ALBERT

Description: Background: Diffuse Large B Cell Lymphoma (DLBCL) is the most common form of lymphoma in adults. Gene expression profiling has demonstrated that DLBCL can be classified into two distinct subgroups – activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL. These subgroups arise through distinct normal cells of origin, activate different oncogenic pathways and display markedly different clinical outcomes. Deregulation of the transcriptome is believed to play a key role in the malignant transformation of B cells that culminates in the development of either ABC or GCB DLBCL. Here we describe global differences in RNA expression, mutation and splicing in relation to the pathogenesis of these subgroups of DLBCL. Methods: RNA sequencing (RNAseq) has emerged as a powerful tool for defining the cancer transcriptome. While mRNA sequencing is the most widely applied method for RNAseq, it overlooks non-coding RNAs, requires high-quality RNA and lacks strand-specificity. To overcome these limitations, we developed a method for strand-specific total RNA sequencing (ssRNAseq) to characterize the transcriptomes of 112 DLBCL tumors. Results: Through this work, we defined the entire spectrum of coding and non-coding RNAs expressed in DLBCLs including hundreds of lincRNAs, snoRNAs and microRNAs in addition to mRNAs. We found that the strand-specificity of our method was greater than 95% in all cases. This strand-specific sequencing strategy allowed us to maintain the orientation of the transcript to enable more accurate transcript annotation and better prediction of novel transcripts. Furthermore, we showed that our method had equal efficacy on frozen and FFPE tumor specimens from the sample patient in 24 cases. In addition, through simultaneous measurement of expression of diverse RNA types combined with mutations in MYD88, GNA13, EZH2, and BCL2, we demonstrated that we could distinguish the clinically important subgroups of DLBCL. Finally, we applied ssRNAseq to distinct training and validation sets of DLBCL cases (N=86 and N=112) to define alternative splicing events in DLBCL and found 1,021 genes that were preferentially spliced in a subgroup-specific manner. These alternatively spliced genes were selectively enriched in a number of different pathways important in lymphomas including those related to immune function, cell cycle progression and focal adhesion pathways, suggesting that alternative splicing regulates a number of important oncogenic processes in DLCBL. Conclusions: Strand-specific total RNA sequencing is a powerful method for defining the transcriptome and alternative splicing events in DLBCL. Here we define a complete coding and non-coding transcriptome of DLBCL and report the first characterization of subgroup-specific alternative splicing in DLBCL using high throughput sequencing. Our data demonstrate the power of our ssRNAseq method in defining the molecular patterns underlying DLBCLs and provide a starting point for defining the role of alternative splicing in this complex and heterogeneous disease. Disclosures Mann: Quiagen: Research Funding.

Description: Background: The proto-oncogene Myc is a key regulator of cell growth and survival, and aberrant Myc expression plays a significant role in various tumors, including non-Hodgkin lymphoma (NHL). Myc-associated lymphoma is clinically aggressive, more resistant to standard therapies, and associated with a significantly higher rate of mortality. Novel treatment paradigms are needed to improve survival of patients with Myc-associated NHL. Expression of Aurora Kinase (Aurk) has been associated with Myc, and Aurk is thought to be essential for the maintenance of Myc-driven lymphoma. Aurk is required for assembly of the mitotic spindle and plays key roles in cell proliferation. Amplification and overexpression of Aurk have been observed in various human tumors, including lymphoma, and are frequently associated with tumor progression as well