of the Flt-1 or KDR receptors, or both. In situ hybridization confirmed the presence of VEGF mRNA in the neoplastic monocytes. In acute myelogenous leukemia (AML) and other MDS subtypes, intense co-expression of VEGF and one or both receptors was detected in myeloblasts and immature myeloid elements, whereas erythroid precursors and lymphoid cells lacked VEGF and receptor expression. Foci of abnormal localized immature myeloid precursors (ALIP) co-expressed VEGF and Flt-1 receptor, suggesting autocrine cytokine interaction. Antibody neutralization of VEGF inhibited colony-forming unit (CFU)-leukemia formation in 9 of 15 CMML and RAEB-t patient specimens, whereas VEGF stimulated leukemia colony formation in 12 patients. Neutralization of VEGF activity suppressed the generation of tumor necrosis factor-α and interleukin-1β from MDS BM–mononuclear cells and BM–stroma and promoted the formation of CFU-GEMM and burst-forming unit-erythroid in methylcellulose cultures. These findings indicate that autocrine production of VEGF may contribute to leukemia progenitor self-renewal and inflammatory cytokine elaboration in CMML and MDS and thus provide a biologic rationale for ALIP and its adverse prognostic relevance in high-risk MDS.

Amifostine (WR-2721, Ethyol) is a phosphorylated aminothiol that affords broad cytoprotection from the myelosuppressive effects of antineoplastics. To further characterize its hematopoietic activities, we investigated the effects of amifostine and its dephosphorylated metabolite, WR1065, on the in vitro growth of human bone marrow progenitors. Preincubation exposure to amifostine or WR1065 stimulated the growth of colony-forming units granulocyte, erythroid, macrophage, megakaryocyte (CFU-GEMM) and erythroid bursts (BFU-E) from bone marrow mononuclear cells in a dose-dependent fashion. Over the concentration range tested (0.1-1000 microM), pretreatment with the aminothiols enhanced formation of CFU-GEMM up to five-fold and BFU-E nine-fold, compared to a three-fold increase in myeloid colony recovery. In CD34+ selected cells, preincubation with amifostine increased formation of CFU-GEMM up to 38-fold and produced macroscopic colonies, exceeding colony number in cultures initiated with optimal concentrations of interleukin-1 (IL-1), IL-3, or kit ligand (KL). When compared with recombinant human cytokines, amifostine enhanced IL-1 and IL-3 induced colony formation, although its stimulatory effect was less than additive. In contrast, pretreatment with amifostine antagonized the stimulatory effects of KL, whereas synergy was observed with concurrent exposure. Ex vivo expansion studies showed that amifostine alone supported and augmented the production of myeloid progenitors in secondary cultures. Similarly, under cytokine-deficient conditions, amifostine promoted cell survival and delayed apoptosis as measured by nucleosome generation. These data indicate that amifostine is a novel multipotent hematopoietic stimulant that augments the formation and survival of bone marrow progenitors.

The aminothiol, amifostine (Ethyol; U.S. Bioscience, West Conshohocken, PA), is a cytoprotective agent that ameliorates the toxicities of anticancer therapy. In vitro, amifostine promotes the formation and survival of primitive hematopoietic progenitors derived from myelodysplastic bone marrow (BM) specimens. To evaluate the hematological effects of amifostine, 18 patients with myelodysplastic syndrome (MDS) and one or more refractory cytopenias received treatment with amifostine in a Phase I/II study. Four cohorts received intravenous treatment with 100, 200, or 400 mg/m2 amifostine three times a week, or 740 mg/m2 weekly for three consecutive weeks followed by 2 weeks observation. Nonresponding patients received a second course of therapy at the next higher dose level depending upon drug tolerance. Bone marrow (BM) progenitor growth was assessed before treatment and after day 21. Diagnoses included refractory anemia (7), refractory anemia with ringed sideroblasts (5), refractory anemia with excess blasts (RAEB) (4), and RAEB-in transformation (RAEB-t) (2). Single- or multi-lineage hematologic responses occurred in 15 patients (83%) treated with the three-times-a-week dose schedule. Fourteen patients had a 50% or greater increase in absolute neutrophil count with amifostine treatment (range, 426 to 11,348/μL). Platelet count increased in 6 (43%) of 14 patients with thrombocytopenia (absolute increase, 16,000 to 110,000/μL), and 5 of 15 red blood cell transfusion-dependent patients had a 50% of greater reduction in transfusion needs. Assayable hematopoietic progenitors increased in 13 of 15 evaluable patients; including CFU-GEMM (12), BFU-E (8), and CFU-GM (6). Amifostine doses less than or equal to 200 mg/m2 were well tolerated, whereas grade II nausea, vomiting, and fatigue was limiting at higher doses. Three patients with excess blasts before enrollment experienced an increase in BM blast percentage and two patients had evolution to acute leukemia that persisted after treatment withdrawal. We conclude that amifostine administered at doses ≤200 mg/m2 three times a week is well tolerated and has hematologic activity in patients with MDS.

