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Van Treeck, Benjamin J., Mira Lotfalla, Thomas W. Czeczok, Taofic Mounajjed, Roger K. Moreira, Daniela S. Allende, Michelle D. Reid, et al. "Molecular and Immunohistochemical Analysis of Mucinous Cystic Neoplasm of the Liver." American Journal of Clinical Pathology 154, no. 6 (September 3, 2020): 837–47. http://dx.doi.org/10.1093/ajcp/aqaa115.
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Abstract Objectives Mucinous cystic neoplasm of the liver is characterized by neoplastic mucinous and/or biliary epithelium surrounded by ovarian-type stroma. Immunohistochemical studies have shown that the ovarian-type stroma expresses estrogen receptor, suggesting potential hormonal responsiveness. The molecular biology of mucinous cystic neoplasm of the liver remains poorly studied. Methods Transcriptome sequencing and immunohistochemistry were performed on a series of mucinous cystic neoplasms. Results Mucinous cystic neoplasm of the liver exhibited significantly increased RNA expression of ovarian stromal markers WT1, PR, and ER2 and sex cord stromal markers SF-1, inhibin-α, and calretinin compared with nonneoplastic liver. Immunohistochemistry confirmed the RNA-level data. Evidence for sex hormone biosynthesis was identified by significant overexpression of multiple estrogen biosynthetic enzymes. Expression of 17β-hydroxysteroid dehydrogenase 1 was confirmed immunohistochemically. Pathway analysis also identified significant upregulation of the hedgehog and Wnt pathways and significant downregulation of T-helper 1 and T-helper 2 pathways. Conclusions Mucinous cystic neoplasm of the liver recapitulates ovarian stroma at the morphologic, DNA, RNA, and protein levels. These data support the concept that this tumor likely arises from ectopic primitive gonadal tissue and/or stromal cells with capacity to transdifferentiate to ovarian cortical cells.
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Jan, Max, Daniel E. Grinshpun, Julian A. Villalba, Paola Dal Cin, David B. Sykes, A. John Iafrate, Benjamin L. Ebert, Gabriela S. Hobbs, and Valentina Nardi. "A cryptic imatinib-sensitive G3BP1-PDGFRB rearrangement in a myeloid neoplasm with eosinophilia." Blood Advances 4, no. 3 (January 30, 2020): 445–48. http://dx.doi.org/10.1182/bloodadvances.2019001182.
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Key Points Targeted RNA sequencing detected a cryptic G3BP1-PDGFRB rearrangement in a myeloid neoplasm with eosinophilia and normal FISH studies. Consistent with the patient’s response to imatinib, we demonstrate this rearrangement is oncogenic and sensitive to TKI in cell culture.
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Jamieson, Catriona, Qingfei Jiang, Frida Holm, Jane Isquith, Adam Mark, Cayla Mason, Wenxue Ma, et al. "Inflammatory Cytokine Responsive Enzymatic Mutagenesis Fuels Myeloproliferative Neoplasm Pre-Leukemia Stem Cell Evolution." Blood 134, Supplement_1 (November 13, 2019): 3780. http://dx.doi.org/10.1182/blood-2019-131510.
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Innate immune anti-viral adenosine to inosine (A-to-I) base editing enzymes (editases) promote hematopoietic stem cell (HSC) self-renewal and protect the human genome from retroviral integration in response to inflammatory cytokine signaling. However, hyper-editing has been linked to therapeutic resistance and cancer progression. Because myeloproliferative neoplasm (MPN) progression is typified by increased JAK2/STAT-mediated cytokine signaling, we investigated the cell type and context specific role of adenosine deaminase acting on RNA1 (ADAR1) editaseactivity in MPN pre-leukemia stem cell (pre-LSC) evolution into acute myeloid leukemia stem cells (LSCs). Here we show by whole transcriptome sequencing (RNA-seq) of 113 FACS-purified hematopoietic stem cells and progenitors from 78 individuals, including 54 MPN and AML patients and 24healthy young and aged individuals, that anti-viral signaling pathway activation and splice isoform switching from ADAR1p110 to JAK2/STAT-inducible ADAR1p150 RNA editase activation contributes to MPN progression. Pre-LSC evolution to LSC was characterized by ADAR1p150 upregulation, distinctive RNA editome patterns, STAT3 hyper-editing, increased replating as a measure of self-renewal. Moreover, LSC generation was typified by beta-catenin self-renewal pathway upregulation, which was recapitulated by lentiviral ADAR1p150 overexpression and reversed by lentiviral ADAR1p150 shRNA knockdown. Our studyunderscores the importance of inflammatory-cytokine fueled enzymatic mutagenesis in human MPN pre-LSC evolution to LSC. Thus, this study sets the stage for developing predictive RNA editome biomarkers of LSC generation to guidetherapeutic strategies aimed at preventing progression of hematopoietic malignancies. Disclosures Crews: Ionis Pharmaceuticals: Research Funding.
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Wang, Sam C., Ibrahim Nassour, Shu Xiao, Shuyuan Zhang, Xin Luo, Jeon Lee, Lin Li, et al. "SWI/SNF component ARID1A restrains pancreatic neoplasia formation." Gut 68, no. 7 (October 12, 2018): 1259–70. http://dx.doi.org/10.1136/gutjnl-2017-315490.
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ObjectiveARID1A is commonly mutated in pancreatic ductal adenocarcinoma (PDAC), but the functional effects of ARID1A mutations in the pancreas are unclear. Understanding the molecular mechanisms that drive PDAC formation may lead to novel therapies.DesignConcurrent conditional Arid1a deletion and Kras activation mutations were modelled in mice. Small-interfering RNA (siRNA) and CRISPR/Cas9 were used to abrogate ARID1A in human pancreatic ductal epithelial cells.ResultsWe found that pancreas-specific Arid1a loss in mice was sufficient to induce inflammation, pancreatic intraepithelial neoplasia (PanIN) and mucinous cysts. Concurrent Kras activation accelerated the development of cysts that resembled intraductal papillary mucinous neoplasm. Lineage-specific Arid1a deletion confirmed compartment-specific tumour-suppressive effects. Duct-specific Arid1a loss promoted dilated ducts with occasional cyst and PDAC formation. Heterozygous acinar-specific Arid1a loss resulted in accelerated PanIN and PDAC formation with worse survival. RNA-seq showed that Arid1a loss induced gene networks associated with Myc activity and protein translation. ARID1A knockdown in human pancreatic ductal epithelial cells induced increased MYC expression and protein synthesis that was abrogated with MYC knockdown. ChIP-seq against H3K27ac demonstrated an increase in activated enhancers/promoters.ConclusionsArid1a suppresses pancreatic neoplasia in a compartment-specific manner. In duct cells, this process appears to be associated with MYC-facilitated protein synthesis.
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Seibel, NL, and IR Kirsch. "Tumor detection through the use of immunoglobulin gene rearrangements combined with tissue in situ hybridization." Blood 74, no. 5 (October 1, 1989): 1791–95. http://dx.doi.org/10.1182/blood.v74.5.1791.1791.
