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Krizsan, Andor, Daniel Knappe, and Ralf Hoffmann. "Influence of theyjiL-mdtMGene Cluster on the Antibacterial Activity of Proline-Rich Antimicrobial Peptides Overcoming Escherichia coli Resistance Induced by the Missing SbmA Transporter System." Antimicrobial Agents and Chemotherapy 59, no. 10 (July 13, 2015): 5992–98. http://dx.doi.org/10.1128/aac.01307-15.
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ABSTRACTIn view of increasing health threats from multiresistant pathogens, antimicrobial peptides (AMPs) and, specifically, proline-rich AMPs (PrAMPs) have been investigated in animal models. PrAMPs enter bacteria via the ABC transporter SbmA and inhibit intracellular targets. We used phage transduction (Tn10insertion) to screen by random mutagenesis for alternative uptake mechanisms for analogs of apidaecin 1b, a honeybee-derived PrAMP. All 24 apidaecin-resistant mutants had the Tn10insertion in thesbmAgene. ThesesbmA::Tn10insertion mutants and theEscherichia coliBW25113 ΔsbmA(JW0368) strain were still susceptible to the bactenecin PrAMP Bac7(1-35) and oncocin PrAMPs Onc18 and Onc112, as well as to Chex1-Arg20, despite significantly reduced internalizations. In a second round of random mutagenesis, the remaining susceptibility was linked to theyjiL-mdtMgene cluster.E. coliBW25113 and its ΔyjiLnull mutant (JW5785) were equally susceptible to all PrAMPs tested, whereas the BW25113 ΔmdtMmutant was less susceptible to oncocins. The JW0368yjiL::Tn10transposon mutant (BS2) was resistant to all short PrAMPs and susceptible only to full-length Bac7 and A3-APO. Interestingly, PrAMPs appear to enter bacteria via MdtM, a multidrug resistance transporter (drug/H+antiporter) of the major facilitator superfamily (MFS) that can efflux antibiotics, biocides, and bile salts. In conclusion, PrAMPs enter bacteria via ABC and MFS transporters that efflux antibiotics and cytotoxic compounds from the cytoplasm to the periplasm.
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Hansen, Anne, Ingo Schäfer, Daniel Knappe, Peter Seibel, and Ralf Hoffmann. "Intracellular Toxicity of Proline-Rich Antimicrobial Peptides Shuttled into Mammalian Cells by the Cell-Penetrating Peptide Penetratin." Antimicrobial Agents and Chemotherapy 56, no. 10 (July 30, 2012): 5194–201. http://dx.doi.org/10.1128/aac.00585-12.
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ABSTRACTThe health threat caused by multiresistant bacteria has continuously increased and recently peaked with pathogens resistant to all current drugs. This has triggered intense research efforts to develop novel compounds to overcome the resistance mechanisms. Thus, antimicrobial peptides (AMPs) have been intensively studied, especially the family of proline-rich AMPs (PrAMPs) that was successfully tested very recently in murine infection models. PrAMPs enter bacteria and inhibit chaperone DnaK. Here, we studied the toxicity of intracellular PrAMPs in HeLa and SH-SY5Y cells. As PrAMPs cannot enter most mammalian cells, we coupled the PrAMPs with penetratin (residues 43 to 58 in the antennapedia homeodomain) via a C-terminally added cysteine utilizing a thioether bridge. The resulting construct could transport the covalently linked PrAMP into mammalian cells. Penetratin ligation reduced the MIC for Gram-negativeEscherichia colionly slightly (1 to 8 μmol/liter) but increased the activity against the Gram-positiveMicrococcus luteusup to 32-fold (MIC ≈ 1 μmol/liter), most likely due to more effective penetration through the bacterial membrane. In contrast to native PrAMPs, the penetratin-PrAMP constructs entered the mammalian cells, aligned around the nucleus, and associated with the Golgi apparatus. At higher concentrations, the constructs reduced the cell viability (50% inhibitory concentration [IC50] ≈ 40 μmol/liter) and changed the morphology of the cells. No toxic effects or morphological changes were observed at concentrations of 10 μmol/liter or below. Thus, the IC50values were around 5 to 40 times higher than the MIC values. In conclusion, PrAMPs are in general not toxic to mammalian cells, as they do not pass through the membrane. When shuttled into mammalian cells, however, PrAMPs are only slightly cross-reactive to mammalian chaperones or other intracellular mammalian proteins, providing a second layer of safety forin vivoapplications, even if they can enter some human cells.
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Sola, Riccardo, Mario Mardirossian, Bertrand Beckert, Laura Sanghez De Luna, Dennis Prickett, Alessandro Tossi, Daniel N. Wilson, and Marco Scocchi. "Characterization of Cetacean Proline-Rich Antimicrobial Peptides Displaying Activity against ESKAPE Pathogens." International Journal of Molecular Sciences 21, no. 19 (October 6, 2020): 7367. http://dx.doi.org/10.3390/ijms21197367.
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Proline-rich antimicrobial peptides (PrAMPs) may be a valuable weapon against multi-drug resistant pathogens, combining potent antimicrobial activity with low cytotoxicity. We have identified novel PrAMPs from five cetacean species (cePrAMPs), and characterized their potency, mechanism of action and in vitro cytotoxicity. Despite the homology between the N-terminal of cePrAMPs and the bovine PrAMP Bac7, some differences emerged in their sequence, activity spectrum and mode of action. CePrAMPs with the highest similarity with the Bac7(1-35) fragment inhibited bacterial protein synthesis without membrane permeabilization, while a second subgroup of cePrAMPs was more membrane-active but less efficient at inhibiting bacterial translation. Such differences may be ascribable to differences in presence and positioning of Trp residues and of a conserved motif seemingly required for translation inhibition. Unlike Bac7(1-35), which requires the peptide transporter SbmA for its uptake, the activity of cePrAMPs was mostly independent of SbmA, regardless of their mechanism of action. Two peptides displayed a promisingly broad spectrum of activity, with minimal inhibiting concentration MIC ≤ 4 µM against several bacteria of the ESKAPE group, including Pseudomonas aeruginosa and Enterococcus faecium. Our approach has led us to discover several new peptides; correlating their sequences and mechanism of action will provide useful insights for designing optimized future peptide-based antibiotics.
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Zhu, Yanyu, James C. Weisshaar, and Mainak Mustafi. "Long-term effects of the proline-rich antimicrobial peptide Oncocin112 on the Escherichia coli translation machinery." Journal of Biological Chemistry 295, no. 38 (July 28, 2020): 13314–25. http://dx.doi.org/10.1074/jbc.ra120.013587.
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Proline-rich antimicrobial peptides (PrAMPs) are cationic antimicrobial peptides unusual for their ability to penetrate bacterial membranes and kill cells without causing membrane permeabilization. Structural studies show that many such PrAMPs bind deep in the peptide exit channel of the ribosome, near the peptidyl transfer center. Biochemical studies of the particular synthetic PrAMP oncocin112 (Onc112) suggest that on reaching the cytoplasm, the peptide occupies its binding site prior to the transition from initiation to the elongation phase of translation, thus blocking further initiation events. We present a superresolution fluorescence microscopy study of the long-term effects of Onc112 on ribosome, elongation factor-Tu (EF-Tu), and DNA spatial distributions and diffusive properties in intact Escherichia coli cells. The new data corroborate earlier mechanistic inferences from studies in vitro. Comparisons with the diffusive behavior induced by the ribosome-binding antibiotics chloramphenicol and kasugamycin show how the specific location of each agent's ribosomal binding site affects the long-term distribution of ribosomal species between 30S and 50S subunits versus 70S polysomes. Analysis of the single-step displacements from ribosome and EF-Tu diffusive trajectories before and after Onc112 treatment suggests that the act of codon testing of noncognate ternary complexes (TCs) at the ribosomal A-site enhances the dissociation rate of such TCs from their L7/L12 tethers. Testing and rejection of noncognate TCs on a sub-ms timescale is essential to enable incorporation of the rare cognate amino acids into the growing peptide chain at a rate of ∼20 aa/s.
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Brakel, Alexandra, Andor Krizsan, Renke Itzenga, Carl N. Kraus, Laszlo Otvos, and Ralf Hoffmann. "Influence of Substitutions in the Binding Motif of Proline-Rich Antimicrobial Peptide ARV-1502 on 70S Ribosome Binding and Antimicrobial Activity." International Journal of Molecular Sciences 23, no. 6 (March 15, 2022): 3150. http://dx.doi.org/10.3390/ijms23063150.
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Proline-rich antimicrobial peptides (PrAMPs) are promising candidates to treat bacterial infections. The designer peptide ARV-1502 exhibits strong antimicrobial effects against Enterobacteriaceae both in vitro and in vivo. Since the inhibitory effects of ARV-1502 reported for the 70 kDa heat-shock protein DnaK do not fully explain the antimicrobial activity of its 176 substituted analogs, we further studied their effect on the bacterial 70S ribosome of Escherichia coli, a known target of PrAMPs. ARV-1502 analogues, substituted in positions 3, 4, and 8 to 12 (underlined) of the binding motif D3KPRPYLPRP12 with aspartic acid, lysine, serine, phenylalanine or leucine, were tested in a competitive fluorescence polarization (FP) binding screening assay using 5(6)-carboxyfluorescein-labeled (Cf-) ARV-1502 and the 70S ribosome isolated from E. coli BW25113. While their effect on ribosomal protein expression was studied for green fluorescent protein (GFP) in a cell-free expression system (in vitro translation), the importance of known PrAMP transporters SbmA and MdtM was investigated using E. coli BW25113 and the corresponding knockout mutants. The dissociation constant (Kd) of 201 ± 16 nmol/L obtained for Cf-ARV-1502 suggests strong binding to the E. coli 70S ribosome. An inhibitory binding assay indicated that the binding site overlaps with those of other PrAMPs including Onc112 and pyrrhocoricin as well as the non-peptidic antibiotics erythromycin and chloramphenicol. All these drugs and drug candidates bind to the exit-tunnel of the 70S ribosome. Substitutions of the C-terminal fragment of the binding motif YLPRP reduced binding. At the same time, inhibition of GFP expression increased with net peptide charge. Interestingly, the MIC values of wild-type and ΔsbmA and ΔmdtM knockout mutants indicated that substitutions in the ribosomal binding motif altered also the bacterial uptake, which was generally improved by incorporation of hydrophobic residues. In conclusion, most substituted ARV-1502 analogs bound weaker to the 70S ribosome than ARV-1502 underlining the importance of the YLPRP binding motif. The weaker ribosomal binding correlated well with decreased antimicrobial activity in vitro. Substituted ARV-1502 analogs with a higher level of hydrophobicity or positive net charge improved the ribosome binding, inhibition of translation, and bacterial uptake.
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TRAHAN, JERRY L., and SUNDARARAJAN VEDANTHAM. "ANALYSIS OF PRAM INSTRUCTION SETS FROM A LOG COST PERSPECTIVE." International Journal of Foundations of Computer Science 05, no. 03n04 (December 1994): 231–46. http://dx.doi.org/10.1142/s0129054194000128.
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The log cost measure has been viewed as a more reasonable method of measuring the time complexity of an algorithm than the unit cost measure. The more widely used unit cost measure becomes unrealistic if the algorithm handles extremely large integers. Parallel machines have not been examined under the log cost measure. In this paper, we investigate the Parallel Random Access Machine under the log cost measure. Let the instruction set of a basic PRAM include addition, subtraction, and Boolean operations. We relate resource-bounded complexity classes of log cost PRAMs to complexity classes of Turing machines and circuits. We also relate log cost PRAMs with different instruction sets by simulations that are much more efficient than possible in the unit cost case. Let LCRCWk(CRCWk) denote the class of languages accepted by a log cost (unit cost) basic CRCW PRAM in O( log k n) time with the polynomial in n number of processors. We position the log cost PRAM in the hierarchy of parallel complexity classes as: ACk=CRCWk⊆(NCk+1, LCRCWk+1)⊆ACk+1=CRCWk+1.
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GEORGIOU, CHRYSSIS, ALEXANDER RUSSELL, and ALEXANDER A. SHVARTSMAN. "FAILURE-SENSITIVE ANALYSIS OF PARALLEL ALGORITHMS WITH CONTROLLED MEMORY ACCESS CONCURRENCY." Parallel Processing Letters 17, no. 02 (June 2007): 153–68. http://dx.doi.org/10.1142/s0129626407002946.
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The abstract problem of using P failure-prone processors to cooperatively update all locations of an N-element shared array is called Write-All. Solutions to Write-All can be used iteratively to construct efficient simulations of PRAM algorithms on failure-prone PRAMS. Such use of Write-All in simulations is abstracted in terms of the iterative Write-All problem. The efficiency of the algorithmic solutions for Write-All and iterative Write-All is measured in terms of work complexity where all processing steps taken by the processors are counted. This paper considers determinitic solutions for the Write-All and iterative Write-All problems in the fail-stop synchronous CRCW PRAM model where memory access concurrency needs to be controlled. A deterministic algorithm of Kanellakis, Michailidis, and Shvartsman [16] efficiently solves the Write-All problem in this model, while controlling read and write memory access concurrency. However it was not shown how the number of processor failures f affects the work efficiency of the algorithm. The results herein give a new analysis of the algorithm [16] that obtain failure-sensitive work bounds, while retaining the known memory access concurrency bounds. Specifically, the new result expresses the work bound as a function of N, Pandf. Another contribution in this paper is the new failure-sensitive analysis for iterative Write-All with controlled memory access concurrency. This result yields tighter bounds on work (vs. [16]) for simulations of PRAM algorithms on fail-stop PRAMS.
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BRUDA, STEFAN D., and YUANQIAO ZHANG. "COLLAPSING THE HIERARCHY OF PARALLEL COMPUTATIONAL MODELS." International Journal of Foundations of Computer Science 21, no. 03 (June 2010): 441–57. http://dx.doi.org/10.1142/s0129054110007350.
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We investigate the computational power of parallel models with directed reconfigurable buses and with shared memory. Based on feasibility considerations present in the literature, we split these models into "heavyweight" (directed reconfigurable buses, the Combining PRAM, and the BSR) and "lightweight" (all the other PRAMs) and then find that the heavyweight class is strictly more powerful than the lightweight class, as expected. On the other hand, we contradict the long held belief that the heavyweight models form a hierarchy, showing that all of them are identical in computational power with each other. We start the process by showing that the Collision write conflict resolution rule is universal on models with reconfigurable buses (in the sense that complex conflict resolution rules such as Priority and Combining can be simulated with constant overhead by Collision).
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Ulla, Bente. "Reconceptualising sleep: Relational principles inside and outside the pram." Contemporary Issues in Early Childhood 18, no. 4 (December 2017): 400–408. http://dx.doi.org/10.1177/1463949117742781.
