bluuebunny/crossref_metadata_2025_split huggingface dataset:
Plasma Membrane Proteomics Identifies Biomarkers Associated with T(4;14) Multiple Myeloma
Authors: Xie Zhigang , Gunaratne Jayantha , Cheong Lip Lee , Liu Shaw Cheng , Koh Tze Loong , Huang Gaofeng , Blackstock Walter , Chng Wee-Joo 
Published: October 2019

Abstract
Abstract 1314
Multiple myeloma (MM) is characterized by recurrent chromosomal translocations. Patients with t(4;14) have very poor prognosis. MMSET, identified by its fusion to the IgH locus in t(4;14) MM, is universally overexpressed in t(4;14) MM. In order to identify cell surface biomarkers associated with t(4;14) MM for small molecule or antibody based therapies, we knocked down MMSET expression with shRNA and generated a cell line pair from KMS11, a t(4;14) MM cell line. We used quantitative mass spectrometry based on stable isotope labelling by amino acids in cell culture (SILAC) to identify plasma membrane proteins associated with MMSET overexpression. Using this approach, 55 cell surface proteins were identified as differentially expressed between KMS11 and KMS11/shMMSET. Western blot and flow cytometry analysis indicated SLAMF7 was universally over-expressed in t(4;14) MM cell lines and down-regulated by MMSET shRNAs. Quantitative RT-PCR (qPCR) analysis indicated MMSET shRNAs resulted in significant reduction of SLAMF7 mRNA, suggesting MMSET might regulate the transcription level of SLAMF7. ChIP followed by qPCR analysis indicated MMSET protein binding was concentrated in an upstream region (near −1,500 bp) of SLAMF7 transcript start site. Furthermore, SLAMF7 shRNA could induce G1 arrest or apoptosis and reduce clonogenetic capacity in t(4;14) MM cells. SLAMF7 is a self-ligand receptor, and we found that SLAMF7 extracellular domain (ECD) could specifically recognize t(4;14) MM cells. Overall, these results illustrated SLAMF7 might be a novel cell surface protein associated with t(4;14) MM, and SLAMF7 ECD might be an alternative approach for targeting SLAMF7 on t(4;14) MM.

Disclosures:
No relevant conflicts of interest to declare.
Geminin Is a Newly Identified Cks1 Interaction Partner and Overexpressed in a Murine Model During Lymphomagenesis
Authors: Hoellein Alexander , Bassermann Florian , Schoeffmann Stephanie , Graf Steffi , Peschel Christian , Gloeckner Johannes , Ueffing Marius , Keller Ulrich 
Published: October 2019

Abstract
Abstract 1328
Cks1 is an activator of the SCFSkp2 ubiquitin ligase complex that targets the cell cycle inhibitor p27Kip1 for degradation. Loss of Cks1 results in p27Kip1 accumulation and decreased proliferation. Cks1 expression is elevated in various B cell malignancies including Burkitt lymphoma and multiple myeloma. We have previously shown that loss of Cks1 results in elevated p27Kip1 levels and delayed tumor development in a mouse model of Myc-induced B cell lymphoma. Surprisingly, loss of Skp2 in the same mouse model also resulted in elevated p27Kip1 levels but exhibited no impact on tumor onset. Analysis of Cks1−/− murine embryonic fibroblasts (MEFs) unveiled a significant growth defect and comparison with double knock out Cks1−/−;p27Kip1−/− MEFs revealed a p27Kip1 independent defect at G1/S transition. These effects were associated with suppressed Cdk1 protein and Cdk2 activity in Cks1−/− cells.
We hypothesized that Cks1 recruits further cell cycle regulators or facilitators of proliferation, which may explain the dramatic growth defect and the prolonged latency of lymphomagenesis upon loss of Cks1. Using Strep-Flag tagged Cks1 and tandem affinity purification followed by mass spectrometry we identified Geminin as a new Cks1 binding partner. Geminin is an inhibitor of rereplication and loss of Geminin leads to recurrent origin firing in S phase. The physical interaction of Cks1 and Geminin was present in cells of human and murine origin and was associated with Cdk2 binding to Geminin. Detailed analysis proved Cks1 dispensibel for cell cycle controlled Geminin degradation or phosphorylation.
Surprisingly when mining public databases and RNA microarray data we found Geminin overexpressend in aggressive human lymphoma subtypes associated with high Ki-67 expression, and in murine Myc-induced lymphoma. Real time PCR revealed increased expression of Geminin during lymphomagenesis in Eμ-Myc mice, which correlated with protein expression. In conclusion, we established the physical Geminin-Cks1 protein interaction, present in human and murine cells, which could act in control of rereplication and genomic stability. Geminin is overexpressed in human and murine lymphoma revealing a potential role in lymphomagenesis.