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Abstract Leukemias and lymphomas can now be classified according to the particular immunoglobulin, T-cell receptor, or growth-affecting genes they are expressing. Recognition of the structural alterations of lymphoid DNA has been used to identify neoplasms of previously uncertain lineage, to aid in diagnosis, and to define the state of differentiation of the neoplasm. We have developed a procedurally simple, rapid turnaround technique for using tumor-specific gene alterations as tumor-specific markers. Probes can be constructed that will recognize only the gene expressed in the tumor and not those in any of the normal cells when used with tissue in situ hybridization. We demonstrate the application of direct sequencing of a specific gene of interest from total RNA from a patient with multiple myeloma. A probe is then generated from this sequence and applied directly to patient material.
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Seibel, NL, and IR Kirsch. "Tumor detection through the use of immunoglobulin gene rearrangements combined with tissue in situ hybridization." Blood 74, no. 5 (October 1, 1989): 1791–95. http://dx.doi.org/10.1182/blood.v74.5.1791.bloodjournal7451791.
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Leukemias and lymphomas can now be classified according to the particular immunoglobulin, T-cell receptor, or growth-affecting genes they are expressing. Recognition of the structural alterations of lymphoid DNA has been used to identify neoplasms of previously uncertain lineage, to aid in diagnosis, and to define the state of differentiation of the neoplasm. We have developed a procedurally simple, rapid turnaround technique for using tumor-specific gene alterations as tumor-specific markers. Probes can be constructed that will recognize only the gene expressed in the tumor and not those in any of the normal cells when used with tissue in situ hybridization. We demonstrate the application of direct sequencing of a specific gene of interest from total RNA from a patient with multiple myeloma. A probe is then generated from this sequence and applied directly to patient material.
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Nambiar, P. R., S. R. Boutin, R. Raja, and D. W. Rosenberg. "Global Gene Expression Profiling: A Complement to Conventional Histopathologic Analysis of Neoplasia." Veterinary Pathology 42, no. 6 (November 2005): 735–52. http://dx.doi.org/10.1354/vp.42-6-735.
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Transcriptional profiling of entire tumors has yielded considerable insight into the molecular mechanisms of heterogeneous cell populations within different types of neoplasms. The data thus acquired can be further refined by microdissection methods that enable the analyses of subpopulations of neoplastic cells. Separation of the various components of a neoplasm (i.e., stromal cells, inflammatory infiltrates, and blood vessels) has been problematic, primarily because of a paucity of tools for accurate microdissection. The advent of laser capture microdissection combined with powerful tools of linear amplification of RNA and high-throughput microarray-based assays have allowed the transcriptional mapping of intricate and highly complex networks within pure populations of neoplastic cells. With this approach, specific “molecular signatures” can be assigned to tumors of distinct or even similar histomorphology, thereby aiding the desired objective of pattern recognition, tumor classification, and prognostication. This review highlights the potential benefits of global gene expression profiling of tumor cells as a complement to conventional histopathologic analyses.
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Pronier, Elodie, Carole Almire, Hayat Mokrani, Aparna Vasanthakumar, Audrey Simon, Barbara da Costa Reis Monte Mor, Aline Massé, et al. "Inhibition of TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine disturbs erythroid and granulomonocytic differentiation of human hematopoietic progenitors." Blood 118, no. 9 (September 1, 2011): 2551–55. http://dx.doi.org/10.1182/blood-2010-12-324707.
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Abstract TET2 converts 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC) in DNA and is frequently mutated in myeloid malignancies, including myeloproliferative neoplasms. Here we show that the level of 5-hmC is decreased in granulocyte DNA from myeloproliferative neoplasm patients with TET2 mutations compared with granulocyte DNA from healthy patients. Inhibition of TET2 by RNA interference decreases 5-hmC levels in both human leukemia cell lines and cord blood CD34+ cells. These results confirm the enzymatic function of TET2 in human hematopoietic cells. Knockdown of TET2 in cord blood CD34+ cells skews progenitor differentiation toward the granulomonocytic lineage at the expense of lymphoid and erythroid lineages. In addition, by monitoring in vitro granulomonocytic development we found a decreased granulocytic differentiation and an increase in monocytic cells. Our results indicate that TET2 disruption affects 5-hmC levels in human myeloid cells and participates in the pathogenesis of myeloid malignancies through the disturbance of myeloid differentiation.
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Gupta, Sanjeev Kumar, Nitin Jain, Guilin Tang, Andrew Futreal, Sa A. Wang, Joseph D. Khoury, Richard K. Yang, et al. "A Cryptic BCR-PDGFRB Fusion Resulting in a Chronic Myeloid Neoplasm With Monocytosis and Eosinophilia: A Novel Finding With Treatment Implications." Journal of the National Comprehensive Cancer Network 18, no. 10 (October 2020): 1300–1304. http://dx.doi.org/10.6004/jnccn.2020.7573.
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RNA-seq was used to identify the partner gene and confirm the presence of a BCR-PDGFRB fusion. Identification of this fusion product resulted in successful treatment and long-term remission of this myeloid neoplasm. Based on our results, we suggest that despite current WHO recommendations, screening for PDGFRB rearrangement in cases of leukocytosis with eosinophilia and no other etiologic explanation is necessary, even if the karyotype is normal.
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Lim, Ha Jin, Jun Hyung Lee, Ju-Hyeon Shin, Seung Yeob Lee, Hyun-Woo Choi, Hyun Jung Choi, Seung Jung Kee, Jong Hee Shin, and Myung-Geun Shin. "Diagnostic Validation of a Clinical Laboratory-Oriented Targeted RNA Sequencing System As a Comprehensive Assay for Hematologic Malignancies." Blood 136, Supplement 1 (November 5, 2020): 38–39. http://dx.doi.org/10.1182/blood-2020-142264.