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This article explores sleep among kindergarten infants and toddlers. Although the collective order of sleep in kindergarten makes it a relational issue, the search here is for relations that extend beyond human actors and beyond the idea of the pram as a sleep container used by a sleeping subject. Here, sleep is seen as entangled with bodies and prams; it has a rhythm and a tempo, as well as the power to challenge the capitalist call for productivity. The article addresses sleep in terms of spatial configurations and contextualises it within a web of political relations rather than as a leftover of life. Informed by Foucault’s notions of heterotopia, the article characterises sleep as a world within a world, drawing attention to relational principles and material-discursive spaces that are characterised as ‘different’, on the understanding that sleep is not an intermission from life or relationships. Moving beyond the conceptualisation of sleep as a health and medical issue, it is reframed as embodied and embedded, enabling exploration of sleep in kindergarten as relational.
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Greiner, Jochen, Lars Bullinger, Krzysztof Giannopoulos, Anita Schmitt, Marlies Goetz, Lena Kienle, Hartmut Döhner, and Michael Schmitt. "The Leukemia-Associated Antigen PRAME Is Overexpressed in Myeloid Leukemias and Inhibits Cell Differentiation by Blocking the Receptor for Retinoic Acid (RAR)-Signaling in Vitro and Is Therefore a Interesting Candidate for Targeted Immunotherapies." Blood 112, no. 11 (November 16, 2008): 1524. http://dx.doi.org/10.1182/blood.v112.11.1524.1524.
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Abstract The leukemia-associated antigen PRAME (preferentially expressed antigen of melanoma) is frequently expressed in several solid tumors. Earlier, we reported (Greiner et al., Blood 2006) that co-expression of leukemia-associated antigens including PRAME constituted a favorable prognostic parameter for AML patients. By microassay analysis and conventional RT-PCR, we detected over-expression of PRAME in more than 60% of AML and of CML patients. In contrast, PRAME is expressed neither in normal CD34-positive hematopoietic stem cells nor in normal tissues (other than testis and placenta). Here, we describe for the first time that retinoic acid-induced cell proliferation and differentiation of several AML cell lines is dependent on PRAME expression: PRAME negative cell lines treated with all-trans retinoic acid (ATRA) in cell culture showed lower cell proliferation than PRAME positive cells as assessed by cell counts and FACS analysis for BrdU, but an increase of cell differentiation detected by FACS analysis for CD66b in contrast with PRAME positive leukemia cell lines. The leukemia-associated antigen PRAME seems to be responsible for the resistance of PRAME-positive AML to ATRA treatment. We detected no differences in induction of apoptosis in PRAME-positive or PRAME-negative cell lines treated with ATRA in FACS analysis for annexin. Over-expression of the antigen and this critical role of PRAME in cell differentiation might be the reason for specific T cell induction against PRAME-derived peptides. To detect specific T cell responses against PRAME-derived peptides we examined samples from healthy volunteers and AML patients. Positive results in ELISPOT assays for IFN-g and Granzyme B secretion were observed in 70% of AML cases analyzed for the peptide PRAME-P3. In chromium release assays, we found a PRAME-specific cell lysis of PRAME-positive AML blasts. Taken together, PRAME is involved in a crucial mechanism for cell growth of leukemic cells, induces specific T cell responses in a high frequency of AML patients, and this constitutes an appropriate target structure for specific immunotherapies or other targeted therapies in AML.
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Hoang, Tsvetalina, Aaron Foster, Jeannette Crisostomo, An Lu, Annemarie Moseley, Mirjam H. M. Heemskerk, Kevin M. Slawin, and David M. Spencer. "Inducible MyD88/CD40 Enhances Proliferation and Survival of PRAME-Specific TCR-Engineered T Cells and Increases Anti-Tumor Effects in Myeloma." Blood 126, no. 23 (December 3, 2015): 1886. http://dx.doi.org/10.1182/blood.v126.23.1886.1886.
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Abstract Introduction: Use of T cells engineered to express antigen-specific T cell receptors (TCRs) has shown promise as a cancer immunotherapy treatment; however, durable responses have been limited by poor T cell persistence and expansion in vivo. Additionally, MHC class I downregulation on tumor cells weakens T cell recognition, further reducing therapeutic efficacy. To address these deficiencies, we co-expressed in human T cells a novel, small molecule (rimiducid)-dependent T cell activation switch, inducible MyD88/CD40 (iMC), along with PRAME-specific TCR to allow control of T cell expansion and activation, while upregulating MHC class I expression on tumor cells. Methods: Human T cells were activated with anti-CD3/CD28 and transduced with retrovirus encoding TCR α and β chains recognizing PRAME-derived, HLA-A*201-restricted peptide SLLQHLIGL (SFG-PRAME) or a polycistronic vector encoding the PRAME-specific TCR along with tandem rimiducid (AP1903)-binding domains (FKBP12v36) cloned in-frame with MyD88 and CD40 signaling domains (SFG-iMC-PRAME). Proliferation, cytokine production and cytotoxicity of modified T cells was assessed using peptide-pulsed T2 cells or against PRAME-expressing, HLA-A2+ U266 myeloma tumor cells with or without rimiducid (10 nM) stimulation. MHC class I expression on tumor cells was measured by flow cytometry using a transwell assay. In vitro tumor killing was analyzed using T cell and tumor coculture assays with various effector to target ratios over a 7-day period. In vivo efficacy was determined using immune-deficient NSG mice engrafted s.c. with U266 cells and treated i.v. with 1x107 transduced T cells. iMC was activated in vivo by weekly i.p. injections of 5 mg/kg rimiducid. Tumor size and T cell expansion was measured using in vivo luciferase bioluminescence imaging and flow cytometric phenotyping. Results: Both PRAME and iMC-PRAME retroviral vectors efficiently transduced activated human T cells (81±2.1% and 89±2.8%, respectively) and showed antigen-specific IFN-g production and cytolytic function against peptide-pulsed T2 cells and PRAME+ U266 myeloma cells. However, both TCR ligation and rimiducid-dependent costimulation were required for IL-2 production (PRAME, 217±256 pg/ml; iMC-PRAME, 23±56 pg/ml; iMC-PRAME + rimiducid, 5417±2599 pg/ml) against peptide-pulsed T2 cells. Coculture assays against PRAME-expressing U266 myeloma cells showed that tumor elimination was optimized with concurrent rimiducid-driven iMC activation, and this effect was accompanied by increased IL-2 secretion and robust T cell proliferation (PRAME, 0.18-fold; iMC-PRAME, 0.28-fold; iMC-PRAME + rimiducid, 7.7-fold). Further, iMC activation produced IFN-g independently of TCR ligation, which significantly increased MHC class I expression on tumor cells (no T cells, 61±3 MFI; PRAME, 1256±493 MFI; iMC-PRAME, 6747±656 MFI; iMC-PRAME + rimiducid, 9096±1583 MFI). In NSG mice engrafted with PRAME+ U266 myeloma tumors, PRAME TCR-modified T cells showed significant tumor control compared to non-transduced control T cells (p-value = 0.01, 0.01 and 0.0001 for PRAME, iMC-PRAME and iMC-PRAME + rimiducid, respectively) and rimiducid activation of iMC-PRAME-modified T cells showed significant tumor control compared to T cells transduced with only the PRAME TCR (p = 0.005). Importantly, weekly injections of rimiducid dramatically expanded PRAME TCR-expressing T cell numbers by 473-fold 4 weeks post-injection compared to T cells expressing the PRAME TCR only (p = 0.02). Summary: iMC is a novel "Go" switch that utilizes rimiducid, a small molecule dimerizer, to drive activation and expansion of PRAME-specific TCR-engineered T cells while sensitizing tumor to TCR-mediated recognition by upregulating MHC class I via IFN-g, thereby increasing antitumor efficacy and durability. Thus, iMC-PRAME is the prototype of a class of novel "Go-TCR" engineered T cell therapies that may increase efficacy, safety and durability of adoptive T cell therapies. Disclosures Hoang: Bellicum Pharmaceuticals: Employment. Foster:Bellicum Pharmaceuticals: Employment. Crisostomo:Bellicum Pharmaceuticals: Employment. Lu:Bellicum Pharmaceuticals: Employment. Moseley:Bellicum Pharmaceuticals: Employment, Equity Ownership. Slawin:Bellicum Pharmaceuticals: Employment, Equity Ownership. Spencer:Bellicum Pharmaceuticals: Employment, Equity Ownership.
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Zhu, Hanzhang, Jingrui Wang, Junjie Yin, Bei Lu, Qijun Yang, Yafeng Wan, and Changku Jia. "Downregulation of PRAME Suppresses Proliferation and Promotes Apoptosis in Hepatocellular Carcinoma Through the Activation of P53 Mediated Pathway." Cellular Physiology and Biochemistry 45, no. 3 (2018): 1121–35. http://dx.doi.org/10.1159/000487353.
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Background/Aims: The expression of PRAME and its role in hepatocellular carcinoma (HCC) remain unknown. The aim of this study was to examine the functional role of PRAME in HCC development and exploring the molecular mechanism. Methods: We first detected PRAME expression in 96 human HCC tissue samples and correlated with clinicopathological characteristics and prognosis of the patients. We then established stable HCC cell lines with PRAME overexpression and knockdown followed by functional analysis in vitro. Further, we examined the relationship between PRAME and p53 pathway in vitro by using Western blotting. Finally, PRAME expression was detected to evaluate its correlation with p-p53 and p53 pathway related apoptotic proteins in xenograft tumor mouse model using immunohistochemistry. Results: PRAME expression was significantly higher in HCC tissues than in adjacent non-tumor tissues and their expression was positively correlated with alpha fetoprotein levels and tumor size. In addition, PRAME expression was associated with AJCC stage and is a potential biomarker of poor prognosis regarding 5-year overall survival in HCC. In vitro studies, we found that PRAME expression was higher in HCC cell lines than in normal hepatic cell line. Inhibited cell proliferation and increased cell apoptosis was observed in PRAME knockdown HCC cells. Futher, increased cell apoptosis was correlated with the proportion of cells in G0/G1 stage, activated p53 mediated apoptosis, and increased cyclin p21 expression. Xenograft analysis in nude mice also found that PRAME knockdown inhibited tumorigenesis while PRAME overexpression had opposite effect. Conclusions: In HCC, PRAME serves as a potential biomarker for poor prognosis and novel therapeutic target in treating this cancer. PRAME is a potential biomarker of poor prognosis in HCC. PRAME surpresses HCC cell death in vitro and in vivo by regulating p53 apoptotic signaling and may serve as a potential therapeutic target in HCC.
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Finashutina, Yu P., N. A. Lyzhko, N. N. Kasatkina, L. A. Kesaeva, V. V. Tikhonova, V. A. Misyurin, M. A. Baryshnikova, and A. V. Misyurin. "Recombinant human PRAME immunization reducesPRAME-expressing tumor growth in mice." Russian Journal of Biotherapy 17, no. 3 (November 25, 2018): 36–42. http://dx.doi.org/10.17650/1726-9784-2018-17-3-36-42.
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Intrоduction.Human antigen PRAME is preferentially expressed in a number of different tumor types and may be a potent target for anti-tumor immunotherapy.Purpose.To study anti-tumor action of immunogenic mix recombinant PRAME protein and adjuvant in mice with innate immunity.Materials and methods.C57BL/6 female mice were used for immunization with purified human recombinant protein PRAME. Human PRAME gene coding sequence was cloned in mammalian expressing vector pCEP4 and resulting plasmid was introduced in mouse melanoma B16F10 cells by transfection followed by RQ-PCR, Western blot and flow-cytometry analysis. Then stably PRAME-transfected melanoma cells were injected in mice.Results.The mouse melanoma B16F10 cells stably expressing human PRAME protein were obtained. We demonstrate the 10-fold decreased tumor volume in mice with melanoma B16F10 expressing human PRAME after preventive immunization series with recombinant PRAME protein. The tumor volume reducing was correlated with high titer (6.14 × 10 5) of anti-PRAME antibodies in mice sera.Conclusion.These data indicate that recombinant protein PRAME is immunogenic and may be a potent antigen for immunotherapuetics studies.
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Oehler, Vivian, Carrie Cummings, Kathleen Sabo, Brent Wood, Katherine Guthrie, Ted Gooley, Era Pogosova-Agadjanyan, et al. "Preferentially Expressed Antigen in Melanoma (PRAME) Expression in Normal and CML CD34+ Progenitor Cells Impairs Myeloid Differentiation." Blood 112, no. 11 (November 16, 2008): 1071. http://dx.doi.org/10.1182/blood.v112.11.1071.1071.
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Abstract PRAME, or the Preferentially Expressed Antigen in Melanoma, is an attractive therapeutic target in cancer treatment. It has been described as a tumor-associated antigen and more recently as a co-repressor of retinoic acid signaling in the presence of all-trans retinoic acid (ATRA) in tumor cell lines. However, the function of PRAME in hematopoietic cells remains unclear. We and others have reported that while PRAME mRNA expression is absent or low in CD34+ hematopoietic progenitors, it is heterogeneously over-expressed in a number of hematologic malignancies including acute myeloid leukemia (AML) and chronic myeloid leukemia (CML). In our previously published microarray studies, PRAME was one of the most statistically significantly differentially expressed genes with CML progression from chronic phase (CP) to blast crisis (BC). In independent validation studies PRAME was expressed in all but on BC patient and was expressed at only very low levels in 50% of CP patients. We then hypothesized that PRAME expression in diagnostic CP CML patients may be a marker of more advanced disease, and may therefore predict poorer response to therapy. Thirty-nine patients with a good response to imatinib mesylate (IM) were compared to 17 patients who progressed or who achieved suboptimal cytogenetic or molecular responses on IM. There was a trend towards increased PRAME expression in the poor responders. The median PRAME expression was 86.3 copies in poor responders vs. no expression in the remaining patients. In order to examine PRAME function in primary hematopoietic cells PRAME was aberrantly expressed in normal CD34+ selected mobilized peripheral blood cells from healthy donors and silenced in primary CD34+ cells from a CML patient in myeloid blast crisis. Forced expression of PRAME in normal cells in three independent experiments resulted in impaired myeloid differentiation. PRAME cells demonstrated increased CD34, CD117, and decreased CD11b expression in culture over time. The ability of progenitors to form CFU-GM and CFU-G colonies was also significantly decreased in PRAME expressing cells. Myeloid differentiation was also inhibited in PRAME expressing cells exposed to ATRA. Correspondingly, when PRAME was silenced in primary CML CD34+ cells, proliferation was increased and PRAME silenced cells demonstrated increased CFU-G and CFU-GM formation both in the presence and absence of ATRA. Microarray data comparing gene expression profiles in PRAME expressing primary progenitors vs. control cells suggested that EZH2 may mediate PRAME effects. The polycomb group protein EZH2 has been described as mediating the repressive effects of PRAME on RARA signaling in the presence of ATRA in solid tumor cells lines. PRAME effects on myeloid differentiation in the presence of ATRA, however, are cell type and context dependent. PRAME function was also examined in CML and AML cell lines. PRAME is highly expressed in the CML cell line K562, which is capable of erythroid differentiation. PRAME is expressed at much lower levels in the AML cell line HL60. Cells from the HL60 cell line can be differentiated into neutrophils after exposure to ATRA. PRAME expressing HL60 cells, as compared to control cells, exhibited decreased CD11b expression after ATRA exposure. In comparison, differentiation of K562 cells was not affected by PRAME silencing. A K562 cell line which expresses an increased number of RARA receptors (equal to HL60 cells) was also examined, but PRAME silencing in these cells had no effect on differentiation. In conclusion, PRAME is expressed primarily in advanced phase CML and its expression at diagnosis in CP disease is associated with poorer treatment response to IM. PRAME expression in normal CD34+ progenitors inhibited myeloid differentiation at the promyelocyte/myelocyte level, and this phenotype was reversed when PRAME was silenced in CML progenitor cells. PRAME function, however, is dependent on lineage commitment. It blocks differentiation in a retinoic acid dependent manner only in cells with myeloid differentiation potential. These data suggest that increasing PRAME expression as patients progress from CP to myeloid BC plays a role in inhibiting myeloid differentiation, but that other genetic events, such as BCR-ABL, are needed to drive proliferation in these cells.