Disclosures:
No relevant conflicts of interest to declare.
A Novel Combination Therapy Based On the Genetics of Follicular Lymphoma
Authors: Wendel Hans-Guido 
Published: October 2019

Abstract
Abstract 1330
Follicular lymphoma (FL) is a common and incurable form of Non-Hodgkin's lymphoma characterized by the translocation t(14;18) that results in increased expression of the anti-apoptotic BCL2 protein. Exactly what drives expansion of the malignant B-cells in FL is not clear. Here we report recurrent genetic lesions targeting cell cycle controls in FL. These include mutations in the CYCLIN D3 gene (6%), chromosomal gains of CDK4 (31%), and losses of CDKN2a/b (9%), and RB1 (10%). Consistent with the genetic data, 46% of FLs show abundant RB phosphorylation, and together these lesions identify patients with advanced tumours who fare badly with standard therapies. In a murine FL model loss of cell cycle control acts as an oncogenic driver and cooperates with BCL2. Moreover, xenografted FLs respond to CDK4/6 inhibition and combinations with BH3 mimetic drugs cause extensive apoptosis and consolidate the therapeutic effect. Hence, loss of cell cycle control is an oncogenic event in ∼50% of FL and the simultaneous blockade of CDK4 and BCL2 is an effective combination therapy for FL.

Disclosures:
No relevant conflicts of interest to declare.
Tregs Specific for Minor Histocompatibility Antigens
Authors: Veerapathran Anandharaman , Pidala Joseph , Beato Francisca , Janssen William E. , Yu Xue-Zhong , Anasetti Claudio 
Published: October 2019

Abstract
Abstract 1891

Background:
The risk of acute GVHD after HSCT is increased in male recipients of female grafts. Disparities for the male-associated H-Y and other minor histocompatibility antigens (mHAs) have the capacity to sensitize alloreactive donor T cells and cause GVHD in HLA-matched recipients. These mHAs are polymorphic proteins that differ between donor and recipient and are presented as peptides by HLA molecules on recipient or donor antigen-presenting cells to donor immune cells. Currently, there is no evidence that minor histocompatibility antigen specific Tregs exist. Earlier in our laboratory, we have measured the frequency, growth requirements, and function of human blood Tregs specific for allo-MHC. In the present study, we sought to detect the frequency, expansion kinetics and characteristics of the minor antigens specific Tregs in the blood of HLA-matched sibling pair.

Methods:
CD4+CD25+CD127− Tregs were isolated by immunoabsorption from sibling donors, and cultured with HLA-matched sibling recipient antigen-presenting cells in the presence of IL-2, IL-15 and rapamycin. We detected 30–50 fold increase in H-TdR uptake at 6 days in Treg cultures stimulated by HLA-identical sibling compared to self DC. The precursor frequency of mHA-specific Tregs are between 7 and 43 (median - 13) cells per one million blood Tregs. The frequency of mHA-specific conventional CD4 T cells among total blood CD4 T cells is similar in HLA-matched sibling donors. Ex vivo expanded mHA-specific Tregs maintained higher levels of Foxp3 expression, retained the lymphoid homing receptor CD62L and a chemokine receptor, CCR7, suggesting that they are functional and are able to migrate to lymphoid tissue in vivo. Split well assay on day 12 demonstrated the mHA specificity, since Treg responded to restimulation with DC from the original HLA-identical sibling, but not self DC. The mHA-specific Tregs expanded to more than 100 fold in vitro, and exhibited antigen specific suppression. When Tregs were cultured at limiting dilution, we obtained 6 mHA-specific Treg clones that retained TGF-beta secretion in response to the sibling's mHA-disparate DC but not self DC.

Conclusion:
We demonstrated for the first time that it is possible to detect and expand mHA specific Tregs from HLA-matched sibling pairs, immunotherapy with mHA-specific Tregs may prevent GVHD.