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Introduction Targeted RNA sequencing (RNA-seq) is a highly accurate method for sequencing transcripts of interest and can overcome limitations regarding resolution, throughput, and multistep workflow. However, RNA-seq has not been widely performed in clinical molecular laboratories due to the complexity of data processing and interpretation. We developed a customized targeted RNA-seq panel with a data processing protocol and validated its analytical performance for gene fusion detection using a subset of samples with different hematologic malignancies. Additionally, we investigated its applicability for identifying transcript variants and expression analysis using the targeted panel. Methods The target panel and customized oligonucleotide probes were designed to capture 84 genes associated with hematologic malignancies. Libraries were prepared from 800 to 1,500 ng of total RNA using GeneMediKit NGS-Leukemia-RNA kit (GeneMedica, Gwangju, Korea) and sequenced using Miseq reagent kit v3 (300 cycles) and MiseqDx (Illumina, San Diego, CA, USA). The diagnostic samples included one reference DNA (NA12878), one reference RNA (Cat no. 740000, Agilent Technologies), 14 normal peripheral blood (PB) samples, four validation bone marrow (BM) samples with known gene fusions, and 30 clinical BM or PB samples from seven categories of hematologic malignancies. The clinical samples included 27 BM aspirates and three PB samples composed of six acute myeloid leukemia, nine B-lymphoblastic leukemia/lymphoma, four T-lymphoblastic leukemia/lymphoma, three mature B-cell neoplasms, six MPN, one myelodysplastic/myeloproliferative neoplasm, and one myeloid/lymphoid neoplasm with eosinophilia and gene rearrangement. For the analytical validation of fusion detection, target gene coverage, between-run and within-run repeatability, and dilution tests (1:2 to 1:8 dilution) were performed. For the comparative analysis of fusion detection, the RNA-seq data were analyzed by STAR-Fusion and FusionCatcher and processed with stepwise filtering and prioritization strategy (Figure 1), and the result was compared to those of multiplex RT-PCR (HemaVision kit; DNA Technology, Aarhus, Denmark) or FISH (MetaSystems Gmbh, Althusseim, Germany) using 30 clinical samples. The RNA-seq data from clinical samples were additionally analyzed by FreeBayes for variant detection and by StringTie for expression profiling (Figure 1). Results First, the analytical validation showed reliable results in target gene coverage, between-run and within-run repeatability, and linearity tests. The uniformity of coverage (% of base pairs higher than 0.2 × total average depth) was calculated to be 99.8%, which revealed even coverage for the target genes in the panel using the reference DNA. Both in the within-run and between-run tests, the read counts and FFPM (fusion fragments per million) of all replicates showed reliable repeatability (r2 = 0.9655 and 0.9874, respectively). The FFPM of the diluted analytical samples including BCR-ABL1 and PML-RARA showed linear log2-fold-changes (r2 = 0.9852 and 0.9447, respectively). Second, compared to multiplex RT-PCR and FISH using 30 clinical samples, targeted RNA-seq combined with filtering and prioritization strategies detected all 13 known fusions and newly detected 17 fusions. Finally, 16 disease- and drug resistance-associated variants on the expressed transcripts of ABL1, GATA2, IKZF1, JAK2, RUNX1, and WT1 were simultaneously designated and expression analysis showed distinct four clusters of clinical samples according to the cancer subtypes and lineages. Conclusions Our customized targeted RNA-seq system provided a stable analytical performance and a more sensitive identification of gene fusions than conventional molecular methods in various clinical samples. In addition, clinically significant variants in the transcripts and expression profiling could be simultaneously identified directly from the RNA-seq data without the need for additional parallel testing. Our study identified the advantages of the clinical laboratory-oriented targeted RNA-seq system to enhance the diagnostic yield for gene fusion detection and to simplify the diagnostic steps as providing a comprehensive tool for analyzing hematologic malignancies in the clinical laboratory. Figure 1 Disclosures Lee: National Research Foundation of Korea: Research Funding.
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Slater, E. P., S. M. Diehl, P. Langer, B. Samans, A. Ramaswamy, A. Zielke, and D. K. Bartsch. "Analysis by cDNA microarrays of gene expression patterns of human adrenocortical tumors." European Journal of Endocrinology 154, no. 4 (April 2006): 587–98. http://dx.doi.org/10.1530/eje.1.02116.
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Objectives: Adrenocortical carcinoma (ACC) is a rare malignant neoplasm with extremely poor prognosis. The molecular mechanisms of adrenocortical tumorigenesis are still not well understood. The comparative analysis by cDNA microarrays of gene-expression patterns of benign and malignant adrenocortical tumors allows us to identify new tumor-suppressor genes and proto-oncogenes underlying adrenocortical tumorigenesis. Design and methods: Total RNA from fresh-frozen tissue of 10 ACC and 10 benign adrenocortical adenomas was isolated after histologic confirmation of neoplastic cellularity of at least 85%. The reference consisted of pooled RNA of 10 normal adrenal cortex samples. Amplified RNA of tumor and reference was used to synthesize Cy3- and Cy5-fluorescently labeled cDNA in a flip-color technique. D-chips containing 11 540 DNA spots were hybridized and scanned and the images were analyzed by ImaGene 3.0 software. Results: The comparative analysis of gene expression revealed many genes with more than fourfold expression difference between ACC and normal tissue (42 genes), cortical adenoma and normal tissue (11 genes), and ACC and cortical adenoma (21 genes) respectively. As confirmed by real-time PCR, the IGF2 gene was significantly upregulated in ACCs versus cortical adenomas and normal cortical tissue. Genes that were downregulated in adrenocortical tumors included chromogranin B and early growth response factor 1. Conclusions: Comprehensive expression profiling of adrenocortical tumors by the cDNA microarray technique is a very powerful tool to elucidate the molecular steps associated with the tumorigenesis of these ill-defined neoplasms. To evaluate the role of identified genes, further detailed analyses, including correlation with clinical data, are required.
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Mograbi, Madison, Michael S. Stump, David T. Luyimbazi, Mohammad H. Shakhatreh, and Douglas J. Grider. "Pancreatic Inflammatory Pseudotumor-Like Follicular Dendritic Cell Tumor." Case Reports in Pathology 2019 (December 5, 2019): 1–5. http://dx.doi.org/10.1155/2019/2648123.
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Follicular dendritic cell sarcoma (FDCS) is a rare and underdiagnosed malignant neoplasm which characteristically presents as a solitary, slow-growing mass with no discrete symptoms. Histologically, lymphocytes and spindle cells featuring large nucleoli in a whorled pattern are usually seen. FDCS is classically found in cervical and axillary lymph nodes, with occasional involvement of extranodal sites. Inflammatory pseudotumor-like follicular dendritic cell tumor (IPT-like FDCT) is an uncommon subcategory of this neoplasm, demonstratively linked to the Epstein-Barr virus (EBV). This neoplasm can present similarly to FDCS, but systemic symptoms may be seen. Although, often found in the spleen and occasionally the liver, IPT-like FDCT has not previously been described within the pancreas. Presented, is an IPT-like FDCT of the pancreas and spleen of a 70 years old woman. Histologic features include variably sized geographic suppurative granulomas with chronic inflammatory cells and an atypical spindle cell proliferation with prominent nucleoli. Positivity for CD45 and CD68 in the larger spindled cells points to an inflammatory pseudotumor subtype and co-expression of CD21, CD23, and CD35 were indicative of follicular dendritic differentiation. The pseudotumor additionally demonstrated EBV-encoded RNA (EBER) positivity typical of IPT-like FDCT. Differentiation between inflammatory pseudotumor (IPT) and inflammatory myofibroblastic tumor (IMT) is additionally discussed.
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Nomburg, Jason, Susan Bullman, Sun Sook Chung, Katsuhiro Togami, Mark A. Walker, Gabriel K. Griffin, Elizabeth A. Morgan, et al. "Comprehensive metagenomic analysis of blastic plasmacytoid dendritic cell neoplasm." Blood Advances 4, no. 6 (March 17, 2020): 1006–11. http://dx.doi.org/10.1182/bloodadvances.2019001260.