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Rezvani, Katayoun, Agnes S. M. Yong, Abdul Tawab, Behnam Jafarpour, Rhoda Eniafe, Stephan Mielke, Bipin N. Savani, et al. "Ex vivo characterization of polyclonal memory CD8+ T-cell responses to PRAME-specific peptides in patients with acute lymphoblastic leukemia and acute and chronic myeloid leukemia." Blood 113, no. 10 (March 5, 2009): 2245–55. http://dx.doi.org/10.1182/blood-2008-03-144071.
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Abstract Preferentially expressed antigen of melanoma (PRAME) is aberrantly expressed in hematologic malignancies and may be a useful target for immunotherapy in leukemia. To determine whether PRAME is naturally immunogenic, we studied CD8+ T-cell responses to 4 HLA-A*0201–restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML), and healthy donors. CD8+ T cells recognizing PRAME peptides could be detected ex vivo in 4 of 10 ALL, 6 of 10 AML, 3 of 10 CML patients, and 3 of 10 donors by HLA-A2 tetramer analysis and flow cytometry for intracellular interferon-γ. The frequency of PRAME-specific CD8+ T cells was greater in patients with AML, CML, and ALL than healthy controls. All peptides were immunogenic in patients, while responses were only detected to PRA300 in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to greater than or equal to 2 PRAME epitopes compared with low PRAME expression levels (4/7 vs 0/23, P = .001), suggesting a PRAME-driven T-cell response. PRAME-specific T cells were readily expanded in short-term cultures in donors and patients. These results provide evidence for spontaneous T cell reactivity against multiple epitopes of PRAME in ALL, AML, and CML. The potential for developing PRAME as a target for immunotherapy in leukemia deserves further exploration.
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Takata, Katsuyoshi, Lauren C. Chong, Avinash Thakur, Tomohiro Aoki, Anja Mottok, Elizabeth Chavez, Pedro Farinha, et al. "PRAME Expression Is Correlated with Treatment Outcome and Specific Features of the Tumor Microenvironment in Classical Hodgkin Lymphoma." Blood 134, Supplement_1 (November 13, 2019): 1509. http://dx.doi.org/10.1182/blood-2019-129730.
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Background: The tumor-associated antigen PRAME is over-expressed in several types of cancer and is currently investigated as a therapeutic target for T-cell immunotherapy. Our previous integrative genomic study in diffuse large B-cell lymphoma (DLBCL) identified PRAME deletion to be correlated with patient outcome and an immunologically "cold" tumor microenvironment. However, it remains an open question whether PRAME expression significantly contributes to differential treatment outcomes and tumor microenvironment crosstalk across various B-cell lymphoma subtypes. Material and Methods: We performed an immunohistochemical (IHC) screen in a large cohort of B-cell lymphomas (de novo DLBCL; N=347, follicular lymphoma (FL); N= 166, mantle cell lymphoma (MCL); N= 180), and classical Hodgkin lymphoma (HL); N= 166) to assess PRAME expression as a prognostic biomarker. Moreover, to investigate PRAME-expression associated tumor microenvironment composition and function, we correlated PRAME IHC results with single cell RNA sequencing data of more than 127,000 cells from 22 HL tissue specimens. Results: PRAME IHC analysis revealed frequent PRAME over-expression in HL (115/166, 69%), followed by DLBCL (104/319, 33%), FL (13/166, 8%), and MCL (14/180, 8%). Interestingly, only HL showed a significant treatment outcome correlation, whereas other B-cell lymphoma subtypes did not. Specifically, using a previously published HL cohort (Steidl et al, NEJM 2010) PRAME-negative Hodgkin Reed Sternberg (HRS) cells indicated significantly shorter overall survival (P = 0.008) and disease-specific survival (P = 0.042 ). To characterize PRAME-specific microenvironment composition and function in HL, we analyzed T-, B-, NK-cell, and macrophage subsets in PRAME-positive (17 of 22 cases) vs -negative (5 of 22 cases) tumor samples using single cell RNA sequencing data. From 22 expression-based microenvironment cell clusters that were annotated and assigned to a cell type based on gene expression, all three CD4 helper T-cell clusters were de-enriched in PRAME-negative samples, and the CD4 non-Treg proportion was significantly lower in PRAME-negative samples (P = 0.049). Strikingly, when focusing on phenotypic features of cells within the CD4 non-Treg T-cell cluster, CXCL13 was identified as the most up-regulated gene in PRAME-negative samples. When interrogating published HRS cell transcriptome data (Steidl et al, Blood 2012), immune response pathways including chemokine receptors and chemokine ligands were up-regulated in PRAME-negative HRS cell samples. Of specific interest, CXCR5, the cognate receptor for CXCL13, was significantly upregulated as a member of the chemokine pathway (P = 0.0086) in PRAME-negative HRS cell samples. These results suggest that crosstalk between CXCL13 (produced in the microenvironment) and CXCR5 (expressed on HRS cells) contributes to tumor maintenance in PRAME-negative HL. Finally, to explore potential therapeutic approaches for PRAME-negative HL cells, we focused on 3 HL-derived cell lines (L540, L591, DEV) with low PRAME expression and exposed these lines to DNMT or HDAC inhibitors. DNMT inhibitor treatment showed clear restoration of PRAME expression in a dose dependent manner, but no restoration was found by HDAC inhibitor treatment. To investigate the effect of DNA methylation in transcriptional regulation of PRAME in HL cells, we performed bisulfite sequencing in the PRAME CpG promoter region in PRAME down-regulated (L540, L591, DEV) and up-regulated (HD-LM2, KMH-2, L1236) cell lines and found hypermethylation in PRAME low vs high cell lines. Moreover, the CpG promoter region was significantly demethylated by DNMT inhibitor treatment in cell lines with low PRAME expression. Conclusion: We discovered that PRAME protein expression was correlated with outcome in HL and identified specific T-cell subsets in PRAME-negative patients. PRAME restoration by DNMT inhibitors might represent a new therapeutic avenue in combination with modern immunotherapies, such as PRAME-specific T-cell therapy or PD1 inhibition. Disclosures Scott: Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding; Celgene: Consultancy. Steidl:Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Bristol-Myers Squibb: Research Funding; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.
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de Figueiredo-Pontes, Lorena L., Fabio M. do Nascimento, Rodrigo S. de Abreu e Lima, Rodrigo Proto-Siqueira, Aglair B. Garcia, Daniel M. Matos, Ivan T. Gout, et al. "Analysis of PRAME Protein Expression in Normal Lymphoid Tissues and during B Ontogenesis." Blood 106, no. 11 (November 16, 2005): 3924. http://dx.doi.org/10.1182/blood.v106.11.3924.3924.
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Abstract PRAME (Preferentially Expressed Antigen in Melanoma) gene was originally isolated in melanoma. A significant increase in the number of PRAME transcripts has been demonstrated in hematologic malignancies such as acute myeloid and lymphoid leukemias, multiple myeloma and chronic lymphoproliferative diseases. Furthermore, our group generated an anti-PRAME monoclonal antibody (MoAb) and by quantitative flow cytometry has demonstrated that PRAME protein was aberrantly expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma. However, the expression of this antigen in normal lymphoid tissues and during B cells ontogeneis has not been characterized. To address this question, PRAME protein expression was studied by flow cytometry in peripheral blood (PB, n=15) and bone marrow (BM, n=6) from healthy donors, lymphonodes (n=4) and spleen (n=4) from patients submitted to lymphonode excision or splenectomy for non malignant diseases. First, we determined in which hematopoietic lineage PRAME was expressed by concomitantly staining PB, BM, lymphonode and spleen mononuclear cells (MCs) with anti-PRAME and a panel of MoAbs specific to B(CD19)/ T(CD3)/ NK(CD16/56), monocytic(CD14) and granulocytic(CD33) markers. PRAME was detected exclusively in CD19+ cells. The median percenatge of PRAME positive cells was 5,31% (2,55–12,34%), 13,01% (8,47–38,15%), 12,79% (3,15–23,06%) and 17,5% (12,67–27,43%) in PB, BM, lymphonode and spleen MCs, respectively. Amongst CD19+ cells, we have observed that PRAME was expressed by 42,39% (16,16–75,72%), 16% (13–69,5%), 15,16% (5,49–41,20%) and 48,82%(12,67–58,89%) in PB, BM, lymphonode and spleen, respectively. To establish in which stage of B ontogenesis PRAME was expressed on, cell suspensions stained with anti-CD19 were submitted to positive magnetic separation and labeled with anti-PRAME, CD5, CD27, CD38, CD34, CD10 and IgD MoAbs. PRAME+/CD19+ cells were CD5−, CD27+, CD38+, CD34−, CD10− and IgD+, thus suggesting that PRAME is expressed by the memory B cell compartment of the normal lymphoid tissues. This study defines PRAME as a B cell antigen that may accompany the neoplastic clone proliferation of mature B cell neoplasms. Although PRAME is mainly an embryonic antigen, expressed by carcinomas of immature phenotype, it is expressed by mature B cells in normal and pathological lymphoid tissues. Our findings suggest that maturational events occurring at the germinal center of lymphoid follicles affects PRAME expression.
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Rezvani, Katayoun, Agnes S. M. Yong, Abdul Tawab, Behnam Jafarpour, Rhoda Eniafe, Stephan Mielke, Bipin N. Savani, et al. "Ex-Vivo Characterization of Polyclonal Memory CD8+ T-Cell Responses to PRAME-Specific Peptides in Patients with Acute Leukemia and Chronic Myeloid Leukemia." Blood 112, no. 11 (November 16, 2008): 2910. http://dx.doi.org/10.1182/blood.v112.11.2910.2910.
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Abstract PRAME (Preferentially expressed antigen of melanoma) is aberrantly expressed in hematological malignancies and may be a useful target for immunotherapy in leukemia. We studied CD8+ T-cell responses to four HLA-A*0201-restricted PRAME-derived epitopes (PRA100, PRA142, PRA300, PRA425) in HLA-A*0201-positive patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic myeloid leukemia (CML) and healthy donors, using PRA300/HLA-A*0201 tetramer staining, intracellular cytokine (IC) assay and ex-vivo and cultured ELISPOT analysis. CD8+ T-cells recognizing PRAME peptides were detected directly ex-vivo in 4/10 ALL, 6/10 AML, 3/10 CML patients and 3/10 donors. The frequency of PRAME-specific CD8+ T-cells was greater in patients with AML, CML and ALL than in healthy controls. All peptides were immunogenic in patients, whilst PRA300 was the only immunogenic peptide in donors. High PRAME expression in patient peripheral blood mononuclear cells was associated with responses to two or more PRAME epitopes (4/7 vs. 0/23 in individuals with low PRAME expression, P = 0.001), suggesting a PRAME-driven T-cell response. In 2 patients studied PRA300/HLA-A*0201+ CD8+T-cells were found to be a mixture of effector and central memory phenotypes. To determine the functional avidity of the PRAME T-cell response, the response of CD8+ T-cells to stimulation with 2 concentrations of peptide was measured by IC-IFN-γ staining. High-avidity CD8+ T-cells were defined as those capable of producing IFN-γ in response to the lower concentration of peptide (0.1μM), while low-avidity CD8+ T-cells were those that only produced IFN-γ in response to the higher concentration of peptide (10 μM). Both high and low-avidity CD8+ T-cell responses could be detected for all peptides tested (median 1.05, 0.90, 0.52, 0.40 high/lowavidity ratios for PRA100, PRA142, PRA300 and PRA425 respectively). In patients with high PRAME expression (>0.001 PRAME/ABL) low-avidity CD8+ T-cell responses to PRAME peptides were more prominent than high-avidity responses, suggesting selective deletion of high-avidity T-cells. In contrast, in some patients with levels <0.001 PRAME/ABL, we could detect the presence of high-avidity CD8+ T-cell responses to PRAME. PRAME-specific CD8+ T-cells were further characterized by IC staining for IL-2, IL-4 and IL-10 production and CD107a mobilization (as a marker of cytotoxicity). Following stimulation with the relevant PRAME peptide, there was no significant production of IL-2, IL-4 or IL-10, suggesting a Tc1 effector response but no significant CD107a mobilization was detected despite significant CD107a mobilization in the same patient in response to CMVpp65495. This finding suggests that patients with leukemia have a selective functional impairment of PRAME-specific CD8+ T-cells, consistent with PRAME-specific T cell exhaustion. However, PRAME-specific T-cells were readily expanded in the presence of cytokines in short-term cultures in-vitro to produce IFN-γ, suggesting that it may be possible to improve the functional capacity of PRAME-specific T-cells for therapeutic purposes. These results provide evidence for spontaneous T-cell reactivity against multiple epitopes of PRAME in ALL, AML and CML and support the usefulness of PRAME as a target for immunotherapy in leukemia. The predominance of low-avidity PRAME-specific CD8+ T-cells suggests that achievement of a state of minimal residual disease may be required prior to peptide vaccination to augment T-cell immune surveillance.
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Ercolak, Vehbi, Semra Paydas, Emine Bagir, Melek Ergin, Gulsah Seydaoglu, Hikmet Celik, Basak Yavuz, et al. "PRAME Expression and Its Clinical Relevance in Hodgkin's Lymphoma." Acta Haematologica 134, no. 4 (2015): 199–207. http://dx.doi.org/10.1159/000381533.
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Objectives: Although Hodgkin's lymphoma (HL) is one of the most curable cancers in adult patients, new targets have to be defined in cases resistant to traditional chemotherapy. The preferentially expressed antigen of melanoma (PRAME) is a cancer testis antigen and its expression is very scarce or absent in normal tissues. For this reason PRAME is a promising candidate for tumor immunotherapy. The aim of this study is to understand the correlation of PRAME expression with prognostic factors in HL, to determine the utility of PRAME as a targeted molecule for immunotherapy and to compare real-time polymerase chain reaction (real-time PCR) and immunohistochemistry (IHC) for the detection of PRAME. Methods: In 82 patients, PRAME was studied using real-time PCR and IHC. Data analyses were performed using statistical methods such as t test, Mann-Whitney U test, χ2 test, Kaplan-Meier method, log-rank test and Cox regression analysis. Results: PRAME was detected in 15 (18.3%) patients using IHC and in 8 (9.8%) patients using real-time PCR. A correlation was found between PRAME positivity and higher International Prognostic Score (p = 0.039). PRAME positivity detected using real-time PCR was found to be correlated with shorter disease-free survival (DFS) and overall survival (OS, p = 0.0005). Discussion: The demonstration of PRAME especially in histiocytes and Reed-Sternberg cells may provide guidance for immunotherapy. Although PRAME positivity increases the risk for death (3.56), independent risk factors that affected DFS and OS occurred in advanced age and high-risk groups. Conclusion: Although real-time PCR is sensitive in the detection of PRAME, IHC can be another useful method. Despite the need for studies conducted on larger patient samples, PRAME expression is considered as a poor prognostic parameter in HL.