Disclosures:
No relevant conflicts of interest to declare.
Ascertainment of Iron Depletion and Deficiency in Blood Donors Through Screening Questions for Pica and Restless Legs Syndrome.
Authors: Bryant Barbara J. , Yau Yu Ying , Arceo Sarah M. , Hopkins Julie A. , Leitman Susan F. 
Published: October 2019

Abstract
Abstract 2283

Background:
Pica and restless legs syndrome (RLS) are associated with iron depletion and deficiency. Pica refers to the compulsive craving for and persistent consumption of non-nutritive substances. Pagophagia, the pathological consumption of ice, is a specific pica strongly associated with iron deficiency. RLS is a neurological movement disorder characterized by uncomfortable sensations in the lower extremities, with a compelling urge to move the extremities to relieve the discomfort. Primary RLS is a central nervous system disorder; however, secondary RLS may be caused or exacerbated by iron deficiency. The purpose of this study was to prospectively assess the prevalence of pica and RLS in blood donors presenting to a hospital-based donor center, to correlate the findings with donor hemoglobin and iron levels, and to study the effects of oral iron replacement on the resolution of symptoms.

Methods:
During a 39-month period, 1236 blood donors deferred for fingerstick hemoglobin <12.5 g/dL and 400 non-deferred “control” donors underwent health screening and laboratory testing (CBC, ferritin, iron, transferrin). Iron deficiency was defined as a ferritin level below the institutional reference range of 9 mcg/L in females and 18 mcg/L in males. Iron depletion was defined as a ferritin of 9 – 19 mcg/L in females and 18 – 29 mcg/L in males. Pica and RLS were assessed by direct questioning. Deferred donors and iron-deficient control donors were given ferrous sulfate 325 mg daily for 60 days. Reassessments were performed and additional iron tablets dispensed at subsequent visits.

Results:
Pica was reported in 11% of donors with iron depletion/deficiency, compared with 4% of iron-replete donors (p<0.0001). The prevalence of pica rose to 21% in females with ferritin <9 mcg/L. Pagophagia (ice pica) was most common and was often of extraordinary intensity, with characteristics of an addiction. Donors with pagophagia given iron reported a marked reduction in the desire to eat ice by day 5–8 of therapy, with disappearance of symptoms by day 10–14. RLS was reported in 16% of subjects with iron depletion/deficiency compared with 11% of iron-replete donors (p=0.012). Iron replacement generally resulted in improvement of RLS symptoms, however, at least 4–6 weeks of iron therapy was necessary. The positive predictive values of pica and RLS in iron deficient/depleted blood donors were 73% and 58%, respectively.

Conclusion:
The presence of pica is highly predictive of iron depletion/deficiency in blood donors; however, RLS lacks a strong correlation in this population. Screening questions for pica, in particular pagophagia, may be useful in the ascertainment of iron depletion and deficiency in blood donors and may identify those who would benefit from oral iron therapy.

Disclosures:
No relevant conflicts of interest to declare.
Phase II Study of Pomalidomide (Pom) in Genomically Defined High Risk Relapsed and Refractory Multiple Myeloma (RRMM)
Authors: Usmani Saad Z , Hansen Emily , Steward Doug , Waheed Sarah , Panozzo Susan B. , Petty Nathan M , Atrash Shebli , Abdallah Al-Ola , Ahmad Nisar , Hoering Antje , Crowley John , Barlogie Bart 
Published: October 2019

Abstract
Abstract 4083

Background:
Pom is a third generation immunomodulatory drug (IMiD) which has demonstrated safety and efficacy in RRMM with prior exposure/resistant to other IMiDs and bortezomib. This is the first report on a phase II clinical trial employing Pomalidomide (Pom) in genomically defined high risk RRMM.

Methods:
First cycle Pom was given at 4mg orally Days 1–21 every 28 days; dexamethasone (DEX) or bortezomib (BOZ) could be given in absence of PR or better from Cycle #3. In the absence of at least PR, Pom dose was escalated to 5mg. Cox regression modeling was employed for univariate and multivariate analyses, whereas Kaplan-Meier curves were used for overall survival (OS) and progression free survival (PFS).

Results:
30 patients with RRMM were enrolled. Baseline characteristics included age >=65yr in 47%, ISS stage >=II was seen in 74% of patients, cytogenetic abnormalities (CA) within 6 months in 79%, and GEP-defined high risk in 66% by GEP70-gene model, 97% by GEP80-gene model, 13% had extramedullary disease (EMD), and 7% had secondary plasma cell leukemia (SPCL). 26/30 patients (87%) had prior autologous stem cell transplant, 23/30 patients (77%) had had at least 2 transplants. 22 patients (73%) had disease progression while on lenalidomide, bortezomib or both within 60 days of enrollment. At least 2 cycle of treatment were administered to 28 (93%) patients enrolled, 20 (67%) patients received >4 cycle of treatment and 16 (53%) received > 6 cycles. 15 patients (50%) discontinued therapy primarily due to progression or death. 5 (17%) patients achieved PR or better as best response. PFS benefit was observed in older patients (HR=0.29, p=0.008), with high LDH presenting an increased risk for OS (HR=14.64, p=0.013.) Most common toxicities, counting all toxicities (>=grade 3) were: thrombocytopenia (63%), leukopenia (63%), anemia (40%), hypophosphatemia (20%) and hypocalcemia (23%). Overall and progression- free survival at 12 months were 64% and 20%, respectively.