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Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a hematologic malignancy believed to originate from plasmacytoid dendritic cells (pDCs), the immune cells responsible for producing type 1 interferons during infection. Nearly all patients with BPDCN have prominent skin involvement, with cutaneous infiltration occupying the dermis and subcutis. One half of patients present with BPDCN cells only in the skin, with no evidence of disease elsewhere. Because normal pDCs are rare or absent in cutaneous sites, and they only traffic to the skin after activation by pathogen or inflammation, our aim was to determine if a microorganism is associated with BPDCN. We performed RNA sequencing in BPDCN skin and bone marrow, with cutaneous T-cell lymphoma (CTCL) and normal skin as controls. GATK-PathSeq was used to identify known microbial sequences. Bacterial reads in BPDCN skin were components of normal flora and did not distinguish BPDCN from controls. We then developed a new computational tool, virID (Viral Identification and Discovery; https://github.com/jnoms/virID), for identification of microbial-associated reads remaining unassigned after GATK-PathSeq. We found no evidence for a known or novel virus in BPDCN skin or bone marrow, despite confirming that virID could identify Merkel cell polyomavirus in Merkel cell carcinoma, human papillomavirus in head and neck squamous cell carcinoma, and Kaposi’s sarcoma herpesvirus in Kaposi’s sarcoma in a blinded fashion. Thus, at the level of sensitivity used here, we found no clear pathogen linked to BPDCN.
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Iwashita, Takuji, Yohei Shirakami, Shinya Uemura, Naoki Mita, Yuhei Iwasa, and Masahito Shimizu. "Mo1405 – Micro-Rna Analysis of Cyst Fluid for Diagnosing Malignant Alteration of Intra Ductal Papillary Neoplasm." Gastroenterology 156, no. 6 (May 2019): S—765. http://dx.doi.org/10.1016/s0016-5085(19)38857-2.
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Chong, Charing, and Patrick Tang. "Circulating Long Non-coding RNA from Cystic Neoplasm of Pancreas for Early Cancer Diagnosis and Treatment." HPB 21 (2019): S430. http://dx.doi.org/10.1016/j.hpb.2019.10.2170.
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Iwakura, Hiroshi, Kiminori Hosoda, Ryuichiro Doi, Izumi Komoto, Haruo Nishimura, Choel Son, Junji Fujikura, et al. "Ghrelin Expression in Islet Cell Tumors: Augmented Expression of Ghrelin in a Case of Glucagonoma with Multiple Endocrine Neoplasm Type I." Journal of Clinical Endocrinology & Metabolism 87, no. 11 (November 1, 2002): 4885–88. http://dx.doi.org/10.1210/jc.2002-020882.
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Abstract Ghrelin is a 28-amino acid peptide that regulates GH release together with GHRH and somatostatin. The expression of ghrelin has been detected in the stomach, small intestine, hypothalamus, pituitary gland, kidney, placenta, and testis. Recently it was reported that ghrelin is present in pancreatic α-cells and that it stimulates insulin secretion. In this study, we examined the ghrelin expression in two cases of glucagonoma and two cases of insulinoma by Northern blot analysis and immunohistochemistry. Ghrelin expression was identified in a case of glucagonoma associated with multiple endocrine neoplasm type I both by Northern blot analysis using total RNA and by immunohistochemistry, although the plasma ghrelin level was not elevated. This is the first case of tumor in which ghrelin gene expression was detected by Northern blot analysis using total RNA.
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Togami, Katsuhiro, Sun Sook Chung, Vikas Madan, Christopher M. Kenyon, Lucia Cabal-Hierro, Justin Taylor, Sunhee Kim, et al. "Male-Biased Spliceosome Mutations in Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Impair pDC Activation and Apoptosis." Blood 136, Supplement 1 (November 5, 2020): 13–14. http://dx.doi.org/10.1182/blood-2020-137727.
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Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive, male-biased (>3:1 M:F) hematologic malignancy in which some patients have bone marrow involvement at diagnosis (50%) and most have tumor formation in the skin (~90%), often preceding marrow disease. The prognosis is poor (median survival of 12-24 months) and there is unmet need for biological insight. TET2, ASXL1, and RNA splicing genes (SRSF2, SF3B1, and ZRSR2) are recurrently mutated in BPDCN. The X chromosome gene ZRSR2 was the most frequently mutated spliceosome gene reported in a prior BPDCN cohort (7 of 24, 29.2%; Taylor, ASH 2013). Our goal was to define the functional consequences of ZRSR2 mutations in BPDCN. First, we confirmed the frequency of ZRSR2 mutations in a larger cohort from the US and Europe; we found ZRSR2 mutations in 24 of 93 (25.8%). Notably, ZRSR2 mutations were almost exclusively in males (23/73 males vs 1/20 females, P=0.019). Next, we compared the global mutation pattern to 30 predefined signatures from >7000 cancers in COSMIC. Analysis of all somatic single nucleotide variants in 11 tumor-normal pairs using whole exome sequencing (tumor was sorted BPDCN cells from marrow) revealed that BPDCN had an ultraviolet (UV)-induced mutation signature (score >0.25 in 6/11 or 55%; Figure 1A). For comparison, we detected the UV signature in melanoma but not in AML from The Cancer Genome Atlas. These data suggest that mutations acquired in the skin stage of BPDCN evolution are retained in subsequent leukemic disease. Next, we performed RNA-sequencing from sorted BPDCN and normal plasmacytoid dendritic cells (pDCs). Differentially expressed genes between BPDCN and pDCs (BCL2, MYB, IRF4, CEP70, IGLL1, GZMB) were similar to those that distinguish BPDCNs from pDCs by bulk and single cell RNA-sequencing. By gene set enrichment analysis (GSEA), BPDCNs were enriched for overexpression of MYC/E2F targets and PI3K/AKT/MTORC1 signaling pathway-associated genes. BPDCNs transcriptomes were also enriched for gene sets associated with RNA splicing machinery and RNA nonsense mediated decay (NMD). To link RNA splicing with functional consequences of ZRSR2 mutations, we generated ZRSR2-knockout BPDCN cells (CAL1) using CRISPR/Cas9. This models primary tumors because ZRSR2-mutant BPDCNs have complete loss of ZRSR2 protein. Activation marker (CD80) upregulation and type 1 interferon secretion after Toll-like receptor (TLR) stimulation with lipopolysaccharide (LPS) or R848 were reduced in ZRSR2-deficient cells. We found similar defective cytokine production in stimulated primary BPDCN cells compared to normal pDCs. After activation, normal pDCs undergo apoptosis in a negative feedback process. In contrast, ZRSR2-knockout, but not control cells, were protected from TLR activation-induced apoptosis. Reexpression of wild-type ZRSR2 in knockout cells restored activation-induced apoptosis (Figure 1B). These data suggested that ZRSR2-mutant BPDCNs have defects downstream of TLR stimulation. By RNA-sequencing, we found that IRF7 mRNA was mis-spliced in all ZRSR2- (2/2), SRSF2- (4/4), and SF3B1- (1/1) mutant BPDCNs compared to those with no mutated splicing gene (4/4). IRF7 (interferon regulatory factor 7) is a transcription factor activated by TLR signaling that is important for pDC activation and apoptosis. The IRF7 mRNA transcript contains a "weak intron" (intron 4) that is subject to intron retention, which leads to NMD and reduced IRF7 protein level in stimulated dendritic cells (Luke, Mol Cell 2019). IRF7 intron 4 was mis-spliced in ZRSR2-, SRSF2-, and SF3B1-mutant BPDCNs. ZRSR2-knockout CAL1 cells had severely impaired ability to upregulate IRF7 after LPS stimulation, which was partially rescued by reepxression of wild-type ZRSR2 (Figure 1C). Expression of constitutively activated IRF7 inhibited