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Chen, Xin, Mengying Jiang, Shengjie Zhou, Hong Chen, Gendi Song, Yichen Wu, and Xueqiong Zhu. "PRAME Promotes Cervical Cancer Proliferation and Migration via Wnt/β-Catenin Pathway Regulation." Cancers 15, no. 6 (March 16, 2023): 1801. http://dx.doi.org/10.3390/cancers15061801.
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A significant burden is placed on the lives of females due to cervical cancer, which is currently the leading cause of cancer death among women. Preferentially expressed antigen in melanoma (PRAME) belongs to the CTA gene family and was found to be abnormally expressed among different types of cancers. Our previous research also indicated that PRAME was highly expressed in cervical cancer compared with normal tissues. However, the roles and detailed mechanisms of PRAME have not been explored in cervical cancer. In the present study, the expression of PRAME in cervical tissues and cells was detected by immunohistochemistry (IHC), qRT-PCR, and Western blotting. Additionally, CCK-8, BrdU, scratch, transwell, and flow cytometry assays were conducted to explore the function of PRAME in regulating the malignant biological behaviors of cervical cancer cells. Nude mice were used to confirm the role of PRAME in tumor growth in vivo. Furthermore, the Wnt inhibitor MSAB was used to verify the role of PRAME in regulating the Wnt/β-catenin pathway both in vitro and in vivo. The results of IHC, qRT-PCR, and Western blotting showed that PRAME was highly expressed in cervical cancer tissues and cells. PRAME knockdown attenuated cell growth, migration, and invasion; induced G0/G1 arrest; and increased cell apoptosis in C33A and SiHa cells through Wnt/β-catenin signaling regulation. However, the upregulation of PRAME exhibited the opposite effects accordingly, which could be partly reversed via MSAB treatment. The growth rate of xenograft tumors was enhanced when PRAME was overexpressed via Wnt/β-catenin signaling activation. Taken together, PRAME is associated with cervical cancer occurrence and progression mediated by Wnt/β-catenin signaling, suggesting that PRAME might be a factor in manipulating cervical carcinogenesis and a potential therapeutic target.
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Shiseki, Masayuki, Mayuko Ishii, Kenjiro Mitsuhashi, Norina Tanaka, Kentaro Yoshinaga, Naoki Mori, and Junji Tanaka. "Prognostic Implications Of PRAME Expression Levels In Myelodysplastic Syndromes." Blood 122, no. 21 (November 15, 2013): 2814. http://dx.doi.org/10.1182/blood.v122.21.2814.2814.
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Abstract The preferentially expressed antigen of the melanoma (PRAME) gene was first identified in melanoma tissue as a tumour-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. While expression level of PRAME is quite low in most of normal tissues of human, including bone marrow normal CD34+ cells, except for testis, PRAME overexpression is observed in various human cancers, including hematological malignant disorders. Although clinical and biological significance of PRAME expression in human cancers has not been fully elucidated, it has been demonstrated that high PRAME expression is associated poor clinical outcomes in a number of solid cancers. Association between PRAME expression and clinical outcomes in hematological malignancies has not been clarified. In acute myeloid leukemia, high expression of PRAME was shown to be associated with worse progression-free survival or overall survival by some research groups but not by other groups. Our previous study demonstrated that inhibition of PRAME expression in leukemic cells results in cell cycle arrest and induction of apoptosis, suggesting oncogenic function of PRAME expression in leukemogenesis. Clinical significance of PRAME expression in myelodysplastic syndromes (MDS) also has not been fully elucidated. In the present study, we examined PRAME expression of bone marrow cells in MDS patients to clarify clinical significance of PRAME expression. Bone marrow samples of MDS patients were used for analysis. Samples were taken at the time of diagnosis with written informed consent from patients. Total RNA extraction, cDNA synthesis and quantitative real-time RT-PCR by the TaqMan probe method using an ABI 7500 real-time PCR system (Applied Biosystems) with co-amplification of the endogenous control gene, human GAPDH (Applied Biosystems), were performed. Expression levels were obtained using the standard curve method in each experiment, after normalization with the GAPDH gene for each sample in duplicate wells. The human PRAME primer-probe sets were from Applied Biosystems (assay ID: Hs00196132_m1). Data including patients’ demographic, disease status, medical history, clinical and laboratory findings, and outcome, were collected from medical records and laboratory data base. A total of 111 MDS patients, 68 males and 43 females with median age of 69 years (range: 20-91 years) were included in the present study. They were classified as RCUD (n=15), RCMD (n=61), RARS (n=8), RAEB-1 (n=15), and RAEB-2 (n=12) according to WHO classification. Based on the IPSS, they were categorized in four risk groups, low risk (n=29), intermediate-1 risk (n=55), intermediate-2 risk (n=23), and high risk(n=4). Expression level of PRAME was varied among the MDS patients analyzed. Median value of relative PRAME expression level of 111 MDS patients and that of 19 control subjects were 0.073 and 0.07, and there was no significant difference in distribution of expression level of PRAME. However, when we compared expression patterns of PRAME among five WHO-subtypes, statistical difference was observed (P=0.0116). Relative PRAME expression level in WHO-subtypes with high blast counts (RAEB-1 and RAEB-2) was significantly higher than that in WHO-subtypes with less blast counts (RCUD, RCMD, RARS) (median value: 0.44 vs. 0.05, P=0.0012). To investigate prognostic implication of PRAME expression in MDS, we analyzed impact of PRAME expression on overall survival (OS). Based on PRAME expression level, 111 patients were divided into two categories, ‘high expression group’ (above median value) and ‘low expression group’ (below median value). Kaplan-Meier analysis demonstrated that high expression group showed significantly poorer overall survival than low expression group (P=0.0165). The estimated 5-year OS rates in high expression group and low expression group were 63.5% and 78.4%, respectively. The present study demonstrated that high PRAME expression is associated with poorer clinical outcome, indicating that PRAME expression could be a useful prognostic marker in MDS. Biological significance of PRAME expression in MDS is unclear. Expression level of PRAME was higher in WHO-subtypes with high blasts counts, suggesting that PRAME may play role in disease progression in MDS. Further studies should be necessary to clarify clinicopathological and biological significance of PRAME expression in MDS. Disclosures: No relevant conflicts of interest to declare.
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आचार्यः Acharya, पुरुषोत्तम Purushottam. "प्रमास्वरूपं तद्भेदाश्च [Pramaswaroopam tadbhedaashcha]." Haimaprabha 20 (July 30, 2021): 46–58. http://dx.doi.org/10.3126/haimaprabha.v20i0.38610.
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नेपाली भाषामा सारंश लेख संगै उपलब्ध छ [Nepali language abstracts available with article] [The presented article includes an in-depth discussion of Prama form the perspective of NyayaDarshan and VaisheshikDarshan in Eastern Philosophy. In the process of differentiating the form and aspects of Prama, NyayaDarshan mentions major four types of Prama; Pratyaksha, Anumiti, Upamiti, and Shaabda while VaisheshikDarshan mentions two types of Prama; Pratyakshaand Anumiti. This paper has been prepared following the existing theoretical bases and qualitative research methodology. Article concludes TadwatiTatprakarkaanubhava is itself Prama and in-depth discussion of Prama as the form of true knowledge has been mentioned. From the perspective of VaisheshikDarshan, Upamiti and ShaabaPramahavebeen assumed as the Anumiti and integrated within Anuman. This article is expected to be useful for the readers interested in Prama mentioned in NyayaDarshan and Vaisheshik Darshan.]
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Quintarelli, Concetta, Gianpietro Dotti, Biagio De Angelis, Valentina Hoyos, Martha Mims, Luigia Luciano, Helen E. Heslop, Cliona M. Rooney, Fabrizio Pane, and Barbara Savoldo. "Cytotoxic T lymphocytes directed to the preferentially expressed antigen of melanoma (PRAME) target chronic myeloid leukemia." Blood 112, no. 5 (September 1, 2008): 1876–85. http://dx.doi.org/10.1182/blood-2008-04-150045.
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Abstract The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed in many hematologic malignancies, including chronic myeloid leukemia (CML). The sensitivity of CML to donor lymphocyte infusion after allogeneic stem cell transplantation suggests this tumor can be highly susceptible to cellular immunotherapy targeted to tumor associated antigens. We therefore tested whether functional PRAME-specific cytotoxic T lymphocytes (PRAME CTLs) could be generated and expanded from healthy donors and CML patients, or whether the limited immunogenicity of this CTA coupled with tumor-associated anergy would preclude this approach. Using optimized culture conditions and HLA-A*02–restricted PRAME-peptides, we have consistently generated PRAME CTLs from 8/9 healthy donors and 5/6 CML patients. These CTLs released IFNγ in response to PRAME peptides (between 113 ± 8 and 795 ± 23 spot forming cells/105 T cells) and lysed PRAME peptide–loaded cells (45 ± 19% at an effector:target [E:T] ratio of 20:1) in a MHC-restricted fashion. Importantly, these CTLs recognized and had cytotoxic activity against HLA-A*02+/PRAME+ tumor cell lines, and could recognize and respond to primary CML cells. PRAME CTLs were generated almost exclusively from the naive T-cell compartment, and clonal analysis showed these cells could have high αβTCR-peptide avidity. PRAME CTLs or vaccines may thus be of value for patients with CML.
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Qin, Yazhen, Honghu Zhu, Yan-rong Liu, Ya-zhe Wang, Hong-Xia Shi, Yue-yun Lai, Lan-Ping Xu, Daihong Liu, and Xiaojun Huang. "The PRAME and WT1 Transcripts Constitute a Good Molecular Marker Combination for Monitoring Minimal Residual Disease in Myelodysplastic Syndromes." Blood 120, no. 21 (November 16, 2012): 3853. http://dx.doi.org/10.1182/blood.v120.21.3853.3853.
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Abstract Abstract 3853 Background With the incorporation of more aggressive therapies such as hematopoietic stem cell transplantation (HSCT), more myelodysplastic syndromes (MDS) patients fall into a status of minimal residual disease (MRD). The sensitive molecular markers are required to monitor MRD and predict relapse. The Wilms' tumor gene (WT1) is now a widely accepted molecular marker of MDS. However, WT1 itself could not fully meet the demands because a subset of MDS patients does not overexpress WT1, and the increase of WT1 expression compared with the normal control of MDS patients was mainly within 2-log. We wondered if the preferentially expressed antigen of melanoma (PRAME) gene could supplement WT1. Methods The PRAME and WT1 transcript levels were simultaneously measured in 312 bone marrow samples collected from newly diagnosed MDS patients and 27 samples from non-malignant cytopenia patients. Bone marrow samples from 14 MDS patients after their disease progression were also detected. To evaluate the value of combined detection of WT1 and PRAME transcripts, one hundred and eleven BM samples collected from 17 MDS patients during their treatment were tested them simultaneously (chemotherapy alone: 1 patient; HSCT:16 patients). Bone marrow samples from six MDS patients and five normal controls were sorted into the blasts (CD34+), nucleated erythrocytes (CD71+), immature myeloid cells (CD33+CD34-), and lymphocyte (CD45+high, low SSC) fractions by flow cytometry and measured the PRAME and WT1 transcript levels, respectively. We had previously established that the upper limits of the PRAME and WT1 transcript levels tested in normal bone marrow samples were 0.28% and 0.50%, respectively. Results None of the 27 non-malignant cytopenia patients overexpressed PRAME (median 0.085%, range 0.01%-0.28%) and WT1 (median 0.095%, range 0.0089%-0.36%). Both WT1 and PRAME were commonly overexpressed in MDS. Both the overexpression frequency and the >1-log increase expression frequency of PRAME were similar to those of WT1 (74.4 % vs 77.6%; 51.6% vs 49.0%; p>0.05), and 88.1% of the patients overexpressed at least one marker. Moreover, the frequencies of PRAME expression with higher degrees of increase were significantly higher compared with those of WT1 expression (>2-log increase: 30.8% vs 3.8%; >3-log increase: 9.0% vs 0%; all p<0.001). PRAME had a higher log increase than WT1 in 53.3% of the patients with overexpressed WT1. For samples collected from newly diagnosed MDS patients, both the WT1 and PRAME transcript levels were significantly correlated with the percentage of blasts in bone marrow (r=0.35 and 0.22, all p<0.001). After disease progression, both WT1 and PRAME expressions prominently increased in six patients, only WT1 markedly increased in six patients, only PRAME markedly increased in one patient, and neither WT1 nor PRAME increased in one patient. Both PRAME and WT1 transcript levels generally fluctuated within the normal range after HSCT in all 10 patients in continuous complete remission (PRAME: 58/64 samples, WT1: 58/64 samples). Six out of seven patients were predicted relapse by the combined detection: sustained overexpression or significant increase over the normal range of both WT1 and PRAME in three patients, earlier by PRAME than WT1 or by PRAME alone in three patients. The expression patterns of WT1 differed from those of PRAME in the sorted cell fractions: WT1 is overexpressed in blasts and is significantly decreased in relative mature cells. Whereas, PRAME is mainly overexpressed not only by stem cells and blasts but also by the relative mature cells. Conclusion The combined detection of WT1 and PRAME transcripts in newly diagnosed MDS patients could find more suitable and sensitive molecular marker for them compared to detecting WT1 alone. The PRAME and WT1 transcripts constitute a good molecular marker combination for monitoring minimal residual disease in MDS. Disclosures: No relevant conflicts of interest to declare.
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Gassenmaier, Maximilian, Matthias Hahn, Gisela Metzler, Jürgen Bauer, Amir Sadegh Yazdi, Ulrike Keim, Claus Garbe, Nikolaus Benjamin Wagner, and Stephan Forchhammer. "Diffuse PRAME Expression Is Highly Specific for Thin Melanomas in the Distinction from Severely Dysplastic Nevi but Does Not Distinguish Metastasizing from Non-Metastasizing Thin Melanomas." Cancers 13, no. 15 (July 31, 2021): 3864. http://dx.doi.org/10.3390/cancers13153864.
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Background: PReferentially expressed Antigen in MElanoma (PRAME) immunohistochemistry is increasingly used as diagnostic adjunct in the evaluation of melanocytic tumors. The expression and prognostic significance of PRAME in melanomas ≤1.0 mm and its diagnostic utility in the distinction from severely dysplastic compound nevi (SDN) have not been studied. Methods: We investigated and compared the immunohistochemical PRAME expression in 70 matched thin metastasizing and non-metastasizing melanomas and 45 nevi from patients with long-term follow-up (35 SDN and 10 unequivocally benign compound nevi). Results: Diffuse PRAME staining in >75% of lesional epidermal and dermal melanocytes identified 58.6% of thin melanomas but did not distinguish metastasizing from non-metastasizing melanomas (p = 0.81). A superficial atypical melanocytic proliferation of uncertain significance, in which the final diagnostic interpretation favored a SDN was the only nevus with diffuse PRAME expression (1/45). Melanomas and SDN with PRAME immunoreactivity exhibited different staining patterns. Most melanomas (67.6%) showed uniform PRAME expression in the in situ and invasive component, whereas most SDN (81.0%) showed a decreasing gradient with depth. Conclusion: Diffuse intraepidermal and dermal PRAME staining is highly specific for melanomas in the distinction from SDN. PRAME expression is not a prognostic biomarker in melanomas ≤1.0 mm.