The diffuse large B-cell lymphoma (DLBCL) cell-of-origin (COO) distinction into germinal center B cell (GCB) and activated B cell (ABC) subtypes, as molecularly described by our group, has profound biological, prognostic, and potential therapeutic implications. New therapeutic agents with selective activity in ABC and GCB DLBCL are under development. An accurate diagnostic assay is urgently needed to qualify patients for clinical trials using targeted agents and as a predictive biomarker. Although the subtypes were originally defined using gene expression profiling on snap-frozen tissues (frozen-GEP), it has become common practice to use less precise but relatively inexpensive and broadly applicable immunohistochemical (IHC) methods in formalin-fixed paraffin-embedded tissue (FFPET). We sought to create a robust, highly accurate molecular assay for COO distinction using new GEP techniques applicable to FFPET. Studies were performed on centrally reviewed DLBCL FFPET biopsies using cases that had “gold standard” COO assigned by frozen-GEP using Affymetrix U133 plus 2.0 microarrays. The training cohort consisted of 51 cases comprising 20 GCB, 19 ABC and 12 Unclassifiable (U) cases. An independent validation cohort, consisting of 68 cases (28 GCB, 30 ABC, 10 U) drawn from the validation cohort of Lenz et al (NEJM 2008) had the typical proportions of COO subtypes seen in DLBCL populations. Nucleic acids were extracted from 10um FFPET scrolls. Digital gene expression was performed on 200ng of RNA using NanoString technology (Seattle, WA). All FFPET GEP studies were performed in parallel at two independent sites (BC Cancer Agency, Vancouver, and NCI, Frederick, MD) using different FFPET scrolls to determine inter-site concordance and assess the robustness and portability of the assay. To assign COO by IHC, tissue microarrays were made using 0.6mm duplicate cores from 60/68 validation cohort cases, and stained for CD10, BCL6, MUM1, FOXP1, GCET1 and LMO2. Two hematopathologists independently assessed the proportion of tumor cells stained, with consensus on discordant cases reached with a third hematopathologist. For the validation studies, those producing and analyzing the GEP and IHC data were blinded to the “gold standard” COO. All 119 FFPET biopsies yielded sufficient RNA. A pilot study using the training cohort identified 20 genes (15 genes of interest and 5 house keeping genes) whose expression, measured using NanoString, would allow accurate replication of the COO assignment model of Lenz et al (NEJM 2008). NanoString was then used to quantitate these 20 genes in the training cohort, allowing the COO model to be optimized. Despite the age of the FFPET blocks (6-32 years old), 95% (49/51) of the training samples gave gene expression data of sufficient quality. The model, including coefficients, thresholds and QC parameters was then “locked” and applied to the independent validation cohort. Ninety-nine percent (67/68) of the samples from the validation cohort (5-12 years old) provided gene expression of adequate quality. Three cases did not give interpretable IHC results. When considering the “gold standard” ABC and GCB cases, the COO assignments by the NanoString assay at the NCI site were 93% concordant, with 5% labeled U and 1 ABC misclassified as GCB (see table). This 2% rate of misclassification of ABC and GCB cases compares favorably with the 9%, 6% and 17% rates for the interpretable cases from the Hans, Tally and Choi algorithms, respectively. Furthermore, the 98% concordance of COO assignment (95% if “gold standard” U cases are also included) between the NCI and BC Cancer Agency sites indicates that, in contrast to the IHC algorithms, the assay is reproducible.TableNanoString GEP assay - NCIHans algorithmTally algorithmChoi algorithmGCBUABCGCBNon-GCBGCBABCGCBABCFrozen GEPGCB2800210183192U721552864ABC1325422026620 In summary, 119 well-characterized DLBCL cases from the LLMPP, previously subtyped by our published disease-defining algorithm using frozen-GEP, were used to develop a highly accurate and robust NanoString 20 gene assay, applicable to RNA from FFPET that is routinely obtained for diagnosis. This new assay shows excellent performance in archival FFPET, and the rapid turn-around time ( Disclosures: No relevant conflicts of interest to declare.