growth of both ZRSR2-knockout and control cells, confirming that the inability to activate IRF7 is important for the effect of ZRSR2 loss on TLR agonist-induced growth inhibition. In conclusion, male-biased ZRSR2 mutations are frequent in BPDCN and impair pDC activation and apoptosis, at least in part via TLR-IRF7. These data may explain why BPDCNs have an impaired activation state (Bierd, BCJ 2019). They also suggest that splicing factor mutations affect cell type-specific pathways to promote transformation, underscoring the importance of studying cancer genes in relevant contexts. Figure Disclosures Griffin: Moderna Therapeutics: Consultancy. Ghandi:Monte Rosa Therapeutics: Consultancy; Cambridge Data Science LLC: Current Employment, Current equity holder in private company. Seiler:Remix Therapeutics: Current Employment. Konopleva:Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Eli Lilly: Research Funding; Genentech: Consultancy, Research Funding; Agios: Research Funding; Rafael Pharmaceutical: Research Funding; Sanofi: Research Funding; AbbVie: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding; Calithera: Research Funding; Amgen: Consultancy; F. Hoffmann La-Roche: Consultancy, Research Funding; Cellectis: Research Funding; Ablynx: Research Funding; Kisoji: Consultancy; Stemline Therapeutics: Consultancy, Research Funding. Pemmaraju:Cellectis: Research Funding; Daiichi Sankyo: Research Funding; DAVA Oncology: Honoraria; Plexxikon: Research Funding; Blueprint Medicines: Honoraria; Incyte Corporation: Honoraria; SagerStrong Foundation: Other: Grant Support; Celgene: Honoraria; Pacylex Pharmaceuticals: Consultancy; Affymetrix: Other: Grant Support, Research Funding; MustangBio: Honoraria; Roche Diagnostics: Honoraria; Novartis: Honoraria, Research Funding; LFB Biotechnologies: Honoraria; Stemline Therapeutics: Honoraria, Research Funding; AbbVie: Honoraria, Research Funding; Samus Therapeutics: Research Funding. Abdel-Wahab:H3 Biomedicine Inc.: Consultancy, Research Funding; Merck: Consultancy; Janssen: Consultancy; Envisagenics Inc.: Current equity holder in private company. Lane:Qiagen: Consultancy; Abbvie: Research Funding; Stemline Therapeutics: Research Funding.
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Covach, Adam, Sanjay Patel, Heather Hardin, and Ricardo V. Lloyd. "Phosphorylated Mechanistic Target of Rapamycin (p-mTOR) and Noncoding RNA Expression in Follicular and Hürthle Cell Thyroid Neoplasm." Endocrine Pathology 28, no. 3 (June 28, 2017): 207–12. http://dx.doi.org/10.1007/s12022-017-9490-7.
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Sapienza, Maria Rosaria, Giuseppe Benvenuto, Manuela Ferracin, Saveria Mazzara, Fabio Fuligni, Claudio Tripodo, Beatrice Belmonte, et al. "Newly-Discovered Neural Features Expand the Pathobiological Knowledge of Blastic Plasmacytoid Dendritic Cell Neoplasm." Cancers 13, no. 18 (September 18, 2021): 4680. http://dx.doi.org/10.3390/cancers13184680.
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Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare and highly aggressive hematologic malignancy originating from plasmacytoid dendritic cells (pDCs). The microRNA expression profile of BPDCN was compared to that of normal pDCs and the impact of miRNA dysregulation on the BPDCN transcriptional program was assessed. MiRNA and gene expression profiling data were integrated to obtain the BPDCN miRNA-regulatory network. The biological process mainly dysregulated by this network was predicted to be neurogenesis, a phenomenon raising growing interest in solid tumors. Neurogenesis was explored in BPDCN by querying different molecular sources (RNA sequencing, Chromatin immunoprecipitation-sequencing, and immunohistochemistry). It was shown that BPDCN cells upregulated neural mitogen genes possibly critical for tumor dissemination, expressed neuronal progenitor markers involved in cell migration, exchanged acetylcholine neurotransmitter, and overexpressed multiple neural receptors that may stimulate tumor proliferation, migration and cross-talk with the nervous system. Most neural genes upregulated in BPDCN are currently investigated as therapeutic targets.
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Torres, Harrys A., Georgios Angelidakis, Ying Jiang, Minas Economides, Marcel Yibirin, Robert Orlowski MD, Richard Champlin, Srdan Verstovsek, Issam I. Raad, and Issam I. Raad. "1052. Serologic vs. molecular testing for screening for hepatitis C virus infection in patients with hematologic malignancies with and without prior hematopoietic cell transplant recipients." Open Forum Infectious Diseases 7, Supplement_1 (October 1, 2020): S555—S556. http://dx.doi.org/10.1093/ofid/ofaa439.1238.
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Abstract Background The prevalence of chronic hepatitis C virus (HCV) infection in patients with cancer in the U.S. has been reported to be 1.5% overall and up to 10.6% in specific subgroups. Testing for antibody to HCV (anti-HCV) is a low-cost diagnostic method in widespread use worldwide; however, the optimal screening test for HCV in cancer patients has not been established. We sought to identify the optimal screening test for HCV in patients with hematologic malignancies and/or prior hematopoietic cell transplant (HCT). Methods New patients who were seen at the Lymphoma/Myeloma, Leukemia, and Stem Cell Transplant clinics at MD Anderson Cancer Center (02/11/2019-11/5/2019) were simultaneously screened for HCV with serologic (antibody to HCV [anti-HCV]) and molecular (HCV RNA) assays. Anti-HCV testing was performed by using the ARCHITECT Anti-HCV assay and HCV RNA testing was performed by using the Cobas HCV test. The agreement between the two tests was evaluated using Cohen’s kappa statistic and McNemar’s test. All tests were two-sided with a significance level of 0.05. Results A total of 214 patients were enrolled in the study, of whom 127 (59%) were men (Table), One hundred forty-nine patients (70%) had a lymphoid neoplasm, 65 (30%) had a myeloid neoplasm, and 15 (7%) underwent HCT. Ninety-three patients (43%) had progressive disease. Three patients (1.4%) had positive anti-HCV, and two (0.9%) had positive HCV RNA. The overall percentage agreement was 99.5% (95% CI, 97.4% to 99.9%). Of the 3 patients with positive anti-HCV, 2 had positive and 1 had negative HCV RNA. There were no cases of seronegative HCV infection. The positive percentage agreement was 66.7% (95 CI, 20.8% to 93.9%), and the negative percentage agreement was 100.0% (95% CI, 98.2% to 100.0%). Cohen’s Kappa coefficient was 0·80 (95% CI, 0.41 to 1.00, p < 0·0001), indicating substantial agreement between anti-HCV and HCV RNA tests for diagnosis of HCV infection. Conclusion The diagnostic yield for screening for chronic HCV infection in heavily immunocompromised cancer patients is similar for serologic and molecular testing. The use of anti-HCV, a diagnostic method with low cost, in patients with cancer would contribute to the World Health Organization’s goal of HCV elimination worldwide. Table. Characteristics of the study population (n=214) Disclosures Harrys A. Torres, MD, Merck & Co., Inc. (Grant/Research Support) Issam I. Raad, MD, Citius (Other Financial or Material Support, Ownership interest)Cook Medical (Grant/Research Support)Inventive Protocol (Other Financial or Material Support, Ownership interest)Novel Anti-Infective Technologies (Shareholder, Other Financial or Material Support, Ownership interest)
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Parfenyev, Sergey E., Sergey V. Shabelnikov, Danila Y. Pozdnyakov, Olga O. Gnedina, Leonid S. Adonin, Nickolai A. Barlev, and Alexey G. Mittenberg. "Proteomic Analysis of Zeb1 Interactome in Breast Carcinoma Cells." Molecules 26, no. 11 (May 24, 2021): 3143. http://dx.doi.org/10.3390/molecules26113143.