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Quintarelli, Concetta, Gianpietro Dotti, Sayyeda T. Hasan, Biagio De Angelis, Valentina Hoyos, Santa Errichiello, Martha Mims, et al. "High-avidity cytotoxic T lymphocytes specific for a new PRAME-derived peptide can target leukemic and leukemic-precursor cells." Blood 117, no. 12 (March 24, 2011): 3353–62. http://dx.doi.org/10.1182/blood-2010-08-300376.
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Abstract The cancer testis antigen (CTA) preferentially expressed antigen of melanoma (PRAME) is overexpressed by many hematologic malignancies, but is absent on normal tissues, including hematopoietic progenitor cells, and may therefore be an appropriate candidate for T cell–mediated immunotherapy. Because it is likely that an effective antitumor response will require high-avidity, PRAME-specific cytotoxic T lymphocytes (CTLs), we attempted to generate such CTLs using professional and artificial antigen-presenting cells loaded with a peptide library spanning the entire PRAME protein and consisting of 125 synthetic pentadecapeptides overlapping by 11 amino acids. We successfully generated polyclonal, PRAME-specific CTL lines and elicited high-avidity CTLs, with a high proportion of cells recognizing a previously uninvestigated HLA-A*02–restricted epitope, P435-9mer (NLTHVLYPV). These PRAME-CTLs could be generated both from normal donors and from subjects with PRAME+ hematologic malignancies. The cytotoxic activity of our PRAME-specific CTLs was directed not only against leukemic blasts, but also against leukemic progenitor cells as assessed by colony-forming–inhibition assays, which have been implicated in leukemia relapse. These PRAME-directed CTLs did not affect normal hematopoietic progenitors, indicating that this approach may be of value for immunotherapy of PRAME+ hematologic malignancies.
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Wadelin, Frances R., Keith A. Spriggs, Franco H. Falcone, and David M. Heery. "Transcriptional and Translational Regulation of the Cancer-Testis Antigen PRAME Mediated by Pathogen-Associated Molecular Patterns." Blood 116, no. 21 (November 19, 2010): 2475. http://dx.doi.org/10.1182/blood.v116.21.2475.2475.
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Abstract Abstract 2475 PRAME (preferentially expressed antigen in melanoma) is a germinal tissue-specific gene that is expressed at high levels in a number of haematological malignancies. In AML, PRAME is a positive prognostic indicator, being associated with a favourable response to chemotherapy and increased overall survival, even in the presence of unfavourable karyotypes. In contrast, over-expression of PRAME mRNA is associated with poor prognosis in CML, NHL, HD, CLL and myeloma, being associated with progressive disease and chemo-resistance. Despite proposals to implement PRAME as a tool for monitoring minimal residual disease and as an immunotherapeutic target, the physiological functions of PRAME in normal and tumour cells are unclear. A role in the regulation of retinoic acid signalling has been proposed, although interactions of PRAME with retinoic acid receptors are weak compared to other coregulator proteins. Sequence homology and structural predictions suggest that PRAME is a member of the family of leucine-rich repeat (LRR) proteins that function in cellular immunity, bearing most resemblance to the LRR domains of Toll-like receptors (in particular TLR3 and TLR4) and internalin proteins. LRR proteins can bind pathogen-associated molecular patterns (PAMPs) including proteins, nucleic acids and sugars, and consistent with this PRAME has been reported to interact with OPA proteins from Neisseriae gonorrhoeae. Here we show that expression of PRAME in HL60 leukaemic cells is strongly induced by lipopolysaccharide (LPS) or peptidoglycan in the presence of interferon (IFN)-gamma, whereas no induction was observed after exposure to other PAMPs such as zymosan A. This burst of PRAME expression peaked between 1 and 4 hrs post treatment, but was not mediated by the proximal promoter region. Moreover, we show that treatment of HL60 with LPS and IFN-gamma increases the association of PRAME transcripts with polysomes indicating PRAME is also translationally regulated, and we provide evidence that PRAME 5'UTR contains an internal ribosome entry sequence for CAP-independent translation. Finally, we show that the subcellular localisation of PRAME protein in HL60 and U937 cells is modulated by treatment with LPS and IFN-gamma. In summary, our studies indicate that the LRR protein PRAME is subject to transcriptional and translational regulation following cellular exposure to bacterial PAMPs and interferon, suggesting it may have a role in innate immunity. Disclosures: No relevant conflicts of interest to declare.
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Stevens, Greg, Burkhard Jansen, Terry Arnold, James Rock, Jennifer Wood, Loren Clarke, and Mark Hyde. "Concordance of Preferentially Expressed Antigen in Melanoma by Non-Invasively Collected Polymerase Chain Reaction and Immunohistochemistry on Paraffin Embedded Tissue." SKIN The Journal of Cutaneous Medicine 7, no. 2 (March 13, 2023): s170. http://dx.doi.org/10.25251/skin.7.supp.170.
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Background:
Pigmented lesion evaluation remains a challenging aspect of dermatology. The DermTech Melanoma Test (DMT) is a non-invasive gene-expression test designed to rule-out melanoma. It consists of the pigmented lesion assay, which detects RNA products of Long Intergenic Non-Coding RNA 00518 (LINC00518) and Preferentially Expressed Antigen in Melanoma (PRAME), and an add-on assay for DNA promoter mutations in telomerase reverse transcriptase (TERT). This registry study examines the concordance of PRAME detection by polymerase chain reaction (PCR) in samples obtained non-invasively prior to biopsy and PRAME detection by immunohistochemistry (IHC) on the same lesions after biopsy.
Methods:
Between April 2021 and March 2022, multiple geographically diverse sites throughout the US submitted data to a registry to assess real-world use of the DMT. Approximately 8,000 clinically atypical lesions were tested. After receiving the test result, providers followed their clinical judgement for biopsy decision. When lesions expressed genomic markers (LINC, PRAME, and/or TERT) and were biopsied, pathology reports were also submitted to the registry. The presence or absence of PRAME by immunohistochemistry (IHC) was reviewed and compared to the detection of PRAME by PCR from the DMT on the same lesion.
Results:
At the 1-year mark of the registry, there were roughly 8,000 unique entries. Of those, 1,021 (12.8%) were positive for one or more of the DMT genomic markers. One thousand three lesions (98.2%) had records available. Pathologists used PRAME IHC for 102 lesions (10.2%). Of those, 40 (39.2%) were positive by IHC, and 62 (60.8%) were negative by IHC. PRAME positivity by PCR correlated with PRAME positivity by IHC in 35 of 40 lesions (87.5%). Conversely, PRAME was detected using PCR in 28 of 62 lesions (45.2%) where it was not detected using IHC.
Conclusions:
The higher sensitivity of PCR compared to IHC may explain the higher concordance when PRAME is positive by IHC than when it is negative by IHC. In this data set, when PRAME is positive by IHC it is usually also positive by PCR. When PRAME is negative by IHC, it can still be detected by PCR in a substantial percentage of cases. The increased sensitivity of PCR is likely due to several factors, including its detection of the PRAME mRNA and sampling of the entire lesion. As such, PRAME PCR status may aid pathologists in understanding the risk of melanoma even when IHC is negative. Further research is warranted to understand the clinical implications of PRAME PCR versus IHC positivity.
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Mansouri, Fatima Z., Cleveland A. Gibbon, and Colin A. Higgins. "PRAM." ACM SIGCSE Bulletin 30, no. 3 (September 1998): 166–70. http://dx.doi.org/10.1145/290320.283108.
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Misyurin, V. A., D. V. Kalenichenko, A. A. Rudakova, Yu P. Finashutina, N. A. Lyzhko, V. V. Tikhonova, L. A. Kesaeva, et al. "Chemoresistance of PRAME-expressing melanoma cell can be resolved with help of bortezomib." Advances in molecular oncology 5, no. 4 (January 4, 2019): 131–34. http://dx.doi.org/10.17650/2313-805x-2018-5-4-131-134.
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Background.PRAME gene spontaneous expression is frequently observed in a cancer cell. The protein encoded by this gene increases the viability of tumour cell. NF-κB signalling pathway takes part in PRAME upregulation. It proposes, that stress conditions may increase the expression level of PRAME in the tumour cell and increase cell’s viability after it. We hypothesized that this phenomenon determines chemoresistance of PRAME-expressing cell, which can be overcome by NF-κB inhibitors, such as bortezomib.Materials and methods.We incubated A875 melanoma cells with cisplatin, bortezomib and dexamethasone, as well as with a mixture of cisplatin with bortezomib and cisplatin with dexamethasone within 24 hours. To assess the cytotoxicity of these combinations MTT-test was used. For evaluation of PRAME expression level, real-time polymerase chain reaction was used. All data were analyzed with Wilcoxon test for coupled samples.Results.It was found that cisplatin and dexamethasone increased an expression level of PRAME compared to control (p <0.03). The addition of dexamethasone to cisplatin reduced cytotoxic effect of cisplatin. Bortezomib has a cytotoxic effect, but it did not increase the activity of PRAME gene (p = 0.12). PRAME gene activity in cells incubated with a mixture of cisplatin and bortezomib was observed at a lower level in comparison with cells incubated with cisplatin (p = 0.0277).Conclusion.The results of experiments show that an increase of PRAME expression level reduces the sensitivity of melanoma cells to the cytotoxic effect of cisplatin. PRAME activity increases under stress conditions. Using of bortezomib can inhibit the growth of PRAME expression and makes the tumour cell more vulnerable to cytotoxic agents. On the other hand, dexamethasone may increase a resistance of PRAME-expressing cell to cytotoxic effects.
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Proto-Siqueira, Rodrigo, Lorena L. Figueredo-Pontes, Eduardo M. Rego, Edgar G. Rizzatti, Fabio M. Nascimento, Rodrigo S. Abreu e Lima, Roberto P. Falcao, Valery Filonenko, and Marco Antonio Zago. "PRAME Protein Is Aberrantly Expressed in Chronic Lymphocytic Leukemia and Mantle Cell Lymphoma." Blood 104, no. 11 (November 16, 2004): 958. http://dx.doi.org/10.1182/blood.v104.11.958.958.
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Abstract PRAME (Preferentially Expressed Antigen in Melanoma) gene is located on chromosome 22 (22q11.22) and is expressed at low levels by normal adrenal, ovarian and endometrial cells. In contrast, its expression has been demonstrated at high levels in several types of cancers, such as in acute myeloid and lymphoid leukemias and multiple myeloma . We recently have reported that PRAME is also expressed in lymphoproliferative diseases and its transcripts were detected in 26 out 58 patients with chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). Nevertheless, the expression of PRAME protein in normal and neoplastic tissues is unknown. In order to address this question, we produced a monoclonal antibody against PRAME protein. A 562-nt fragment of the PRAME cDNA that includes the region that codes for the immunogenic 9-peptide was clone in pCR2.1-TOPO vector in DH5 alpha cells and subcloned into a pET24a expression vector, and the 196-amino acid peptide was expressed in BL21(DE3) cells as His-taged protein. The monoclonal antibody (MoAb) against PRAME was generated from splenocytes from mice immunized with the purified peptide. We then analyzed PRAME expression by flow cytometry in samples of peripheral blood (PB, n=15), bone marrow (BM, n=3) from healthy donors; in tonsils (n=3) from patients submitted to tonsilectomy for non malignant diseases and in PB samples from 26 CLL and 7 MCL patients. PRAME positive cells represented less than 15% of cells from normal PB, BM and tonsil cells. In contrast, 25 out of 26 CLL and 6 out of 7 MCL cases presented more than 20% of PRAME + cells [mean 59% (range:20–95%) and 75% (20–94%) for CLL and MCL, respectively]. The lymphoid cells from normal BM and tonsils that expressed PRAME were CD19+CD10+ CD27+ CD38± TdT− cIgM− CD5− suggesting that PRAME is expressed early during lymphoid ontogenesis and that its expression in CLL and MCL cells is aberrant. Furthermore, PRAME expression in normal lymphocytes was dimmer than in CLL and MCL cells. To quantify PRAME expression we evaluated PRAME Specific Antibody Binding Capacity (SABC) by a quantitative flow cytometry (QFC) method. The mean values of SABC were of 10,339 sites per cell (range 3,075 to 24,665) and of 14,191 sites per cell (range 5,059 to 20,679) for CLL and MCL, respectively. In contrast, in normal PB lymphocytes SABC values ranged from 1,628 to 1,781 sites per cell. Even in the two cases of CLL and MCL that had less than 20% PRAME+ cells, PRAME SABC was 3,075 and 12,347 sites per cell respectively, therefore significantly higher than the observed in normal PB lymphocytes. To evaluate the sensitivity of the QFC method, we established a cut off value for PRAME SABC based on the highest value detected in normal PB cells and then serially diluted tumor MCL cells marked with PRAME in normal PB lymphocytes. Based exclusively on PRAME expression, we were able to detected neoplastic MCL cells up to a 1:1000 dilution. In conclusion, PRAME protein is strongly expressed in the neoplastic clone in most patients with CLL and MCL. This finding supports the suggestion that this antigen may be further explored as a target for diagnostic, to detect minimal residual disease detection and for therapeutic approaches.
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Takata, Katsuyoshi, Daisuke Ennishi, Ali Bashashati, Saeed Saberi, Elena Viganò, Shannon Healy, Julie S. Nielsen, et al. "Somatic PRAME Deletions Are Associated with Decreased Immunogenicity, Apoptosis Resistance and Poor Outcomes in Diffuse Large B-Cell Lymphoma." Blood 132, Supplement 1 (November 29, 2018): 667. http://dx.doi.org/10.1182/blood-2018-99-113516.