Conclusions:
The present trial is the first investigation evaluating Pom's clinical activity in high risk RRMM and demonstrates good anti-myeloma activity in this heavily pre-treated patient population. Given the activity in poor prognostic clinical features such as EMD and SPCL, there is precedence to evaluate Pom as an IMiD of choice in combination with other novel agents for this subset of RRMM.

Disclosures:
No relevant conflicts of interest to declare.
Abrogation of Myeloid Derived Suppressor Cell-Like Inhibitory Activity of K562 Restores Antigen Presenting Cell Functions
Authors: Tanimoto Kazushi , Muranski Pawel , Hensel Nancy F. , Keyvanfar Keyvan , Fujiwara Hiroshi , Barrett A. John , Melenhorst J. Joseph 
Published: October 2019

Abstract
Abstract 4118
The human leukemia cell line K562 represents an attractive platform for creating an artificial antigen presenting cell (AAPC): it is readily expandable, does not express HLA class I and II and can be stably transduced with various genes. To generate an AAPC to expand CMV antigen-specific T cells for adoptive immunotherapy, we stably transduced K562 with HLA-A2 in combination with 4-1BB ligand, or CD64 or HLA-DR15. In preliminary experiments, irradiated K562 cells expressing HLA-A2 and 4-1BB ligand pulsed with CMV pp65 and IE-1 HLA-A2 specific peptides failed to elicit antigen-specific CD8+ T cells in HLA-A2+ peripheral blood mononuclear cells (PBMC) or isolated T cells. Since CMV peptides added directly to the PBMC readily expanded antigen-specific CD8+ T cells, we concluded that K562 AAPC inhibited the T cell response. We found that both parental K562 cells and AAPC strongly inhibited T cell proliferation to the bacterial superantigen staphylococcus enterotoxin B (SEB), anti-CD3 stimulation with OKT3, and in MLR. The inhibitory effect of K562 appeared to be T cell specific since K562 cells did not suppress EBV-transformed B cells.
Transwell experiments demonstrated preservation of the inhibition, suggesting that suppression was mediated by a soluble factor. MLR inhibition was not reversed by neutralizing anti-TGFβ antibody or PGE2 inhibitors. Finally, the full abrogation of the suppressive activity of K562 was achieved by a brief fixation of cells with formaldehyde at concentrations as low as 0.1%: The MLR was restored when K562 was fixed, and donor T cell response to SEB- and OKT3-loaded K562 AAPC was significantly higher when using fixed K562 cells compared to unfixed cells. Moreover, fixed pp65 and IE-1 peptide-loaded HLA A2+ AAPC expressing 4-1BB ligand induced robust (3–5 fold improved) expansion of CMV-specific T cells from all tested HLA-A2+ donors when compared with irradiated AAPC control. Thus, fixed K562 cell constructs efficiently presented antigen and stimulated T cells.
Overall, we demonstrate that K562 line can serve as a source of AAPC for cell therapy approaches after abrogation of their suppressive activity using formaldehyde. However, our results also revealed a previously unappreciated feature of K562 biology, clearly indicating that these commonly used cells are potent inhibitors of peptide antigen-, superantigen-, and OKT3- driven T cell proliferation. Thus K562 line displays a myeloid-derived suppressor cell-like functionality. Our findings have implications for broader understanding of the immune evasion mechanisms used by leukemias and other tumors.

Disclosures:
No relevant conflicts of interest to declare.
Azacytidine Compromises NK-Cell Activity in AML and MDS Patients Undergoing MRD-Based Pre-Emptive Treatment After Allogeneic Stem Cell Transplantation
Authors: Sockel Katja , Schönefeldt Claudia , Sopper Sieghart , Wermke Martin , Schmitz Marc , Ehninger Gerhard , Bornhäuser Martin , Wolf Dominik , Platzbecker Uwe 
Published: October 2019

Abstract
Abstract 4122
The hypomethylating agent azacytidine (AZA) represents the standard treatment for many high-risk MDS and AML patients. While the clinical efficacy has been confirmed in several studies, the precise molecular mechanism of action has not been fully understood yet.
Human NK-cells play an important role in the regulation of immune responses against malignant cells. Their function is controlled by a complex interplay of activating and inhibitory receptors - some of them being regulated by methylation of the respective genes. We, therefore explored, whether AZA modulates in vitro NK-cell function as well as in vivo during minimal-residual disease (MRD)-guided treatment of imminent relapse in MDS and AML patients treated within the prospective RELAZA trial (NCT00422890).