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Breast cancer is the most frequently diagnosed malignant neoplasm and the second leading cause of cancer death among women. Epithelial-to-mesenchymal Transition (EMT) plays a critical role in the organism development, providing cell migration and tissue formation. However, its erroneous activation in malignancies can serve as the basis for the dissemination of cancer cells and metastasis. The Zeb1 transcription factor, which regulates the EMT activation, has been shown to play an essential role in malignant transformation. This factor is involved in many signaling pathways that influence a wide range of cellular functions via interacting with many proteins that affect its transcriptional functions. Importantly, the interactome of Zeb1 depends on the cellular context. Here, using the inducible expression of Zeb1 in epithelial breast cancer cells, we identified a substantial list of novel potential Zeb1 interaction partners, including proteins involved in the formation of malignant neoplasms, such as ATP-dependent RNA helicase DDX17and a component of the NURD repressor complex, CTBP2. We confirmed the presence of the selected interactors by immunoblotting with specific antibodies. Further, we demonstrated that co-expression of Zeb1 and CTBP2 in breast cancer patients correlated with the poor survival prognosis, thus signifying the functionality of the Zeb1–CTBP2 interaction.
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Loscocco, Giuseppe G., Giacomo Coltro, Paola Guglielmelli, and Alessandro M. Vannucchi. "Integration of Molecular Information in Risk Assessment of Patients with Myeloproliferative Neoplasms." Cells 10, no. 8 (August 2, 2021): 1962. http://dx.doi.org/10.3390/cells10081962.
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Philadelphia chromosome-negative myeloproliferative neoplasms (MPN) are clonal disorders of a hematopoietic stem cell, characterized by an abnormal proliferation of largely mature cells driven by mutations in JAK2, CALR, and MPL. All these mutations lead to a constitutive activation of the JAK-STAT signaling, which represents a target for therapy. Beyond driver ones, most patients, especially with myelofibrosis, harbor mutations in an array of “myeloid neoplasm-associated” genes that encode for proteins involved in chromatin modification and DNA methylation, RNA splicing, transcription regulation, and oncogenes. These additional mutations often arise in the context of clonal hematopoiesis of indeterminate potential (CHIP). The extensive characterization of the pathologic genome associated with MPN highlighted selected driver and non-driver mutations for their clinical informativeness. First, driver mutations are enlisted in the WHO classification as major diagnostic criteria and may be used for monitoring of residual disease after transplantation and response to treatment. Second, mutation profile can be used, eventually in combination with cytogenetic, histopathologic, hematologic, and clinical variables, to risk stratify patients regarding thrombosis, overall survival, and rate of transformation to secondary leukemia. This review outlines the molecular landscape of MPN and critically interprets current information for their potential impact on patient management.
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Taylor, Justin, Mark Donoghue, Raajit K. Rampal, Roni Tamari, Martin S. Tallman, Darren R. Feldman, Barry S. Taylor, and Omar I. Abdel-Wahab. "Hematologic Malignancies Arising in Patients with Germ Cell Tumors: Secondary Somatic Differentiation of Hematopoietic Malignancies from Germ Cell Precursors." Blood 132, Supplement 1 (November 29, 2018): 87. http://dx.doi.org/10.1182/blood-2018-99-111976.
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Abstract Genomic analyses have recently illuminated our understanding of therapy-associated myeloid neoplasms in patients receiving therapy for other cancers. One of the most intriguing relationships between solid tumors and myeloid neoplasms involves a unique clinical entity of patients with germ cell tumors (GCT) and myeloid neoplasms. One in 17 patients with primary mediastinal germ cell tumor (PMGCT) develops a hematologic malignancy (most commonly AML, MDS, or histiocytosis) and the median survival in such patients is poor at only 5 months. Intriguingly, the presence of isochromosome 12p [i(12p)], a clonal marker common to GCTs but absent in de novo myeloid neoplasms, has been demonstrated as shared across GCTs and myeloid neoplasms in such individuals. While these data suggest a clonal relationship between the two, the exact nature of the shared origin is unknown. There are two competing hypotheses to explain this: (1) an embryonic progenitor with the capacity to differentiate into germ cell and hematopoietic lineages harbors the initiating genetic alterations and drives development of both malignancies or (2) the leukemia is derived directly from GCTs with the capacity to differentiate into hematopoietic lineages. To trace the clonal evolution of these seemingly unrelated cancer types and identify recurrent genomic lesions in leukemias present in GCT patients, we applied whole exome sequencing (WES), targeted genomic analyses, and/or RNA-seq to leukemias, GCTs, and germline DNA in a series of patients with myeloid neoplasms and concurrent GCTs. We collected 12 patients with GCT and synchronously or metachronously occurring myeloid neoplasms (8 AML, 5 MDS/CMML, 2 histiocytic sarcoma (some had >1 hematologic malignancy)) with an average of 4 months between the two diagnoses. Consistent with prior reports, all were young men (median age 26) with PMGCT and nonseminomatous histology and a 3-month median survival from leukemia diagnosis (Fig. A). In each case, at least one copy number alteration or coding mutation was shared across the GCT and hematopoietic neoplasm, demonstrating the shared origin of both lesions. For example, half of the patients (6/12) carried i(12p) in both the GCT and hematopoietic neoplasm. In the i(12p) negative cases, somatic genetic alterations identified in the GCT were also found in the leukemia. The most common genomic alterations in leukemias beyond i(12p) included mutations activating RAS-PI3K-AKT signaling (including PTEN, RAS and PI3K isoform mutations) or inactivating TP53 (Fig. B). The only exception was a testicular-only GCT patient who developed clonally distinct acute promyelocytic leukemia; however, further analysis identified this as a chemotherapy-induced neoplasm with the PML-RARa breakpoint mapped to an etoposide sensitive area and this patient was not counted amongst the 12 cases. We next traced the evolutionary history of clonally related GCTs and leukemias based on cancer cell fraction of all coding mutations and copy number alterations using WES of DNA from each tumor type and finger nails. In each instance, we identified clonal evolution of the hematopoietic malignancies from antecedent precursors within the GCT. To illustrate this, a 19-year-old male developed successive diagnoses of histiocytic sarcoma, CMML, and AML within 18 months of GCT diagnosis. Lineage tracing by WES of each of these four individual cancers revealed that all four were clonally related, and the histiocytic sarcoma, CMML, and AML were all derived from the GCT with a common precursor giving rise to the three hematopoietic malignancies (Fig. C-D). Moreover, the histiocytic sarcoma evolved separately from CMML/AML in this patient, where the AML represented leukemic transformation from the CMML. These data conclusively demonstrate that myeloid neoplasms developing in patients with PMGCT represent secondary somatic differentiation of a hematologic progenitor from totipotent aberrant cells that are present in the GCT. Thus, GCT-associated leukemias have a unique ontogeny from de novo and/or secondary myeloid neoplasms, which originate from progenitors within the bone marrow. As such, GCT-associated leukemias have characteristic genomic alterations hallmarked by frequent i(12p) in combination with mutations activating RAS-PI3K-AKT signaling and inactivating TP53, and these patients do poorly even when treated with aggressive contemporary chemotherapy. Figure Figure. Disclosures Rampal: Jazz: Consultancy, Honoraria; Celgene: Honoraria; Constellation: Research Funding; Incyte: Honoraria, Research Funding; Stemline: Research Funding. Tallman:ADC Therapeutics: Research Funding; Orsenix: Other: Advisory board; AROG: Research Funding; Cellerant: Research Funding; AbbVie: Research Funding; Daiichi-Sankyo: Other: Advisory board; BioSight: Other: Advisory board.