as resistance to chemotherapy. Inhibition of Aurk may overcome resistance to chemotherapy and improve clinical outcomes in patients with Myc-overexpressing lymphoma. Methods: Cytotoxicity assays using MTS and trypan blue were used to compare levels of drug sensitivity in lymphoma cell lines resistant or sensitive to a conventional chemotherapeutic drug cyclophosphamide. Apoptosis and cell cycle assays were performed using Annexin V and Propidium Iodide staining. The Multiplexed Inhibitor Beads and quantitative Mass Spectrometry (MIB/MS) assays were used to profile kinome changes in response to Aurk inhibition. Murine xenograft models were used to assess the efficacy and tolerability of single vs. combined therapy. Results: Two Myc-overexpressing cell lines were identified as resistant (Raji) or sensitive (Ramos) to cyclophosphamide, with IC50 of ~ 400 µM and ~ 250 µM, respectively. Raji cells were characterized by increased expression of multidrug resistant protein 1 (MDR1) and mutated p53. There were no significant differences in baseline Aurk or Myc expressions between Raji and Ramos cells. Both cell lines were sensitive to alisertib, an aurora A kinase inhibitor, with maximum cytotoxicity achieved at ~ 100 nM. Combined treatment with alisertib and cyclophosphamide induced more significant cell growth inhibition as compared to treatment with the single agent alone. The combination index (CI) values were less than 1, indicating that alisertib was synergistic to cyclophosphamide in terms of inhibitory effect on tumor cell viability. Alisertib induced apoptosis and pronounced cell cycle arrest, resulting in polyploidy, in Raji cells. Alisertib had little to no effect on Myc, p53, or the total aurora A kinase protein expression in Raji cells although p-Histone-3-Ser10, a downstream target of Aurk, and p-Src levels were significantly decreased at 24 hours of treatment in vitro. Nocodazole-treated cells had reduced p-Aurk level and increased p-Rb as well as increased Mdm2 when treated with alisertib for 24 hours. Athymic nude mice bearing Ramos or Raji lymphoma xenografts were treated with cyclophosphamide, alisertib, or the combination. As expected, all mice bearing Ramos xenograft had complete tumor regression by day 35 of treatment while all mice bearing Raji xenograft had rapid disease progression with median survival of ~ 35 days when treated with cyclophosphamide alone. In contrast, when treated with the combination of cyclophosphamide and alisertib, all mice bearing Raji xenograft had complete regression of tumor by day 35 and had significant improvement in survival (median survival not reached by day 100) compared to the single agent control (p=0.022). Lastly, kinome analysis of Raji xenograft tumors treated with alisertib showed suppression of various kinases involved in Aurk, Src, and PI3K pathways. Western blot of the Raji tumors treated with a prolonged course (25 days) of alisertib showed significant decrease in p-Src and p53 protein levels. Conclusion: Our data demonstrates that alisertib induces synthetic lethality and overcomes chemoresistance in Myc-overexpressing tumors even in the presence of MDR1 overexpression and p53 mutation. The synergistic effect was largely independent of depletion of cytoplasmic level of Myc. Alisertib, when combined with a conventional chemotherapy drug, induced apoptosis and cell cycle arrest of Myc-overexpressing tumor cells in vitro and showed promising anti-tumor activity in mice bearing chemoresistant Myc-overexpressing lymphoma. Disclosures Park: Janssen: Other: travel; Seattle Genetics: Research Funding; Teva: Research Funding.