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Abstract Background: The current standard of care in diffuse large B-cell lymphoma (DLBCL) consists of chemotherapy and therapeutic monoclonal antibodies that have significantly improved patient outcomes over the past 15 years. However, a large proportion of patients suffer from refractory or relapsed disease. Therefore, the development of new therapeutic strategies for this subgroup of patients, who are threatened by a high chance of disease-related death, represents an important unmet clinical need. Methods: We enrolled into our study 347 de novo DLBCL patients uniformly treated with R-CHOP from the BC Cancer population-based cohort between September 2000 and January 2012. RNAseq and high-resolution copy number analysis were performed and correlated with clinical outcome data and tumor microenvironment composition. We also performed functional studies to investigate PRAME-mediated memory T-cell responses and gene expression changes. Results: We discovered novel, highly focal deletions of 22q11.22, including the PRAME gene in 13% (44/338) of the cases. The deletions cluster in a narrow chromosomal region that includes a very small number of genes (VpreB1, ZNF280A/B, PRAME, GGTLC2, miR-650). Of clinical importance, 22q11.22 deletions were found significantly more frequently in germinal centre B-cell-like (GCB) type DLBCL (17% (31/180) vs. activated B-cell-like (ABC) type: 8% (8/98), P < 0.01), and were also significantly associated with worse outcome, which was specifically observed in GCB-DLBCL (5-year disease specific survival, non-PRAME-deleted: 84.5% vs. PRAME-deleted: 67.2%, P = 0.026). Homozygous deletions were more strongly associated with poor outcome than heterozygous deletions. Interestingly, 90% of PRAME-deleted cases were Ig-lambda restricted (P < 0.001). PRAME is a prominent member of the cancer testis antigen (CTA) family of proteins that are expressed in various types of cancers, but not in normal tissues, including normal mature B-cells, apart from male germinal cells. Due to the cancer-specific expression of CTAs, these molecules are considered promising targets for cancer immunotherapy using cytotoxic T-cells and tumor vaccination approaches. To determine the association with tumor microenvironment composition, we analyzed CD4/CD8 flow cytometry data from DLBCL patient samples. The numbers of CD4 and CD8-positive T cells were significantly lower in PRAME-deleted cases compared to wild type (CD4: P < 0.001, CD8: P = 0.013). Notably, RNAseq analysis revealed that the HLA-A*0201 genotype was seen significantly more often in PRAME deleted cases (PRAME wt: 2.5% vs. PRAME deleted: 10.8%, P = 0.005). In order to functionally characterize its interaction with the immune microenvironment, we utilized enzyme-linked immunoSpot (ELISPOT) assays to investigate memory T-cell reactions of patient-derived T cells to PRAME antigens using patient-derived peripheral blood mononuclear cells (PBMC) and measured IFN-g production (7 control healthy donors, 4 PRAME-deleted and 4-wild type patients). While T cells from PRAME-replete patients had no reaction to PRAME antigens, PRAME-deleted patient-derived T-cells had significant reactions to 4 independent PRAME peptides. These data suggest that PRAME-deleted tumor cells can escape from cytotoxic T-cell attack to gain growth advantage. Next, we performed PRAME knock-out (KO) experiments using CRISPR/Cas9 genome editing to clarify the cell autonomous effects of PRAME deletions. Using 2 different cell lines (Karpas422 and SUDHL-4), we found TNFSF10 (TRAIL) expression was significantly down-regulated in homozygous PRAME-KO cell lines compared to wild type. The soluble form of TRAIL (sTRAIL) was also reduced, as measured with enzyme-linked immunosorbent assays. These results suggest that PRAME downregulated cells may contribute to cell survival via TRAIL and sTRAIL reduction. Conclusion: We identified recurrent PRAME deletions and characterized their clinical and functional role in DLBCL. Our findings contribute to the understanding of cell-autonomous and extrinsic roles of PRAME deletions in lymphomagenesis and may lead to the discovery of new therapeutic avenues to simultaneously treat the tumor and the host. Disclosures Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:Janssen: Research Funding; Roche: Research Funding; NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Celgene: Consultancy, Honoraria. Steidl:Tioma: Research Funding; Seattle Genetics: Consultancy; Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Juno Therapeutics: Consultancy; Nanostring: Patents & Royalties: patent holding.
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Tanaka, Norina, Yan-Hua Wang, Masayuki Shiseki, Minoko Takanashi, and Toshiko Motoji. "Inhibition of PRAME Expression Causes Cell Cycle Arrest and Apoptosis In Leukemic Cells." Blood 116, no. 21 (November 19, 2010): 1695. http://dx.doi.org/10.1182/blood.v116.21.1695.1695.
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Abstract Abstract 1695 Introduction: The preferentially expressed antigen of melanoma (PRAME) was originally described as a tumor-associated antigen recognized by autologous cytotoxic T cells against a melanoma surface antigen. PRAME seems to act as a dominant repressor of retinoic acid receptor (RAR) signaling, but the function of PRAME in leukemia remains unclear. In the present study, we clarified the function of PRAME in leukemia, by the method of small interfering RNA (siRNA)-induced knockdown of PRAME using a leukemic cell line. To elucidate the clinical significance of PRAME expression in acute leukemia, especially its role at the relapse of disease, expression of PRAME mRNA levels and cell cycle profiles were analyzed in acute leukemia at the time of diagnosis and relapse in paired samples. Methods: The K562 cell line was used in siRNA experiments. After PRAME siRNA transfection, the effect on cell growth was examined by colony formation assay and cell counts in liquid culture. Furthermore, cell cycle analysis and apoptotic assays (annexinV assay and caspase-3 activity assay) were performed to assess the time course from day 1 to day 6. At the same time, the possible changes in various gene expressions and protein levels were analyzed by quantitative real-time RT-PCR and western blot analysis. As clinical samples, PRAME mRNA levels were measured in a total of 44 acute leukemia patients. We also examined the relationship between PRAME expression and the percentages of S phase in leukemic cells taken from 35 paired acute leukemia patients from whom sufficient blast cells were obtained. Results: A significant decrease in cell growth was observed in liquid culture and colony formation assay of the PRAME-inhibited cells. At the same time, cell cycle analysis showed a significant decrease of cells in the S phase and increase of cells in the G0/G1 phase in PRAME siRNA-treated cells. Among the cell cycle related genes analyzed with quantitative real-time RT-PCR, a clear increase of p27 expression was observed between day 3 and day 6 in PRAME siRNA-treated cells. Increase of p27 protein expression was also confirmed with western blot analysis. Furthermore, PRAME siRNA-treated cells showed a change of erythroid regulatory genes. Our result observed an increase in GATA-1 protein from day 3 to day 6, a decrease in GATA-2 protein from day 1 to day 5, and a decrease in PU.1 protein from day 2 to day 6, as well as quantitative real-time RT-PCR. On annexin V assay, the percentage of apoptotic cells gradually increased from day 3 to day 6 in PRAME siRNA-treated cells. The total percentage of apoptotic cells on day 6 was 45.5% (early apoptotic cells 33.1%, late apoptotic/necrotic cells 12.4%) in PRAME siRNA-treated cells and only 10.1% (early apoptosis 8.0%, late apoptosis 2.1%) in control cells. Caspase-3 was activated on day 3 in PRAME siRNA-treated cells, then increased gradually with the maximum activity being observed on day 6 (33.4%) using antibody against cleaved caspase-3 by flow cytometory. Western blot analysis showed that a faint band of cleaved caspase-3 protein was detected after day 3, and then an obviously augmented band was observed on days 5–6. In 51.4% of clinical samples in our study, the PRAME expression level was higher at relapse than at diagnosis. In the group in which PRAME expression was higher at relapse, the percentage of S phase cells at relapse was significantly increased compared to that at diagnosis (median, 2.4% at diagnosis vs. 6.8% at relapse, P = 0.02, n = 18). Conclusions: Inhibition of PRAME by siRNA in K562 cells suggested that PRAME expression is associated with cell cycle progression from the G0/G1 phase to S phase, inhibition of apoptosis and blocking of cell differentiation. Furthermore, we found cell cycle progression in leukemia patients in whom PRAME was highly expressed at relapse. The PRAME gene may be one of the important genes influencing proliferation of leukemic cells. Insights into the function of PRAME are expected to provide a new perspective on characteristics at relapse in acute leukemia, making it an attractive molecular target for potential therapy. Disclosures: No relevant conflicts of interest to declare.
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Kern, Chandlar H., Mingyao Yang, and Wan-Sheng Liu. "The PRAME family of cancer testis antigens is essential for germline development and gametogenesis." Biology of Reproduction 105, no. 2 (April 19, 2021): 290–304. http://dx.doi.org/10.1093/biolre/ioab074.
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Abstract Preferentially expressed antigen in melanoma (PRAME) belongs to a group of cancer/testis antigens that are predominately expressed in the testis and a variety of tumors, and are involved in immunity and reproduction. Much of the attention on PRAME has centered on cancer biology as PRAME is a prognostic biomarker for a wide range of cancers and a potential immunotherapeutic target. Less information is available about the PRAME family’s function (s) during gametogenesis and in the overall reproduction process. Here, we review the current knowledge of the PRAME gene family and its function in germline development and gametogenesis. Members of the PRAME family are leucine rich repeat proteins, localized in nucleus and cytoplasm, with multifaceted roles in germ cells. As transcriptional regulators, the PRAME family proteins are involved in germline development, particularly in the maintenance of embryonic stem cell pluripotency, development of primordial germ cells, and differentiation/proliferation of spermatogenic and oogenic cells. The PRAME family proteins are also enriched in cytoplasmic organelles, such as rough endoplasmic reticulum, Golgi vesicle, germinal granules, centrioles, and play a role in the formation of the acrosome and sperm tail during spermiogenesis. The PRAME gene family remains transcriptionally active in the germline throughout the entire life cycle and is essential for gametogenesis, with some members specific to either male or female germ cells, while others are involved in both male and female gametogenesis. A potential molecular mechanism that underlies the function of PRAME, and is shared by gametogenesis and oncogenesis is also discussed.
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Cazzato, Gerardo, Eliano Cascardi, Anna Colagrande, Vincenzo Belsito, Lucia Lospalluti, Caterina Foti, Francesca Arezzo, et al. "PRAME Immunoexpression in 275 Cutaneous Melanocytic Lesions: A Double Institutional Experience." Diagnostics 12, no. 9 (September 9, 2022): 2197. http://dx.doi.org/10.3390/diagnostics12092197.
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In recent years, the preferentially expressed antigen in melanoma (PRAME) has also been used in the histopathological diagnosis of melanocytic lesions, in order to understand if it could constitute a valid, inexpensive, and useful resource in dermatopathological fields. We performed a double-center study to evaluate whether the data on the usefulness and possible limitations of PRAME could also be confirmed by our group. From 1 December 2021 to 29 March 2022, we collected 275 cases of melanocytic lesions that were immunostained with PRAME (Ab219650) and rabbit monoclonal antibody (Abcam). To better correlate the PRAME expression with its nature (benign, uncertain potential for malignancy, or malignant), we categorized PRAME tumor cells’ percentage positivity and intensity of immunostaining in a cumulative score obtained by adding the quartile of positive tumor cells (0, 1+, 2+, 3+, 4+) to the PRAME expression intensity in tumor cells (0, 1+, 2+, 3+). Of these 275 lesions, 136 were benign, 12 were of uncertain potential for malignancy (MELTUMP or SAMPUS or SPARK nevus), and 127 were malignant. The immunoexpression of PRAME was completely negative in 125/136 benign lesions (91.9%), with only a few positive melanocytes (1+) and intensity 1+ in the remaining 11 cases (8.1%). Of the 127 cases of melanoma (superficial spreading, lentigo maligna, and pagetoid histotypes), PRAME was strongly positive in 104/127 cases (81.8%) with intensity 4+ and 3+. In 17 cases (13.3%; melanoma spindle and nevoid cell histotypes), PRAME was positive in percentage 2+ and with intensity ranging from 2+ to 3+. In 7 cases (5.5%) of desmoplastic melanoma, PRAME was 1+ positive and/or completely negative. Of the 12 cases of lesions with uncertain potential for malignancy, the immunoexpression of PRAME was much more heterogeneous and irregularly distributed throughout the lesion. These data are perfectly in agreement with the current literature, and they demonstrate that the reliability of PRAME is quite high, but its use cannot cause physicians to disregard the morphological information and the execution of other ancillary immunohistochemical stains such as Melan-A, HMB-45, MiTF, and SOX-10.
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Lyzhko, N. A., V. A. Misyurin, Y. P. Finashutina, T. V. Akhlynina, L. A. Kesaeva, V. V. Tikhonova, N. N. Kasatkina, O. N. Solopova, M. A. Baryshnikova, and A. V. Misyurin. "DEVELOPMENT OF CYTOSTATIC EFFECT OF MONOCLONAL ANTIBODIES TO THE PROTEINS PRAME." Russian Journal of Biotherapy 15, no. 4 (December 30, 2016): 53–58. http://dx.doi.org/10.17650/1726-9784-2016-15-4-53-58.
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Introduction. PRAME protein is a promising target for cancer immunotherapy. PRAME is not expressed in normal tissues, but active in number of the tumor types. We have developed the mouse monoclonal antibodies 5D3F2 and 6H8F12 against PRAME epitopes. Aim. To determine the effects provided by the monoclonal antibodies 5D3F2 and 6H8F12 against the cells with different levels of PRAME gene expression. Materials and methods. We used different cell lines: NOMO-1 and WI-38 with low levels of expression PRAME; THP-1 with intermediate level of PRAME expression; K562 and WI-38-PRAME with high level of PRAME expression. We incubated these cell lines in the presence of monoclonal antibodies 5D3F2 and 6H8F12. The final concentration of monoclonal antibodies in culture varied from 6 pg/ml to 120 mcg/ml. The live cells were counted at the 24, 48 and 72 hours after incubation. The number of dead cells was evaluated by the MTT-test after 24 hours. Results. Cell growth rate is significanely decreased during incubation with monoclonal antibodies. This effect is correlated with increase of monoclonal antibody concentrations (Pearson coefficient 0,67;p = 0,0219). K562 growth rate was much less compared to the THP-1’s rate (p = 0,0061), NOMO-1 (p = 0,0005) and WI-38 (p = 0,0002) in the presence of the same amount of monoclonal antibody 6H8F12. K562 cell growth rate was lower than the WI-38-PRAME’s rate (p = 0,0027), despite the comparable level of PRAME expression. Effects of monoclonal antibody 5D3F2 and 6H8F12 were similar (p = 0,3946). According to the MTT-test, the comparable number of death cells in K562 and WI-38-PRAME was observed (p = 0,8405). Under the same conditions the amount of death cells in THP-1 was smaller than K562 (p = 0,6335). To compare with K562, fewer cells died in NOMO-1 and WI-38 (p = 0,0026 and p = 0,0005, respectively). Conclusion. It was shown that monoclonal antibody 5D3F2 and 6H8F12 exhibit a significant cytotoxic effect against PRAME-express-ing cells. In case of higher levels of PRAME expression the cytotoxic effect was stronger.
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Misurin, Andrey, Tatyana Gaponova, Larisa Mendeleeva, Elena Parovichnikova, and Valeryi Savchenko. "Expression of PRAME and WT1 in Multiple Myeloma Patients during High Dose Chemotherapy and Auto-SCT." Blood 112, no. 11 (November 16, 2008): 5128. http://dx.doi.org/10.1182/blood.v112.11.5128.5128.