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Kutyna, Monika M., Li Yan A. Wee, Sharon Paton, Dimitrios Cakouros, Agnieszka Arthur, Rakchha Chhetri, Andreas W. Schreiber, et al. "Therapy-Related Myeloid Neoplasm Has a Distinct Pro-Inflammatory Bone Marrow Microenvironment and Delayed DNA Damage Repair." Blood 136, Supplement 1 (November 5, 2020): 37–38. http://dx.doi.org/10.1182/blood-2020-137277.
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Introduction: Therapy-related myeloid neoplasms (t-MN) are associated with extremely poor clinical outcomes in otherwise long-term cancer survivors. t-MN accounts for ~20% of cases of myeloid neoplasms and is expected to rise due to the increased use of chemotherapy/radiotherapy (CT/RT) and improved cancer survivorship. Historically, t-MN was considered a direct consequence of DNA damage induced in normal hematopoietic stem cells (HSC) by DNA damaging cytotoxics. However, these studies have largely ignored the bone marrow (BM) microenvironment and the effects of age and concurrent/previous cancers. Aim: We performed an exhaustive functional study of mesenchymal stromal cells (MSC) obtained from a comparatively large cohort of t-MN patients and carefully selected control populations to evaluate the long-term damage induced by cytotoxic therapy to BM microenvironment and its impact on malignant and normal haematopoiesis. Methods: Four different cohorts were used: (1) t-MN, in which myeloid malignancy occurred after CT/RT for a previous cancer (n=18); (2) patients with multiple cancer and in which a myeloid neoplasm developed following an independent cancer which was not treated with CT/RT (MC-MN; n=10); (3) primary MN (p-MN; n=7) untreated and without any prior cancer or CT/RT; (4) age-matched controls (HC; n=17). Morphology, proliferation, cellular senescence, differentiation potential and γH2AX DNA damage response was performed. Stem/progenitor supportive capacity was assessed by co-culturing haematopoietic stem cells on MSC feeder-layer in long-term culture initiating assay (LTC-IC). Cytokine measurements were performed using 38-plex magnetic bead panel (Millipore) and RNA sequencing libraries were prepared with Illumina TruSeq Total RNA protocol for 150bp paired-end sequencing on a NextSeq500 instrument. Functional enrichment analysis was performed using EnrichR software. Results: MSC cultured from t-MN patients were significantly different from HC, p-MN and MC-MN MSC according to multiple parameters. They exhibited aberrant morphology consisting of large, rounded and less adhesive cells compared to typical spindle-shaped morphology observed with controls. MSC from myeloid neoplasm also showed impaired proliferation, senescence, osteo- and adipogenic differentiation with t-MN MSC showing the greatest differences. DNA repair was dramatically impaired compared to p-MN and HC (Fig.1A). Importantly, these aberrant t-MN MSC were not able to support normal or autologous in vitro long-term haematopoiesis (Fig.1B). The biological characteristic and poor haematopoietic supportive capacity of MSC could be "cell-intrinsic" or driven by an altered paracrine inflammatory microenvironment. Interestingly, several inflammatory cytokines were higher in t-MN compared with marrow interstitial fluid obtained from p-MN patients (Fig.1Ci) and many of these including Fractalkine, IFNα2, IL-7 and G-CSF were also significantly higher in t-MN MSC conditional media (Fig.1Cii). Together, this data suggest that t-MN microenvironment is distinct from p-MN with paracrine production of pro-inflammatory milieu that may contribute to poor HSC supportive capacity. Preliminary whole transcriptome analysis revealed differential gene expression between t-MN and HC (Fig.1Di) and p-MN MSC. Importantly, the deregulated genes play critical role in cell cycle, DNA damage repair, and cellular senescence pathways explaining phenotypical characteristic of t-MN MSC (Fig.1Dii). Moreover CXCL12 expression, a key regulator of haematopoiesis, was significantly lower in t-MN compared to HC (p=0.002) and p-MN MSC (p=0.009), thus explaining poor HSC supportive capacity. The key difference between the p-MN, MC-MN and t-MN is prior exposure to CT/RT. To study this we obtained MSC from two t-MN patients for whom we had samples at the time of their primary cancer, post high-dose chemotherapy and at the time of t-MN. MSC displayed aberrant proliferation and differentiation capacity after high-dose cytotoxic therapy (2 to 4 years prior to developing t-MN) and remained aberrant at t-MN diagnosis (Fig.1E). Conclusions: BM-MSC from t-MN patients are significantly abnormal compared with age-matched controls and typical myeloid neoplasm. Importantly, prior CT/RT leads to long-term irreversible damage to the BM microenvironment which potentially contributes to t-MN pathogenesis. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase:Novartis Australia: Research Funding.
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Liu, Zhen, Zhenming Kang, Yujian Dai, Huiming Zheng, and Yingjun Wang. "Long noncoding RNA LINC00342 promotes growth of infantile hemangioma by sponging miR-3619-5p from HDGF." American Journal of Physiology-Heart and Circulatory Physiology 317, no. 4 (October 1, 2019): H830—H839. http://dx.doi.org/10.1152/ajpheart.00188.2019.