Description: Sickle cell disease (SCD) is highly prevalent in sub-Saharan Africa; however, there are relatively few studies describing the clinical profile for children with laboratory-confirmed SCD. Prior to December 2014, neither neonatal screening nor standardized methods for SCD diagnosis were routinely available in Malawi, as hemoglobin electrophoresis and alternative diagnostic methods were absent. We describe implementation of hemoglobin electrophoresis for children with clinically suspected SCD at Kamuzu Central Hospital, one of two national teaching hospitals in Malawi. Children with clinically suspected SCD were recruited January - May 2015 and underwent comprehensive clinical and laboratory characterization. 137 total patients were recruited and 117 were confirmed to have HbSS disease. Among children who were being cared for as SCD prior to enrollment, 86% had HbSS suggesting generally accurate clinical diagnosis by local providers. Baseline clinical parameters and self-reported SCD complications for the study population are displayed in Table 1. Of those with confirmed SCD, median age was 7.3 years (IQR 2.7-10.4) with 53% males. Prior malaria was reported by 39% of patients, and was higher in the 0-5 age group compared with the over 5 age group (46% vs. 31%, p=0.03). The most commonly reported SCD complications were anemia (72%), joint pain (56%), jaundice (52%), and acute pain episodes (50%). Children with confirmed SCD had median hemoglobin of 7.3 g/dL (IQR 6.9-7.9), total bilirubin of 1.7 mg/dL (IQR 1.1-2.6) and lactate dehydrogenase of 658 IU/L (IQR 527-773). Urinalysis demonstrated 26% of patients with blood and 7% with proteinuria by dipstick. As of May 2015, more than 250 samples for enrolled children as well as routine clinical care had been batch-processed weekly with an average turn-around time of 36 hours for results. Three Malawian laboratory technicians were trained to perform hemoglobin electrophoresis, all of whom have been performing the test independently since April 2015. Our findings highlight a need for wider implementation of resource-appropriate diagnostics as an essential foundation for care and research. Children had substantial clinical and laboratory evidence of SCD-related morbidity. Earlier diagnosis can improve care for this population by facilitating earlier therapeutic interventions, as well as providing a basis for research to better understand SCD-related morbidity in sub-Saharan Africa. These efforts can ultimately inform management strategies to improve outcomes and increase life expectancy among children with SCD in Malawi. Table 1. All (n=117) Male (n= 62) Female (n=55) p value Age years, median (IQR) 7.3 (2.7-10.4) 5.3 (2.3-9.4) 8.9 (4.2-11.9) 0.004 Height cm, median (IQR, n) 115 (88-131, 60) 111 (89-128, 36) 119.5 (93-140, 24) 0.21 Weight kg, median (IQR, n) 19 (13-27, 108) 16.5 (12-23.6, 58) 21 (14-30, 50) 0.01 Blood Pressure Systolic mmHg, median (IQR, n) 103 (98-110, 83) 101 (94-108, 43) 103 (99-110, 40) 0.37 Blood Pressure Diastolic mmHg, median (IQR, n) 60 (55-65, 83) 58 (53-65, 43) 61 (56-68, 40) 0.13 Heart Rate BPM, median (IQR, n) 104 (91-118, 114) 105 (94-123, 61) 104 (88-112, 53) 0.15 O2 Saturation %, median (IQR, n) 93 (88-97, 108) 91 (85-96, 59) 95 (91-98, 49) 0.004 % Hypoxemic (SPO2 〈 90%), n (%) 36 (30.7) 26 (41.9) 10 (18.2) 0.005 Body Temperature Celsius, median (IQR, n) 37 (36.7-37.4, 91) 37 (36.7-37, 46) 37 (36.4-37.2, 45) 0.22 Positive History of: Malaria, n (%) 45 (38.5) 22 23 0.34 0-5 years, n (%) 25 (46.3) - - 0.03 6-18 years, n (%) 20 (31.7) - - Pneumonia, n (%) 29 (24.8) 10 (16.1) 19 (34.5) 0.02 HIV, n (%) 0 0 0 - Anemia, n (%) 84 (71.8) 49 (79.0) 35 (63.6) 0.06 Pallor, n (%) 16 (13.7) 7 (11.3) 9 (16.4) 0.43 Jaundice, n (%) 61 (52.1) 33 (53.2) 28 (50.9) 0.82 Received Blood Transfusion, n (%) 87 (74.4) 47 (75.8) 40 (72.7) 0.47 Days since last transfusion, median (IQR) 316 (133-1144) 240 (111-410) 577 (180-1784) 0.03 Pain episodes, n (%) 58 (49.6) 27 (43.5) 31 (56.4) 0.16 Joint pain, n (%) 66 (56.4) 33 (53.2) 33 (60.0) 0.34 Dactylitis, n (%) 41 (35.0) 19 (30.6) 22 (40.0) 0.29 Leg ulcers, n (%) 5 (4.3) 5 (8.1) 0 0.03 Stroke, n (%) 10 (8.5) 5 (8.1) 5 (9.1) 0.84 Nocturnal Enuresis, n (%) 24 (20.5) 12 (19.4) 12 (21.8) 0.74 Disclosures No relevant conflicts of interest to declare.