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Abstract High dose therapy enables further improvement in the outcome of multiple myeloma (MM) patients. However, it is still necessary to determine prognostic factors that may influence treatment results and provide additional criteria for the precise selection of treatment approaches. There are several tumor associated genes (MAGE, LAGE, GAGE, PRAME) that are over-expressed in different malignancies including MM. These genes are believed to modulate cancer properties and should be taken into account during treatment. Their significance as prognostic factors is under investigation. The aim of our study was to analyze the expression levels of PRAME and WT1 genes in MM patients during high dose chemotherapy following by auto-SCT. After having informed consent 25 primary MM patients were included into this study. The median age was 48 years (range, 31–62). All patients were treated by 3 cycles of VAD, Cyclophosphamide 6g/m2 + G-CSF to mobilize Stem cells, EDAP, melphalan 200 mg/m2 followed by auto-SCT. As second line therapy we used bortezomib+dexamethasone. Quantitative PRAME and WT1 gene expression analysis was performed by means of RQ-PCR. Results were normalized against expression of ABL gene which was used as internal control. Investigation was performed before treatment (n=25), after three VAD cycles (n=12), and before (n=5) and after auto-SCT (n=4). In primary MM patients: PRAME gene expression was found in 68% (n=17), WT1 in 24% (n=6) of patients, all of whom were PRAME-positive. Median expression levels were 0.1% (0.001–132%) for PRAME and 0.01% (0.002–0.07%) for WT1. PRAME and WT1 expression did not correlate with tumor bulk and was independent of the levels of M-protein, beta-2M and albumin. The expression of PRAME significantly decreased after 3 VAD cycles to 0.001–207% (n=8), at the moment of auto-SCT it was 0.05–6.1% (n=3) and after auto-SCT it was 0.013–4.9% (n=3). However for WT1, we observed increased of WT1 expression after 3 VAD cycles to 0.004-0.05% (n=4)and at the moment of auto-SCT it was 0.035–0.4% (n=3) and after auto-SCT – 0.019–2.03% (n=3). In the patients with high primary PRAME expression (>median expression) the frequency of CR+PR was significantly lower then in PRAME-negative primary patients and in patients with low (<median expression) primary PRAME expression (55% vs 84, p = 0.04). It was found also that WT1-positive primary patients were bad responders and they achieved only minimal response after 3 VAD cycles. It should be stressed that during treatment in a small number of initially negative PRAME and WT1 gene patients, we demonstrated detection by PCR. We detected the appearance of gene expression at low levels in 1 of 8 initially negative PRAME (6.1%) and in 5 of 19 initially negative WT1 (range of level 0.01–0.4%). The detection of gene expression did not correlate with disease status. All these patients achieve CR+ VGPR. In one of these secondary positive patients (acquired PRAME and WT1) relapse occurred. Conclusion: Expression of PRAME gene was found in 68% primary patients and the level of PRAME decreased with tumor reduction. High expression level of PRAME turned out to be a factor of unfavorable prognosis. Expression of WT1 was found in 24% of MM patients all of whom were PRAME-positive. WT1 expression increased during treatment in a small group of pts. Some initially negative pts acquired PRAME and WT1 expression during treatment, but clinical relevance of it is not clear so far.
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Oehler, Vivian G., Katherine A. Guthrie, Carrie L. Cummings, Kathleen Sabo, Brent L. Wood, Ted Gooley, Taimei Yang, et al. "The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells." Blood 114, no. 15 (October 8, 2009): 3299–308. http://dx.doi.org/10.1182/blood-2008-07-170282.
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Abstract The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications.
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Quintarelli, Concetta, Gianpietro Dotti, Biagio De Angelis, Valentina Hoyos, Fabrizio Pane, Martha P. Mims, Helen E. Heslop, Cliona M. Rooney, Malcolm K. Brenner, and Barbara Savoldo. "Polyclonal PRAME-Specific Cytotoxic T Lymphocytes Generated Using Protein-Spanning Pools of Overlapping Pentadecapeptides Target Chronic Myeloid Leukemia." Blood 112, no. 11 (November 16, 2008): 3899. http://dx.doi.org/10.1182/blood.v112.11.3899.3899.
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Abstract The cancer testis antigen PRAME is a potential target for adoptive T-cell or vaccine therapy of many hematologic malignancies and solid tumors. PRAME-specific cytotoxic T lymphocytes (PRAME-CTLs) can be detected in patients with hematologic malignancies and we have shown that they can be generated and expanded ex-vivo, using an optimized combination of artificial antigen presenting cells (aAPC) (K562 cell line genetically modified to express the HLA-A*02, CD80, CD40L and OX40L molecules) and cytokines (IL12, IL7 and IL15). Four HLA-A*02 PRAME-derived epitopes (P100, P142, P300 and P425) have previously been identified using a proteosome-mediated digestion analysis. However, this strategy, since relying only on the major cleavage site targeted by the immune-proteosome machinery for epitopes generation, may limit the potential clinical value of the identified peptides. We have now adopted an alternative method that uses a peptide-library consisting of 125 synthetic pentadecapeptides, overlapping by 11 aminoacids, spanning the entire PRAME protein. We evaluated whether novel HLA-A*02 restricted CD8+ T-cell responses to multiple immunogenic epitopes can be identified and used to consistently generate polyclonal PRAME-CTL lines from healthy donors and patients with hematologic malignancies. CD8+ T lymphocytes from 14 HLA-A*02 healthy donors and 3 patients with chronic myelogenenous leukemia (CML) were primed with autologous CD40L-activated B blasts loaded with the PRAME-peptide library in the presence of low doses of IL12, IL7 and IL15, and then expanded by weekly re-stimulation with peptide loaded aAPC and IL-2. The frequency and specificity of PRAME-CTLs were evaluated using IFNg Elispot and 51Cr release assays against PHA-blasts loaded with the PRAME-library. Using this approach we consistently generated PRAME-CTLs in 12 of the 14 HLA-A*02 healthy donors (526±101 SFC/105 cells as assessed by IFNg Elispot assay) compared to an irrelevant peptide-library (7±2 SFC/105). Similarly, PRAME-CTLs were generated from all 3 CML patients (441±250 SFC/105 cells vs 22±10 SFC/105 against an irrelevant peptide-library). These PRAME-CTLs were also able to target autologous tumor blasts (57±6 IFNg SFC/105), demonstrating that the same peptides were processed and presented physiologically. A Cr51 release assay confirmed that the PRAME-reactive T cells were cytotoxic, lysing autologous-PHA blasts loaded with the peptides derived from the PRAME-library (63±14% at a 20:1 E: T ratio), but not with irrelevant peptides (<15%). MHC class-I blocking experiments using specific antibodies showed that both IFNg release and cytotoxic activity were HLA-restricted. Using pentadecapeptides sub-pools, we found that the responses of our expanded PRAME-CTLs were polyclonal, since they consistently released IFNg in response to 1 to 6 pentadecapeptides pools (59% were specific for 1 or 2 pools, 25% to 3 pools, and 16% to 6 pools). Moreover, the approach we describe has allowed us to identify 6 potential new immunogenic 15-mer peptides that are processed and presented by tumor cells, and should facilitate expansion of polyclonal PRAME-CTLs for adoptive transfer or after vaccine administration to patients with PRAME+ hematological malignancy.
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Andrews, Claire N., Karen Murphy, Sinead Moran, Patrick Thornton, Nichola Harten, and Philip T. Murphy. "Prognostic Significance of WT1 and PRAME Gene Expression in Bone Marrow Samples of MDS and AML Patients Treated with Azacytidine." Blood 124, no. 21 (December 6, 2014): 5599. http://dx.doi.org/10.1182/blood.v124.21.5599.5599.
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Abstract Introduction: The Wilms’ tumor gene (WT1) and preferentially expressed antigen of melanoma (PRAME) gene are frequently overexpressed in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) Combined quantitation of PRAME and WT1 transcript levels could provide a powerful molecular marker in newly diagnosed MDS patients, enabling the early identification of relapse through minimal residual disease monitoring and also to enable alternate treatment strategies (Qin et al, 2012). In this study, we investigated the prognostic impact of WT1 and PRAME gene expression on MDS and AML patients treated with azacytidine. Methods: In this ethically approved, retrospective study, we studied WT1 and PRAME gene expression in the bone marrow of 20 patients with MDS and AML (unsuitable for more intensive chemotherapy) prior to starting epigenetic therapy with azacytidine (100mg/M2 sc for 5 days every 28 days). 20 patients with lymphoma not involving the bone marrow were used as a control group. Quantitative assessment of WT1 and PRAME transcript levels was performed on formalin fixed paraffin-embedded trephine biopsies, using the WT1 Ipsogen ProfileQuant and a ‘homebrew’ kit respectively. A control GADPH was used. Overall survival was extimated using the Kaplan-Meier method and compared using the log-rank test. The logistic regression model was used to correlate IPSS-R with WT-1 and PRAME gene expression levels. Results: The median age of the patient group was 71. 16 patients had MDS (RCMD: 3; RARS: 2; RAEB1: 2; RAEB2: 5; CMML: 4) and 4 had AML. Median age of the control group was 61. Median number of cycles of azacytidine was 4 (range 1-21). In 11 of the patient group and in all of the controls, neither PRAME nor WT1 gene expression was detected.. PRAME gene expression without WT1 gene expression was detected in 5 patients, whilst, in the remaining 4 patients, both PRAME and WT1 gene expression was detected. There was no significant difference in mean percentage of bone marrow blasts between patients with detectable PRAME +/- WT1 gene expression (16.67%) and those without (12.64%). Median gene expression levels of PRAME +/- WT1 significantly correlated with IPSS-R (p=0.003). Overall survival (OS) was significantly inferior in patients who were positive for PRAME +/- WT1 (p=0.0103). Median OS for patients positive for both PRAME and WT1 was 270 days and for patients who were PRAME positive and WT1 negative was 300 days, whilst patients negative for both had a median OS of 630 days (figure 1). Conclusions: Our study results show that patients who have detectable WT1 and PRAME expression in their bone marrow prior to azacytidine therapy have more advanced disease in terms of IPSS-R score and have a much inferior OS compared to patients without detectable WT1 and PRAME. Such patients should be considered for either additional or alternate therapy to single agent azacytidine. Disclosures No relevant conflicts of interest to declare.
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Ramchatesingh, Brandon Liam, and Ivan Litvinov. "Abstract 5734: The effect of PRAME on retinoid response and cell proliferation in cutaneous and head and neck squamous cell carcinoma." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5734. http://dx.doi.org/10.1158/1538-7445.am2022-5734.
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Abstract As cells undergo terminal differentiation, they adopt their tissue-specific functions and permanently exit the cell cycle. Inducing differentiation of premalignant cells and malignant cells are proposed strategies for cancer prevention and treatment, respectively. Retinoids, compounds related to retinol, drive terminal differentiation of numerous cell types. These compounds exhibit efficacy for the prevention of cutaneous squamous cell carcinomas (cSCC) and head and neck squamous cell carcinomas (HNSCCs), and their incorporation into treatment plans for these cancers is supported by laboratory and clinical studies. Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer-testis antigen that represses retinoid signaling, and is associated with adverse outcomes in a plethora of malignancies. Although PRAME is known to be expressed in subsets of cutaneous SCC (cSCC) and head and neck SCC (HNSCC) tumors, its functions, prognostic and therapeutic significance have never been investigated in these cancers. We hypothesize that PRAME expression in SCC cells confers resistance to the anti-neoplastic effects of retinoids and supports cell proliferation. PRAME expression was evaluated in human cSCC tumors, and in cSCC and HNSCC cell lines by immunoblotting and qRT-PCR. PRAME-overexpressing immortalized keratinocyte, cSCC and HNSCC cell lines were generated. shRNA-mediated knockdown of PRAME was performed in a cSCC and a HNSCC cell line. Cells were treated with all-trans retinoic acid (ATRA) for 24, 48 or 72 hours. Expression of differentiation markers was assessed by immunoblotting and qRT-PCR of markers of differentiation. Cell counting assays, immunoblot analysis of cell cycle genes and Ki67 immunofluorescence staining were used to assess proliferation. PRAME expression is detected in subsets of cSCC tumors and in select SCC cell lines. Overexpression of PRAME in HNSCC cells enhanced cell proliferation compared to control cells. Treatment with ATRA did not promote differentiation of PRAME-expressing cells. Furthermore, PRAME overexpression attenuated the anti-proliferative effect of ATRA in HNSCC cells. We conclude that PRAME enhances proliferation of malignant keratinocytes in vitro and may confer resistance to retinoid-induced differentiation and proliferation arrest. Investigations to assess the prognostic and therapeutic significance of PRAME expression in SCCs are warranted. Citation Format: Brandon Liam Ramchatesingh, Ivan Litvinov. The effect of PRAME on retinoid response and cell proliferation in cutaneous and head and neck squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5734.
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Fu, Rong, Kai Ding, and Zonghong Shao. "The Expression and Clinical Significance of PRAME Gene in Acute Leukemia." Blood 110, no. 11 (November 16, 2007): 4225. http://dx.doi.org/10.1182/blood.v110.11.4225.4225.
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Abstract Objective To investigate the expression of PRAME (preferentially expressed antigen of melanoma) gene in acute leukemia and its clinical significance in monitoring prognosis, detecting minimal residual disease (MRD) and gene immunotherapy. Methods The expression of PRAME gene mRNA in bone marrow mononuclear cells is measured by reverse transcriptase polymerase chain reaction in 34 patients with acute leukemia and 12 bone marrow samples of health donors. The relationships between PRAME gene expressions and some clinical data, such as gender, age, white blood count, leukemic immunophenotype, the percentage of blast cells, and the karyotype of chromosome, were also estimated. Results PRAME gene was expressed in 38.2% of all the patients, 40.7% of all the AML patients, which was higher than the 28.6% of ALL patients (p >0.05). There was no expression of PRAME gene in healthy donors. In all the sub phenotypes of AML, the expressive rate of PRAME gene in M3 patients is 80%, which is higher than that in M2 (33.3%) and in M5 (28.6%). The expressive rate of PRAME gene was also positively correlated with the expression of CD15, CD33, and the abnormality in the karyotype of chromosome, but not correlated with age, gender, white blood count and percentage of blast cell in bone marrow. Conclusion PRAME gene is highly expressed in acute leukemia, and could be regarded as a useful tool for monitoring MRD. Differential expression in acute leukemia patients vs. healthy donors suggests that the immunogenic antigens PRAME are potential candidates for immunotherapy in acute leukemia.
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Al-Khadairi, Ghaneya, and Julie Decock. "Cancer Testis Antigens and Immunotherapy: Where Do We Stand in the Targeting of PRAME?" Cancers 11, no. 7 (July 15, 2019): 984. http://dx.doi.org/10.3390/cancers11070984.
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PRAME or PReferentially expressed Antigen in Melanoma is a testis-selective cancer testis antigen (CTA) with restricted expression in somatic tissues and re-expression in various cancers. It is one of the most widely studied CTAs and has been associated with the outcome and risk of metastasis. Although little is known about its pathophysiological function, PRAME has gained interest as a candidate target for immunotherapy. This review provides an update on our knowledge on PRAME expression and function in healthy and malignant cells and the current immunotherapeutic strategies targeting PRAME with their specific challenges and opportunities. We also highlight some of the features that position PRAME as a unique cancer testis antigen to target.
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Finashutina, Yu P., A. V. Misyurin, T. V. Akhlynina, N. A. Lyzhko, A. A. Krutov, E. V. Aksenova, V. A. Misyurin, and A. Yu Baryshnikov. "PRODUCTION OF PURIFIED HUMAN RECOMBINANT ANTIGEN PRAME AND SPECIFIC MONOCLONAL ANTIBODIES." Russian Journal of Biotherapy 14, no. 3 (September 30, 2015): 29–36. http://dx.doi.org/10.17650/1726-9784-2015-14-3-29-36.