Abstract 3111 Diffuse Large B cell lymphoma (DLBCL) is the most common form of non-Hodgkin's lymphoma. The Leukemia Lymphoma Molecular Profiling Project (LLMPP) collaboration has done extensive work regarding this disease including gene expression profiling to show that the cell of origin impacts the prognosis of the patient. Those cases which arise from an activated B cell (ABC) have a worse prognosis than those cases which arise from a germinal center B cell (GCB). We have used a subset of cases from our institution which have had gene expression profiling performed on frozen material and assigned ABC (n=18) or GCB (n=24) status (Rosenwald et al NEJM 2002, 346:1937). We have used matched formalin fixed paraffin embedded (FFPE) tissue block from these cases to obtain a microRNA profile utilizing the qNPA technology (High ThroughPut Genomics) as previously published (Roberts et al Lab Invest 2007, 87:979). This novel FFPE based RNAase protection assay measured 688 human microRNAs. Each microRNA was represented twice on the array and the values averaged for a signal value. The data were normalized to the total signal of the microarray. We were able to define a signature of increased microRNA for each DLBCL subtype as shown in Table 1. ABC subtype GCB subtype hsa-miR-155 hsa-miR-28-5p hsa-miR-196a hsa-miR-138 hsa-miR-501-3p hsa-miR-151-3p hsa-miR-656 hsa-miR-151-5p hsa-miR-1247 hsa-miR-182 hsa-miR-210 hsa-miR-613 The hsa-miR-155 is of importance in lymphoma as it regulates the generation of immunoglobulin class-switch in plasma cells (Turner et al Immunity 2007, 27:847) and was previously identified as a characteristic of ABC cell lines (Lossos et al Blood 2009,113:3754). The hsa-miR-210 has been shown to modulate the MYC antagonist MNT which allows for MYC expression (Grandori et all Cell Cycle 2009, 8:2756). This microRNA profile provides an opportunity to further explore the role microRNA plays in lymphoma biology and in particular, in DLBCL subtype determination. The success of the technique in FFPE tissue holds promise for further microRNA studies on archival material. Disclosures: No relevant conflicts of interest to declare.

Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma diagnosed in the U.S. It displays marked heterogeneity, biologically and clinically, with patients demonstrating a wide range of responses to therapy. Major histocompatibility class II (MHCII) molecules are involved in antigen presentation and are vital to the immune system. Decreased MHCII expression is one of the normal changes seen as B-cells differentiate into mature, antibody-secreting plasma cells. This decrease occurs in concert with changes in other proteins and transcription factors which define the phenotype of a B- or plasma cell. Loss of MHCII expression in DLBCL patients is associated with extremely poor prognosis. In this study, we asked whether MHCII loss in DLBCL was associated with a more differentiated phenotype. We hypothesized that malignant cells from MHCII(-) DLBCL cases would be closer to the plasma cell end of the differentiation spectrum than MHCII(+) cases. We previously used a 40-case tissue microarray to demonstrate that although MHCII(-) DLBCL cases did not present a fully plasma cell differentiated phenotype, protein expression of the late B-cell marker, MUM1/IRF4, was inversely associated with MHCII, suggesting that MHCII loss may be associated with cases further along the differentiation continuum (Wilkinson, AACR 2009). Here, we built upon this work using immunohistochemistry (IHC) to score expression of a panel of B- and plasma cell markers. These included B-cell transcription factors Oct2, Bob 1, Pax5 and mature B-cell surface marker CD20 as well as plasma cell markers XBP1, BLIMP1, and CD138. IHC results were semi-quantitatively assessed by scoring both frequency and intensity of staining. The results were compared to MHCII expression as previously determined with gene expression profiling and IHC. Using this routine IHC assessment, the presence of plasma cell markers XBP1, BLIMP1, or CD138 did not correlate with low or absent expression of MHCII, while the B-cell markers were present in most cases. Thus, no clear evidence of a more differentiated phenotype was observed in MHCII(-) DLBCL. These findings could result from lack of association between loss of MHCII expression and plasma cell differentiation or the limitations of the methodology used. To further investigate this question, we have developed protocols with QdotsR (Life Technologies) to more accurately quantify expression of the plasma cell-associated proteins relative to MHCII. By using nanocrystals with narrow emission spectra coupled to monoclonal antibodies, multiple colors can be visualized and specifically measured in each cell. This technique may yield more accurate, quantitative data regarding protein expression of plasma cell markers and MHCII. The results of these studies should lead to a better understanding of MHCII expression in DLBCL with possible implications for therapeutic targeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1769.