Description: Background: The detection of minimal residual disease (MRD) following therapy in acute myeloid leukemia (AML) is associated with increased risk of relapse and poor survival and can influence clinical decisions regarding treatment. Multi-parameter flow cytometry can assess MRD in ~90% of AML cases (due to aberrant surface antigen expression); however, its poor sensitivity typically requires bone marrow aspirates, which are invasive and cannot be obtained frequently. Polymerase chain reaction (PCR) assays are highly sensitive in detecting AML-associated mutations, but significant sample processing and assay development are needed to detect patient-specific mutations, which curbs the broad applicability of PCR for the majority of AML patients. Here we present a microfluidic chip assay (Figure 1) with high sensitivity for detecting low levels of aberrant antigen expressing blasts in peripheral blood, thereby enabling frequent MRD monitoring for high-risk AML patients undergoing allogeneic stem cell transplant (SCT). Early detection of MRD in AML post-SCT can influence clinical decisions, such as adjustments in immunosuppression or administration of donor lymphocytes. Methods: Subjects had 3 mL of peripheral blood drawn into an EDTA tube every 2 weeks following their SCT from day 14 through day 98 and monthly through day 365 post-SCT. The blood was injected into microfluidic chips (Figure 1), each containing 50 flow channelsthat were chemically modified with capture antibodies specific for human CD34, CD33 or CD117 - surface antigens commonly expressed by AML blasts. Following cell selection, fluorescently-labeled antibodies specific for 1) the capture antibody, 2) an aberrant lineage marker (e.g. CD7, CD56) that was known to be present on the patients AML blasts and 3) CD45, were injected into the chips. Cells were then released for off-chip fluorescence microscopy and enumerated with the results compared to conventional MRD assays, ongoing treatments and clinical outcomes. Results: 7 patients were enrolled with 4 having reportable data at the time of submission. Pt#1 had AML with NPM1 and FLT3-ITD mutations. NPM1 PCR classified Pt#1 as MRD(-) 51 days after SCT, while the microfluidic assay was MRD(+) at day 48 with 81 aberrant cells/mL. The aberrant cell count began to steadily increase at day 77 that persisted until Pt#1 was diagnosed with hematologic relapse on day 85 and died at day 95 (Figure 2). Pt#2 is currently 216 days post-SCT and has had persistent low levels (5 - 57 cells/mL) of aberrant cells since day 64. This patient recently developed GVHD and is being treated with corticosteroids and has no evidence of hematologic relapse at this time. Pt#3 developed high level aberrant cells (1066 cells/mL) at day 35 post SCT. These values dropped while the patient was treated with valganciclovirfor CMV viremia (22 cells/mL at day 47), but the high level of AML blasts returned upon completion of therapy (443 cells/mL at day 105). This patient developed multi-organ system failure and at the time of death (day 124), circulating immature cells were detectable in the peripheral blood. Pt#4 has AML with an NPM1 type A mutation. The patient was MRD(+) as determined by NPM1 PCR analysis (checked 3 times) and by the microfluidic assay (checked 7 times); however, on the most recent assessment at day 125, both assays were MRD(-). Conclusion: A microfluidic assay was able to isolate trace quantities of circulating AML blasts (

Description: Background Over 90% of Ph-positive chronic myelogenous leukemia (“typical CML”) patients have breakpoints in the M-bcr, which typically result in b2a2 (e13a2) and/or b3a2 (e14a2) fusion mRNAs, both of which are translated into the p210 BCR-ABL protein. CML patients with the p190 BCR-ABL (m-bcr) or p230 BCR-ABL (μ-bcr) fusion genes have been reported. Atypical BCR breakpoints outside these cluster regions are extremely rare. For instance, only 8 cases have been described of e6a2 fusion CML. Very little is known about the clinical or biological characteristics of this subtype of CML, including the role of collaborating gene mutations in the development of disease. In this study, we defined the gene mutations that occurred in a rare e6a2 CML case and compared the observed gene mutations to those in “typical” chronic phase (CP)-CML cases. To our knowledge, this is the first comparison of the genetic mutations occurring in typical CML and in this rare atypical form of CML. Methodology We identified the index e6a2 CML patient, and eight additional typical CML patients for whom we had bone marrow aspirate, peripheral blood and paired normal tissue. We performed whole-exome sequencing for all of these samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 