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Tumor antigens recognized by CTLs have been identified several years ago and are major targets for creating anticancer vaccines. PRAME is an antigen which is highly expressed in various malignant tumors including melanomas and hematopoietic malignancies such as acute and chronic leukemias (AML, CML). Technology for producing recombinant antigen PRAME is based on creating a bacterial producer strain containing cDNA of human PRAME gene. We have obtained two producers of recombinant PRAME protein and its N-half, the synthesis of the target protein in the producers occurs in the inclusion bodies. The schemes of isolation and purification of soluble proteins have been developed. The protein purity was approximately 95-96%. The monoclonal antibodies raised against truncated recombinant PRAME were used for PRAME protein analysis by Western blot on the various tumor cells. Specific monoclonal antibodies recognized the native PRAME protein in tumor cell lines as well as in tumor samples from patients. Our findings support the suggestion that this recombinant antigen may be further used as a target for diagnostic and therapeutic approaches. The monoclonal antibodies can be used for immunoassays of tumor samples from patients with hematologic malignancies to reveal clinical features and to monitor tumor progression.
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Cosío, Silvia Gutiérrez, Esteban Ballestar, Carlos Santamaría, Belen Blanco, Luis Ignacio Sánchez Abarca, Teresa Caballero Velázquez, Carmen Herrero Sánchez, et al. "Effect of 5-Azacytidine (5-AzaC) In the Expression of PRAME In Acute Myeloid Leukemia (AML)." Blood 116, no. 21 (November 19, 2010): 3615. http://dx.doi.org/10.1182/blood.v116.21.3615.3615.
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Abstract Abstract 3615 Introduction: Preferentially expressed antigen of melanoma (PRAME) was first isolated as a human melanoma antigen by cDNA expression cloning using melanoma-reactive cytotoxic Tcells (CTL). PRAME is a tumor associated antigen (TAA) of particular interest since it is widely expressed by lymphoid and myeloid malignancies and solid tumors. Several studies have associated high PRAME RNA levels with good prognosis in acute myeloid leukemia (AML). In addition, several authors have suggested that PRAME could be used as a target for anticancer T-cell therapy. PRAME expression is regulated at the epigenetic level. For this reason inhibitors of DNA methylation, such as 5-azacytidine, can modulate the expression of this TAAs. In the current study we analyzed the effect of 5-azaC on the expression of PRAME in blasts versus CD34+ cells from healthy donors in an attempt to increase its expression, thus inducing a potential target for therapeutic strategies. Methods: We analyzed PRAME mRNA expression of blast cells from AML patients at diagnosis versus CD34+ stem cells from healthy donors by RT-PCR without treatment or after exposure to 1mM 5-azaC during the four days of culture and correlated the expression of PRAME with the methylation status of the promoter. Results: PRAME is significantly over-expressed in blasts from AML patients (n=11) compared with normal CD34+ cells (n=8) ((700±1102 vs. 1.8±2.5 p=0.002). Interestingly, we found an inverse correlation between PRAME expression and the degree of methylation in the promoter among both AML samples and healthy donors (r=-0.77 p=0.010). In order to evaluate the effect of 5-azaC on PRAME gene expression, we treated blast cells and CD34+ cells from healthy donors with the drug and we observed that the exposure to the drug induced a decrease in the percentage of methylation in the promoter and subsequently increased the expression of PRAME but, interestingly, the higher the basal methylation of the promoter the more intense the effect of the drug among AML cells. By contrast, CD34+ cells from healthy donors were resistant to the effect of the drug so that no significant changes were observed neither in terms of methylation status of the promoter nor in the expression of PRAME prior to or after exposure to the drug among healthy donors. Conclusions: The promoter region is highly methylated in normal CD34+ cells compared to AML cells and this pattern correlates with a higher expression of PRAME in blasts. Furthermore, the level of PRAME methylation was reduced in AML patients after exposure to 5-azaC which correlated with an increase in the expression of PRAME. By contrast, the effect of 5-azaC on the methylation pattern of the promoter was significantly lower in CD34+ cells from healthy donors. Disclosures: Cañizo: Celgene: Membership on an entity's Board of Directors or advisory committees. San Miguel:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jangssen-cilag: Membership on an entity's Board of Directors or advisory committees; millennium: Membership on an entity's Board of Directors or advisory committees. Off Label Use: The drug used in this study is the demethylating agent 5-azacytidine (5-azaC) and the purpose is to increase PRAME expression in blasts from AML patients and generate CTL CD8+ specific response against tumor cells.
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Loeb, Anisha M., Sommer Castro, Cynthia Nourigat-Mckay, LaKeisha Perkins, Laura Pardo, Amanda R. Leonti, Thao T. Tang, et al. "Targeting PRAME with TCR-Mimic CAR T Cells in AML." Blood 138, Supplement 1 (November 5, 2021): 733. http://dx.doi.org/10.1182/blood-2021-148677.
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Abstract Chimeric antigen receptor (CAR) Ts have been effective in pre-B ALL, but their efficacy in AML has yet to be established. A significant barrier to effective CAR T therapy for AML is the substantial overlap of cell surface antigens expressed on AML and normal hematopoietic cells. To overcome this barrier, we profiled the transcriptome of over 3000 AML cases in children and young adults and contrasted this to normal hematopoietic tissues in search for AML-restricted targets (high expression in AML, silence in normal hematopoiesis). This led to the discovery of over 200 AML-restricted genes. Of these, Preferentially Expressed Antigen in Melanoma (PRAME) is among one of the highest expressing AML-restricted genes (Figure 1A) and, given its previous track record as a target for a variety of cancers, we selected this target for further assessment and therapeutic development in AML. However, PRAME is intracellular and therefore is inaccessible for targeting with conventional CAR T. Recently, a novel approach to target intracellular antigens was developed using TCR mimic (mTCR) antibodies, which recognize peptide/human leukocyte antigen (HLA) complexes on the tumor cell surface in a similar mode of recognition as authentic T Cell Receptors (TCRs). The Pr20 antibody was developed to recognize the PRAME ALY peptide in the context of HLA-A*02. Utilizing this Pr20 antibody, we developed a mTCR CAR T targeting PRAME and evaluated its preclinical efficacy in AML. The VL and VH sequences from Pr20 were used to construct the single-chain fragment variable domain of the 41-BB/CD3ζ CAR vector. We evaluated PRAME mTCR CAR T cells against OCI-AML-2 and THP-1 AML cell lines (PRAME +/HLA-A*02 +), K562 CML cell line (PRAME +/HLA-A*02 -) and HEK293T (293T) (PRAME -/HLA-A*02 +). Using a PE-conjugated Pr20 antibody, we confirmed that OCI-AML2 and THP-1 express PRAME ALY: HLA-A*02 but not K562 and 293T by flow cytometry (Figure 1B). As further confirmation, AML blasts in primary patient samples also stained with the Pr20 antibody (Figure 1C). For in-vivo studies, leukemia-bearing mice were treated with unmodified T or PRAME mTCR CAR T cells at 5x10 6 cells (1:1 CD4:CD8) per mouse 1 week following leukemia injection. Leukemia burden was measured weekly by bioluminescence IVIS imaging. Cells were treated with 10ng/mL of IFN-γ prior to co-incubation with T cells for 16 hours. PRAME mTCR CAR T cells demonstrated potent cytolytic activity against OCI-AML2 and THP1 but not against K562 or 293T cells, following co-incubation with target cells for 24 hours (Figure 1D). Consistent with potent, target-specific reactivity against PRAME ALY: HLA-A*02 positive cells, increased levels of IFN-γ, IL-2 and TNF-α were detected in cocultures of CAR T cells with OCI-AML2 and THP1 but not with K562 and 293T cells (Figure 1D). The cytolytic activity of PRAME mTCR CAR T cells extended to primary AML specimens expressing the PRAME ALY: HLA-A*02 antigen (data not shown). In-vivo efficacy of PRAME mTCR CAR T was demonstrated in OCI-AML2 and THP-1 CDX models (Figure 1E). Treatment with CAR T cells induced leukemia clearance and significantly reduced leukemia burden in OCI-AML2 and THP-1 xenograft mice, respectively, while treatment with unmodified T cells exhibited leukemia progression (Figure 1E). The anti-leukemia activity of CAR T cells resulted in enhanced survival in OCI-AML2 (p=0.0035) and THP-1 (p=0.0047) xenografts (Figure 1F). The in-vivo activity of PRAME mTCR CAR T cells was target specific, as treatment with CAR T cells did not affect leukemia burden and survival in K562 xenograft mice (Figure 1F). Given that IFN-γ promotes PRAME presentation, we investigated whether treatment of IFN-γ would enhance cytolytic activity of PRAME mTCR CAR T cells. OCI-AML2 and THP-1 cells pretreated with IFN-γ were more sensitive to cytolysis compared to untreated controls (Figure 1G). In this study, we demonstrate the therapeutic potential of targeting PRAME with mTCR CAR T cells in AML. We show potent, target-specific reactivity of PRAME mTCR CAR T cells against PRAME ALY: HLA-A*02 positive AML cells, both in-vitro and in-vivo. We further demonstrate that the activity of PRAME mTCR CAR T cells can be enhanced with IFN-γ treatment, providing a useful strategy to increase efficacy. Thus, the results presented provide a novel approach to target PRAME with CAR T cells and compelling data to evaluate PRAME mTCR CAR T cells in AML clinical trials. Figure 1 Figure 1. Disclosures Pardo: Hematologics, Inc.: Current Employment. Hylkema: Quest Diagnostics Inc: Current equity holder in publicly-traded company; Moderna: Current equity holder in publicly-traded company. Scheinberg: Eureka Therapeutics: Current equity holder in publicly-traded company.
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Kwon, Jaewoo, Yejong Park, Eunsung Jun, Woohyung Lee, Ki Byung Song, Jae Hoon Lee, Dae Wook Hwang, and Song Cheol Kim. "Clinical Outcome of RAMPS for Left-Sided Pancreatic Ductal Adenocarcinoma: A Comparison of Anterior RAMPS versus Posterior RAMPS for Patients without Periadrenal Infiltration." Biomedicines 9, no. 10 (September 22, 2021): 1291. http://dx.doi.org/10.3390/biomedicines9101291.
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Radical antegrade modular pancreatosplenectomy (RAMPS) is considered an effective procedure for left-sided pancreatic ductal adenocarcinoma (PDAC). However, whether there are differences in perioperative outcomes, pathologies, or survival outcomes between anterior RAMPS (aRAMPS) and posterior RAMPS (pRAMPS) has not been reported previously. We retrospectively reviewed and compared the demographic, perioperative, histopathologic, and survival data of patients who underwent aRAMPS or pRAMPS for PDAC. We also compared these two groups among patients without periadrenal infiltration or adrenal invasion. A total of 112 aRAMPS patients and 224 pRAMPS patients were evaluated. Periadrenal infiltration, neoadjuvant treatment, and concurrent vessel resection were more prevalent in the pRAMPS group. After excluding patients with periadrenal infiltration, 106 aRAMPS patients were compared with 157 pRAMPS patients. There were no significant differences between the aRAMPS and pRAMPS groups in the pathologic tumor size, resection margin, proportion of tangential margin in the R1 resection, and number of harvested lymph nodes. The median overall survival and disease-free survival also did not differ significantly between the two groups. We cautiously suggest that pRAMPS will not necessarily provide more beneficial histopathologic outcomes and survival rates for left-sided PDAC cases without periadrenal infiltration. If periadrenal infiltration is not suspected, aRAMPS alone should be sufficiently effective.
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Bui, Chau M., Sumire Kitahara, Wonwoo Shon, Tatsiana Pukhalskaya, and Bruce R. Smoller. "Lack of PRAME Expression in Cutaneous T-Cell Lymphomas." Dermatopathology 9, no. 1 (December 31, 2021): 11–16. http://dx.doi.org/10.3390/dermatopathology9010002.
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Cutaneous T-cell lymphomas (CTCLs) are rare tumors with no established markers that can reliably distinguish between benign and malignant lesions. Preferentially Expressed Antigen in Melanoma (PRAME) is a cancer/testis antigen that is found in many solid and hematologic malignancies. PRAME overexpression typically portends a poor prognosis and lower chemotherapeutic response. To date, no studies have established a role for PRAME in CTCL. An analysis was performed on 47 cases definitively diagnosed as CTCL: 25 cases of mycosis fungoides, 2 of Sezary syndrome, 5 of CD30+ lymphoproliferative disorder, 7 of primary cutaneous anaplastic large T-cell lymphoma, 3 of primary cutaneous CD4+ small/medium T-cell lymphoproliferative disorder, 1 of subcutaneous panniculitis-like T-cell lymphoma, and 4 of angiocentric T-cell lymphoma. PRAME immunohistochemistry was completely negative in all cases. PRAME expression was not found in any CTCL subtypes, suggesting that the pathogenesis of CTCL is not mediated by PRAME. Further study is required to identify biomarkers that might aid in the diagnosis and prognostication of CTCLs.
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Garnett, Bernice Raveche, Kenneth D. Rosenberg, and Daniel S. Morris. "Consumption of soda and other sugar-sweetened beverages by 2-year-olds: findings from a population-based survey." Public Health Nutrition 16, no. 10 (October 4, 2012): 1760–67. http://dx.doi.org/10.1017/s1368980012004399.
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AbstractObjectiveTo determine risk factors for consumption of soda and other sugar-sweetened beverages (SSB) among 2-year-old children.DesignThe analysis was performed using three linked data sets: the 2004–2005 Oregon Pregnancy Risk Assessment Monitoring Survey (PRAMS); its longitudinal follow-up, 2006–2007 Oregon PRAMS-2; and 2004–2005 Oregon birth certificates.SettingPRAMS is a surveillance programme supported by the federal Centers for Disease Control and Prevention and implemented by participating state health departments. Using mixed methods, PRAMS surveys women 2–6 months after a live birth. Oregon PRAMS-2 re-interviews respondents shortly after the index child's second birthday. Oregon PRAMS oversamples minority women.SubjectsUsing monthly cohorts, we randomly selected 5851 women from the 2004–2005 birth certificates. In total 1911 women completed both PRAMS and PRAMS-2. The weighted response rate of PRAMS-2 was 43·5 %.ResultsAlmost half of mothers (49·9 %) reported that their child drank SSB on at least 1 d/week. Mothers whose children drank SSB at least once weekly were more likely to have low income (adjusted OR = 2·83, 95 % CI 2·09, 3·83) and to eat out on ≥2 d/week (OR = 2·11 %, 95 % CI 1·66, 2·70). Hispanic and non-Hispanic black women were most likely to report that their child drank SSB at least once weekly.ConclusionsHalf of mothers reported that their 2-year-old children drank SSB at least once weekly. Public health interventions and policies should address childhood SSB consumption including educating health-care providers and parents.
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Rodríguez, Marcos Balsa, Marco Agus, Fabio Bettio, Fabio Marton, and Enrico Gobbetti. "Digital Mont’e Prama." Journal on Computing and Cultural Heritage 9, no. 4 (December 19, 2016): 1–23. http://dx.doi.org/10.1145/2915919.
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