Coordinate expression of nuclear and chloroplast genes is necessary for the biogenesis of chloroplast ribosomes. In order to elucidate mechanisms which might regulate their expression, genes from both cellular compartments must be characterized Genes coding for 20 chloroplast DNA-encoded ribosomal proteins of Nicotiana tabacum (tobacco) are known (reviewed in Bonham-Smith and Bourque, 1990). The remaining (over 35; Capel and Bourque, 1982) tobacco chloroplast ribosomal proteins must be coded by as yet uncharacterized nuclear genes. Here some features of deduced amino acid sequences and gene multiplicity for nuclear-encoded tobacco chloroplast ribosomal proteins L12, L24, and L27 are described. Comparisons with related E. coli and plant genes are presented.

As previously demonstrated by gene expression profiling, MHCII is an independent gene expression signature in DLBCL, which when expressed at low levels, is associated with poor patient survival. The mechanism of decreased survival in low- expressing DLBCL patients is presumably loss of tumor immunosurveillance. However, the possibility remains that MHCII expression is merely a marker of another aspect of tumor biology such as chemosensitvity, radiation sensitivity, or proliferation rate. MHCII status could also reflect a change in the cellular redox environment since we have previously found an association between decreased antioxidant defense enzyme expression, increased thioredoxin system function and poor prognosis in DLBCL. We therefore investigated whether there are alterations in these other aspects of tumor biology by using a MHCII (−) DLBCL cell line model in which we restored MHCII expression by transfection with CIITA, the master transactivator of MHCII. To do this, we stably transfected the MHCII (−) DLBCL cell line, DB, (American Tissue Culture Corp), with a CIITA expression vector pcDNA 3.1 (InVitrogen) and isolated MHCII (+) clones. The phenotype was confirmed by RT-PCR and flow cytometry using HLA-DR as the representative molecule of MHCII expression (reported as MnX or mean channel of fluorescence). The parental line DB, clones 1–5, 4–3, 6-1, vector only control (pcDNA 3.1), and a positive cell line (Raji), were then subjected to in vitro chemosensitivity assays with the individual components of the CHOP regimen (cyclophosphamide, doxorubicin, vincristine, and dexamethasone, the latter was used in the place of prednisone). Chemosensitivity was measured with MTS cell survival assay by calculating the EC 50 (molar concentration of drug required to kill 50% of cells). Radiation sensitivity was measured as percent cell survival 48 hours after exposure to 5 Gy. Proliferation rate was assessed by cell doubling time (in hours) using manual cell counts. The potential of the cells to reduce oxidants was assessed using the AOP-490 kit, which measured anti-oxidant biomarkers as uM uric acid EQ/ug protein (Oxis Research Inc, Portland, OR). No significant differences were detected in sensitivity to any of the tested treatment modalities, including the 4 different chemotherapy agents or radiation, between the parental or transfected clones. Nor were reducing potential or doubling times significantly altered with induction of MHCII expression. Representative data for the DB cell line, vector only control, and a representative clone are listed in Table1. Results for the other clones were similar. Our results help to exclude altered chemosensitivity, radiosensitivity, anti-oxidant potential, or proliferation rates as factors contributing to the unfavorable prognosis in patients with MHCII(−) DLBCL. Therefore, diminished tumor immunosurveillance remains the most likely explanation for the poor outcome in these patients. Table 1 DB parental Vector only Clone 1–5 HLA-DR (MnX) 3 2 500 Dox (EC 50 uM) 4.72 3.61 4.19 Radiation (% ctrl) 49.6 49.14 39.41 Redox potential 33.3 31.2 34.8 Doubling time (hr) 7.19 7.68 7.66

Studies have demonstrated the trilineage hemoprotective effects of amifostine (Ami). Recent studies suggested direct stimulation of hematopoiesis. In vitro growth of CFU-GEMM, BFU-E and CFUGM from normal and MDS bone marrows was evaluated after 15 min preincubation with Ami or its active metabolite, WR-1065, glutathione (GSH), or recombinant growth factors. Concentrations of Ami and WR-1065 at 0.1–1000 μM stimulated growth of CFU-GEMM and BFU-E from normal marrow mononuclear cells; > 10 μM GSH was toxic. Ami was the most potent stimulant yielding up to 9-fold greater BFU-E, 4 × CFU-GEMM and 3 × CFU-GM over controls. Compared to kit ligand, IL-I and IL-3 (200 U/ml), Ami was a more potent hemopoietin yielding up to 3 × greater CFU-GEMM and BFU-E recovery. Despite deficient colony growth in MDS controls, Ami preincubation with clinically achievable concentrations (100–500 μM) stimulated growth of CFU-GEMM and BFU-E 2–7-fold and improved colony/cluster ratio in 7 of 8 pts studied. The data indicate that Ami is a potent stimulant of primitive progenitor growth that exceeds that of recombinant cytokines. The profound simulation observed in MDS suggests that Ami may improve hematopoiesis in patients with MDS and warrants testing in clinical trials.