3 GB of sequencing data using high throughput sequencing on the Illumina platform. Results We identified 15 candidate cancer genes that were somatically mutated in our e6a2 CML patient. Commonly implicated biological processes comprising these genes included transcription (STAT5A, TET2, GTF2F1), cellular differentiation (TP73), and signal transduction (GPR116). Interestingly, the majority of these mutations also occurred in typical CML, albeit at lower frequency. Thus, genes mutated common to our atypical case and typical CMLs included STAT5A, TET2, GTF2F1, ABL1 and CYP2A6. Thus, while atypical e6a2 BCR-ABL fusion CML cases are extremely rare, they appear to share many aspects of the biology with typical CMLs. Conclusion This study represents an in-depth analysis of a rare e6a2 CML in combination with one of the first analyses of gene mutations that occur in typical CML. Our data provide a significant first step to identifying genes that play a role in the pathogenesis along with BCR-ABL that perhaps contribute to drug resistance, and ultimately impact overall survival. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Burkitt lymphoma typically involves the bone marrow in advanced stage disease. Pure leukemic presentation of Burkitt lymphoma (pure Burkitt leukemia) with negative radiographic findings is uncommon but considered clinically as stage IV disease. The WHO classification clearly distinguishes Burkitt leukemia (formerly classified as FAB-L3) from B lymphoblastic leukemia, as the former has a mature B-cell phenotype (expresses surface immunoglobulin) and characteristically harbors a MYC translocation with an immunoglobulin partner. There is limited prognostic information on patients with pure Burkitt leukemia (PBL) because they are typically combined with Burkitt lymphoma patients with widespread disease (WBL). Methods: We performed a multicenter retrospective analysis of newly-diagnosed Burkitt lymphoma cases involving the bone marrow at four academic medical centers from 2000-2013. Inclusion criteria included availability of clinical information and follow-up data, MYC rearrangement, and treatment with intensive chemotherapy (Hyper-CVAD or ALL-like regimens). Pediatric patients were excluded (age 〈 21 years). Diagnosis was established by the institutions' expert hematopathologist in conjunction with cytogenetic, flow cytometric, and radiographic findings. Patients were placed in the pure Burkitt leukemia (PBL) group if they had bone marrow involvement but were otherwise radiographically negative (i.e. PET negative). Kaplan-Meier analysis was used to examine the difference in overall survival between the WBL and PBL groups. We also determined prognostic factors associated with survival in univariate and multivariate Cox regression analyses. Results: We identified 51 patients with bone marrow involvement by Burkitt lymphoma. Of these patients, 24 were excluded because of a lack of clinical or follow-up information (15), pediatric age group (7), no treatment initiated (1), or misdiagnosed and given inadequate treatment (1). Of the 27 patients that met our inclusion criteria, 11 patients had pure Burkitt leukemia (PBL) and 16 had tissue involvement with bone marrow disease (WBL). The male-to-female ratio in the PBL group was 4.5:1 and in the WBL 4:1 (p=0.23). The median age for the PBL was 46 years vs 47 years in the WBL (p=0.34). CSF involvement was similar in both groups (30% PBL vs. 38.5% WBL; p=0.14); with elevated LDH levels in all patients in both groups (PBL median 3399 IU/L, WBL 2550 IU/L; p=0.81). In general, the WBL group had more complex karyotypes and a significantly greater number of cases with chromosome 1q abnormalities (5/7, 71%) compared to the PBL group (1/5, 20%). In the survival analysis, patients with PBL exhibited significantly better survival with a 5-year-overall survival of 79.5% (95% CI: 57.7-100%) vs. 18.4% (95% CI: 5.3-63.3%) in the WBL group (p=0.002). Conclusion: To the best of our knowledge, this is the largest series comparing adults with pure Burkitt leukemia and Burkitt lymphoma with widespread disease. Within the limitations of a retrospective analysis, the 5-year overall survival for the widespread Burkitt lymphoma (WBL) group is inferior compared to pure Burkitt leukemia (PBL) treated with intensive chemotherapy. Interestingly, the WBL group had more complex karyotypes compared to the PBL group. Figure 1. Overall Survival of Burkitt Leukemia versus Widespread Burkitt Lymphoma (BL). Figure 1. Overall Survival of Burkitt Leukemia versus Widespread Burkitt Lymphoma (BL). Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1442 Background: Deletions of the long arm of chromosome 20 have conventionally been associated with myeloid neoplasms. The frequency of chromosome 20q deletion (del(20q)) is 1–10% in myeloid diseases and associated with good prognosis in patients with myelodysplastic syndromes (MDS) and poor response to treatment in patients with acute myeloid leukemia (Greenberg 1997; Campel 1994). Previously chromosome 20q deletions were thought to be pathognomonic for myelodysplastic syndrome. Due to the observation that del(20q) may be present in non-malignant clones in patients with non-myeloid cancers, the 2008 WHO Classification (Swerdlow ed.) stated that MDS could not be diagnosed solely on the basis of isolated del(20q). The significance of isolated del(20q) in non-myeloid disorders have yet to be defined. When this chromosomal abnormality is found incidentally in marrows of patients for non-myeloid cancers, some oncologist may consider altering plans for cytotoxic chemotherapy out of concern for developing MDS. The aim of this study was to determine if isolated del(20q) in non-myeloid disorders implies an impending myeloid disease and if treatment should be altered for such patients. Methods: We conducted a retrospective, single institution cohort study among patients who between January 2005 and July 2012 were found to have 20q deletions without other chromosomal alterations per conventional cytogenetics and non-myeloid disorders per clinical history or bone marrow biopsies. Patients with isolated 20q deletion were identified from the clinical cytogenetic laboratory database and results were reviewed per cytogeneticist for accuracy. Pathology reviewed initial and subsequent bone marrow biopsies for histopathologic or immunophenotypic evidence of a myeloid disease. If a subsequent bone marrow exam was not available, the patient's complete blood counts were reviewed to determine if they developed clinical evidence of a myeloid disorder. We defined clinical suspicion for MDS as the development of transfusion dependence (≥1 unit red blood cell transfusion every 8 weeks over 4 months), any grade = 2 anemia with either grade = 2 thrombocytopenia or grade = 2 neutropenia not related to chemotherapy or immunotherapy, or any grade = 3 cytopenia without known etiology. For patients undergoing chemotherapy, MDS was suspected if there was evidence of poor bone marrow reserve as manifest by dose modifications or delays for hematologic toxicity. Results: Thirty nine patients with isolated del(20q) were identified in the cytogenetic database from January 2005 to July 2012. Twelve out of thirty nine (31%) patients were found to have non-myeloid disorders. Three patients with multiple myeloma (25%), two patients with chronic lymphocytic leukemia (17%), two patients with autoimmune disorders (17%) and five others with breast cancer, diffuse large B cell lymphoma, monoclonal gammopathy of undetermined significance, Crohn's disease and melanoma. There were an equal number of men and woman with median age of 60 years (range 30–83 years) at the time of isolated del(20q) detection. Six patients were found to have del(20q) at the initial presentation of their disease and six developed del(20q) after undergoing treatment. Nine patients were treated with standard first line systemic therapies. Six of the nine patients were treated with chemotherapy and four of them did not have to undergo any dose modifications due to myelosuppression. In the patients with chronic lymphocytic leukemia, FCR (Fludarabine,Cyclophosphamide and Rituximab) was dose modified and later discontinued due to persistent neutropenia and thrombocytopenia. After a median follow up of twenty two months (range 2 – 64 months) no patients developed evidence of a myeloid disorder by bone marrow pathology or clinical evidence. Conclusion: Isolated deletion of the long arm of chromosome 20 in patients with non-myeloid disorders does not result in bone marrow failure or myeloid disease, therefore physicians should not alter their treatment plans. Further patient follow up is necessary to provide more insight on the prognosis and treatment of non-myeloid disorders with isolated chromosome 20q deletion. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 5193 Diffuse large B-cell lymphoma (DLBCL) affects ∼25,000 people in the U.S. each year, and fewer than half of them are cured with standard therapy. DLBCL can be divided into two subtypes by gene expression profiling, germinal center B-cell (GCB) type and activated B-cell (ABC) type. ABC-type DLBCL patients have significantly poorer outcomes. To improve therapeutic options, better animal models that accurately mimic human DLBCL (hDLBCL) are needed. Canine DLBCL is one of the most common cancers in veterinary oncology. Similar to human DLBCL patients, dogs with lymphoma are treated with both CHOP-like regimens and autologous stem cell transplants. Morphologically, canine lymphomas are similar to hDLBCL, with shared histologic markers, such as CD20 and PAX5. With recent technologies based on knowledge of the canine genome sequence, it is now possible to evaluate dogs as a potential large-animal model for hDLBCL. We evaluated 58 canine B-cell lymphomas by generating comprehensive gene expression profiles and comparing them to previously published hDLBCL expression profiles. Canine B-cell lymphoma expression profiles were similar in some ways to hDLBCLs. For instance, increased expression of NF-kB pathway genes was noted in a subset of lymphomas, mirroring NF-kB pathway activation in human ABC-type DLBCL. Furthermore, immunoglobulin heavy chain (IGH) mutation status, which is correlated with ABC/GCB cell of origin in hDLBCL, separated canine DLBCL into two groups with statistically different progression-free and overall survival times. However, canine DLBCL differed from hDLBCL in other aspects, including rare immunohistochemical positivity for BCL6 and MUM1/IRF4. Collectively, these results define aspects of canine B-cell lymphomas that resemble hDLBCL, identifying molecular similarities that could allow dogs to be used as a representative model of hDLBCL. Further comparative studies, including therapeutic trials, could potentially improve outcomes in both species. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2135 Hemophagocytic lymphohistiocytosis (HLH) is a multisystem disorder characterized by immune dysregulation and hypercytokinemia. Diagnostic criteria include a genetic mutation consistent with familial HLH or the presence of 5 of 8 defined clinical criteria (fever, splenomegaly, bicytopenia, hypertriglyceridemia and/or hypofibrinogenemia, hemophagocytosis, low/absent NK cell function, hyperferritinemia, and elevated soluble CD25). In pediatrics, a ferritin value of 〉10,000 mχγ/L has been reported to have 90% sensitivity and 96% specificity in defining the presence of HLH (Allen, C. E., Yu, X., Kozinetz, C. A. and McClain, K. L. (2008). Pediatr. Blood Cancer). We examined if hyperferritinemia (all patients with ferritin level 〉10,000 mχγ/L between 2007 and 2012) correlated with diagnosis of HLH in 94 patients (73 adult; 21 pediatric) at our institution. Chart reviews were performed to evaluate the presence or absence of HLH criteria, additional clinical features that may be indicative of HLH, and diagnosis. These data resulted in our classification of patients into four groups (Table 1): (1) “clinically defined HLH” when predetermined criteria were met; (2) “potential HLH” when clinical criteria was suggestive of HLH, but not all criteria were met; (3) “possible HLH” when rheumatologic syndromes, liver disease, or fever/DIC was present of unknown etiology; or (4) “Non-HLH” when the elevated ferritin was a result of a known etiology (Table 2). As expected, 18 (86 %) of pediatric patients with a ferritin 〉 10,000 mχγ/L had clinically defined or potential/possible HLH. Notably, 44 (60 %) of adult patients with a ferritin 〉 10,000 mg/L had clinically defined or potential/possible HLH. Such an incidence of HLH in the adult population with elevated ferritin raises caution for appropriate diagnosis of this population and clearly warrants further study. If patients with sickle cell disease, GVH, or known causes for liver failure are excluded, then HLH should be suspected in 83% of adult patients with ferritins 〉10,000 mcg/L. Table 1: HLH classifications of 94 patients with ferritin 〉 10,000 mχγ/L Adult, n = 73 n (%) Pediatric, n = 21 n (%) Clinically defined HLH 18 (25) 12 (57) Potential HLH 9 (12) 5 (24) Possible HLH 17 (23) 1 (5) Non-HLH 29 (40) 3 (14) Table 2: Diagnoses of non-HLH patients with ferritin 〉 10,000 mcg/L (some diagnoses overlap) Liver failure of clear etiology (10) APAP toxicity (4) Shock liver (4), EtOH, Other Sickle cell disease (9) 7 adult, 2 peds, all with probable iron overload Other tumors (9) CMML+VT+shock liver, prostate ca with bone mets, CMML to AML, MDS/MPD with infection, AML, T lymphoblastic lymphoma with cholestasis, allo txp for AML with iron overload, ALL+ abd wall hematoma Iron overload (11) 9 sickle cell, 1 Castlemans, 1 allo txp for AML Other Pancreatitis, GVH (3) Table 3: Clinical suspicion for HLH Potential HLH (%) Possible HLH (%) Total Adult 9 17 Peds 5 1 HLH suspected? Adult 1 (11) 2 (12) Peds 4 (80) 1 (100) All criteria sent? Adult 1 (11) 1 (6) Peds 2 (40) 1 (100) Hematology involved? Adult 6 (67) 11 (64) Peds 5 (100) 1 (100) Did hematologist note ferritin? Adult 1 (17) 3 (27) Peds 5 (100) 0 Disclosures: Ma: Novo Nordisk Inc.: Consultancy, Speakers Bureau.