Methods:
After purifying NK-cells of healthy donors by MACS (magnetic cell sorting), NK-cells were exposed in vitro to different concentrations of AZA (100nM, 1μM, 3μM) with or without IL-2. In parallel, the NK-cell phenotype of patients (n=12) with AML or MDS, undergoing MRD-guided treatment with AZA after stem cell transplantation was monitored by FACS from peripheral blood samples on day 1, 5 and 7 of the first and second AZA cycle. All patients were still in complete haematological remission at the time of therapy.

Results:
In vitro, we observed a significant reduction (3,1% to 1,8% p=0.028) of the immature and cytokine-regulating CD56bright NK-cell subpopulation with increasing concentrations of AZA. There was a trend towards a reduced expression of the death-ligand TRAIL, the activating receptors NKG2D and NKp46 and for an increased expression of the inhibitory KIR CD158b1/b2, whereas we could not detect any changes in the expression of FAS-L, Perforin, Granzyme B, NKp30, NKp44, CD69, CD57, DNAM-1, CD16, and NKG2A-CD94. Confirmatory, we observed a significant decrease in the expression of TRAIL (p=0.003), NKG2D (p=0.03) and NKp46 (p=0.006) during AZA treatment in-vivo. Interestingly, these changes appeared to be reversible.
The observed reduction of NK-cell activating receptors and TRAIL during AZA treatment correlated with a reduction or stable course of MRD in all analyzed patients.

Conclusion:
In summary these data suggest that the clinical effects of AZA are not mediated by enhancing NK-cell activity. In fact, the drug may have inhibitory effects on NK-cell function which should be considered when applying AZA in the post-transplant setting.

Disclosures:
Platzbecker: Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Why Is Cancer Care So Expensive: Potential Implications of Fraud and Abuse
Authors: Lu Zhiqiang , Qureshi Zaina Parvez , Chen Brian , Xirasagar Sudha , Ray Paul , Rao Gowtham , Lopez Isaac , Felder Tisha , Ablin Richard , Sartor Oliver , Bennett Charles 
Published: October 2019

Abstract
Abstract 4275

Introduction:
Annually, $82 billion to $272 billion is reportedly lost to federal health care fraud. Between 1996 and 2005, 379 federal health care fraud cases initiated by qui tam relators (“whistle blowers”) concluded, resulting in $9.3 billion in recoveries. Of these, pharmaceutical companies accounted for 13 cases (False Claims Act (FCA) cases, the primary statute invoked in health care fraud and abuse), but $3.9 billion of recoveries (4% of the cases and 39% of the financial recoveries). We report concluded FCA cases involving pharmaceutical manufacturers between 2006 and 2011. Oncology accounts for the largest per cent of total pharmaceutical expenditures. Over 90% of all new cancer pharmaceuticals cost > $20,000 for 12-weeks of treatment.

Methods:
Websites for the Department of Justice (DOJ), Taxpayers Against Fraud, Health and Human Services Inspector General's Office, Health Care Fraud and Abuse Control Project, and Lexis/Nexis were queried for pharmaceutical FCA cases (2006 to 2011).

Results:
Between 2006 and 2011, the DOJ closed 54 cases with pharmaceutical FCA violations, 38 with and 16 without qui tam relators, accounting for recoveries of $11.3 billion (mean $296 million) and $2.6 billion (mean, $165 million), respectively. Illegal marketing is the most common fraud allegations invoked against pharmaceutical manufacturers (19 cases). Pharmaceutical manufacturers accounted for 31% of total FCA cases, and 71.5% of total FCA recoveries (Table 1).

Conclusion:
Since the DOJ's shift of focus to pharmaceutical corporations in 2001, the trend has intensified, with virtually every large pharmaceutical corporation settling at least one FCA case. Pharmaceutical cases now account for 31% of the federal fraud cases and 71% of the financial recoveries. Fraud and abuse may be an important component of the high costs of cancer care in the United States. Moreover, unless fundamental changes occur, the pharmaceutical industry will continue to be the main FCA investigative target as this sector has the deepest pockets and is the health care sector most resistant to deterrence.

Disclosures:
No relevant conflicts of interest to declare.