Description: Abstract 2201 Idiopathic thrombotic thrombocytopenic purpura (TTP) is typically associated with severe ADAMTS13 deficiency due to the production of autoantibodies against ADAMTS13. Recent studies have demonstrated that B cell activating factor (BAFF), a TNF family member known to promote activation and survival of autoreactive B cells, is increased in TTP patients (Thomas et al. 2011 155:620 Br J Haematol; Watanaboonyongcharoen et al. 2011, AABB Abstract # 1118421). We hypothesized that high BAFF levels in TTP results in loss of B cell tolerance and the production of autoantibodies. Since defective clearance of apoptotic cells by macrophages has been found in autoimmune diseases, antibodies against MARCO (Macrophage Receptor with Collagenous structure) represented good candidate for study as a potentially pathophysiologically relevant autoantibody. Such anti-MARCO antibodies may lead to defective apoptotic cell clearance and the development of TTP. We measured anti-MARCO antibodies by ELISA and Western blot in 34 idiopathic TTP patients between 1999 and 2012: 25 female and 9 male with a median age of 40 years (range 25–72). All patients were diagnosed on the clinical basis of microangiopathic hemolytic anemia and thrombocytopenia without any other cause. ADAMTS13 activity and the presence of anti-ADAMTS13 inhibitor tests were performed in all patients. Fifty percent of patients had ADAMTS13 activity less than 10%, while 56% had ADAMTS13 inhibitor. All 34 patients underwent therapeutic plasma exchange (TPE) daily until the platelet count was at least 150 × 109/l for two consecutive days. High dose steroids were initiated immediately after first TPE. While direct binding ELISA did not yield specific results due to high background, specific MARCO bands were detected by Western blotting of recombinant MARCO protein with patient plasma IgG. Ninety-seven percent of patients with TTP (33/34) were positive for anti-MARCO IgG antibody compared to forty percent (10/25) of healthy controls, p 〈 0.001 (Table 1). As a surrogate for antibody titer, intensity of each Western blot band was quantified by densitometry using NIH ImageJ software. Patients with TTP had significantly increased anti-MARCO IgG as defined by the densitometric area under the curve (1.3 × 103; range 0–8.2 × 103) compared to healthy controls (0, range 0–4.5 × 103), p 〈 0.001. A cut-off point for high titer anti-MARCO IgG was calculated by using mean + 2SD of the area under the curve (AUC) of anti-MARCO IgG Western blot band density measured in healthy controls. Patients with an increased amount of anti-MARCO IgG (AUC 〉 2.9× 103) tended to have a higher relapse rate compared to those with normal anti-MARCO IgG (4 of 6 patients [67%] vs. 10 of 28 patients [36%], respectively, p = 0.20), although statistical significance was not reached due to the limited number of patients. Thus, for the first time, we identify anti-MARCO IgG in idiopathic TTP, suggesting a role for macrophage inhibition in the pathophysiology of TTP. Studies of anti-MARCO antibodies in larger numbers of patients may lead to development of a novel prognostic marker for TTP patients. Table 1. Presence of anti-MARCO IgG on Western blot Anti-MARCO IgG Population group TTP n (%) Healthy n (%) Yes, (n = 43) 33 (97) 10 (40) No, (n = 16) 1 (3) 15 (60) p 〈 0.001. Disclosures: No relevant conflicts of interest to declare.