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Infantile hemangiomas (IH) are a type of benign vascular neoplasm that may cause permanent scarring. Hemangioma-derived endothelial cells (HemECs) are commonly used as an in vitro model to study IH. Long noncoding RNA is a type of RNA transcript longer than 200 nucleotides that does not encode any protein. LINC00342 was discovered to regulate proliferation and apoptosis in nonsmall cell lung cancer. However, the role of LINC00342 in IH has never been reported before. Expressions of LINC00342 and miR-3619-5p were detected in proliferating versus normal skin tissues. Colony formation and Cell-Couting Kit 8 assays were carried out to study the effects on cell proliferation after knockdown and overexpression of LINC00342, respectively. Meanwhile caspase-3 activity and nucleosomal fragmentation assay were applied to detect cell apoptosis. Micro-RNA binding sites on LINC00342 and hepatoma-derived growth factor (HDGF) were predicted and confirmed via dual-luciferase reporter assay. Biotin RNA pulldown assay was used to verify the direct binding between RNA molecules. LINC00342 enhanced proliferation and inhibited apoptosis in HemECs. MiR-3619-5p targeted both LINC00342 and HDGF, where LINC00342 sponged miR-3619-5p and positively regulated HDGF. HDGF knockdown rescued the effects of LINC00342 on HemECs. The LINC00342-miR-3619-5p-HDGF signaling pathway could regulate cell proliferation and apoptosis in HemECs. NEW & NOTEWORTHY The role of LINC00342 in infantile hemangiomas has not yet been elucidated. This paper highlights the regulatory role of LINC00342 in cell proliferation and apoptosis in hemangioma-derived endothelial cells and the underlying molecular mechanisms. The findings would provide potential target for treatment of infantile hemangiomas.
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Ma, Wenxue, Larissa Balaian, Phoebe Mondala, Yudou He, Cayla Mason, Jessica Pham, Jeremy Lee, et al. "Imetelstat Inhibits Telomerase and Prevents Propagation of ADAR1-Activated Myeloproliferative Neoplasm and Leukemia Stem Cells." Blood 136, Supplement 1 (November 5, 2020): 18. http://dx.doi.org/10.1182/blood-2020-140771.
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BACKGROUND Clonal stem cell derived myeloproliferative neoplasms (MPNs) have a propensity to evolve to acute myeloid leukemia (AML). Deregulation of the innate immune deaminase associated with RNA1 (ADAR1) has been linked to malignant progression and therapeutic resistance. Increased expression of the stem cell gene, human telomerase reverse transcriptase (hTERT), has also been linked with malignant transformation. However, the combinatorial role of ADAR1 and hTERT in the evolution of MPN stem cells to therapy resistant acute myeloid leukemia stem cells (LSCs) and the capacity of a telomerase inhibitor, imetelstat, to prevent survival and self-renewal of pre-LSC and LSC had not been established. Recent clinical trials show early signs of efficacy of imetelstat in treatment of myelofibrosis (MF). However, its role in selectively inhibiting pre-LSC transformation to self-renewing LSC has not been elucidated. Here we show that targeting telomerase activity prevents pre-LSC and LSC maintenance both in vitro and in vivo, suggesting telomerase inhibition as an effective strategy for preventing MPN progression. METHODS To quantify hTERT level and ADAR1 activity in the setting of normal HSPC and MPN stem cell evolution, whole genome sequencing (WGS) analysis was performed on 76 normal and MPN blood CD34+ cells and matching saliva samples. Results were compared with RNA-seq of 100 FACS purified young, aged, MPN and AML CD34+CD38- stem cells and CD34+CD38+ progenitor cells. Confocal fluorescence microscopic evaluation of stem cell ADAR1 and hTERT localization, telomere length by Flow-FISH and telomerase activity by TRAP assays, lentiviral ADAR1 overexpression and shRNA knockdown were performed. In vitro stromal co-cultures, and humanized immunocompromised mouse models were established to determine the impact of imetelstat (a oligonucleotide inhibitor of telomerase) on normal, MPN stem cell and LSC maintenance. RESULTS Combined hTERT overexpression, ADAR1 activation and a significant reduction in telomere length correlated with accelerated stem cell aging during MPN progression to AML. Increased ADAR1 mediated adenosine to inosine (A-to-I) transcript editing coincided with accelerated telomere shortening in high risk MPN stem cells. Moreover, lentiviral ADAR1 overexpression enhanced pre-LSC engraftment. Treatment with imetelstat reduced MPN stem cell and LSC propagation in stromal co-cultures as well as in humanized mouse models commensurate with reduced hTERT expression levels and telomerase activity and decreased ADAR1 editing activity. Specifically, stromal co-culture assays revealed that combined treatment with dasatinib at 1 nM, and imetelstat at 1 µM or 5 µM significantly inhibited survival and replating of blast crisis (BC) CML progenitors compared with aged bone marrow progenitors (p < 0.001, ANOVA). As a single agent, imetelstat (5 µM) inhibited survival and replating of pre-LSC derived from myelofibrosis compared with normal bone marrow progenitor samples (p < 0.001, ANOVA). In pre-LSC MPN NSG-SGM mouse models established from 4 different MF samples, a significant reduction in proliferation of human CD45+ cells (p < 0.01, t test) was observed in bone marrow and spleen, when compared with vehicle control. Treatment of humanized LSC mouse models, established with 5 different BC CML, with 30 mg/kg of imetelstat, 3 times a week for 4 weeks resulted in a significant reduction in proliferation of malignant progenitors and human CD45+ cells (p < 0.001, ANOVA). As measured by a Flow-FISH assay, abnormal telomere length was reversed by imetelstat treatment compared with mismatch control (p < 0.05, ANOVA). In addition, FACS analysis revealed a significant reduction in activated beta-catenin expression after imetelstat treatment of LSC engrafted mice compared with vehicle control (p < 0.01, ANOVA). Finally, RNA-seq analysis performed on human CD34+ cells from imetelstat treated LSC mouse models revealed a significant reduction in LSC harboring malignant ADAR1-mediated A-to-I editing at doses that spared normal hematopoietic stem cells. CONCLUSIONS Combined WGS and RNA-Seq analyses, lentiviral ADAR1 overexpression, stromal co-culture assays and humanized pre-LSC and LSC mouse model studies reveal that pre-LSC evolution into LSC coincides with both ADAR1 and hTERT activation, which can be prevented with imetelstat. Disclosures Rizo: Geron Corp: Current Employment, Current equity holder in publicly-traded company. Huang:Geron Corp: Current Employment, Current equity holder in publicly-traded company. Jamieson:Forty Seven Inc: Patents & Royalties; Bristol-Myers Squibb: Other.
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Tutanov, Oleg, Evgeniya Orlova, Ksenia Proskura, Alina Grigor’eva, Natalia Yunusova, Yuri Tsentalovich, Antonina Alexandrova, and Svetlana Tamkovich. "Proteomic Analysis of Blood Exosomes from Healthy Females and Breast Cancer Patients Reveals an Association between Different Exosomal Bioactivity on Non-tumorigenic Epithelial Cell and Breast Cancer Cell Migration in Vitro." Biomolecules 10, no. 4 (March 25, 2020): 495. http://dx.doi.org/10.3390/biom10040495.
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Exosomes are important intercellular communication vehicles, secreted into body fluids by multiple cell types, including tumor cells. They contribute to the metastatic progression of tumor cells through paracrine signalling. It has been recently discovered that blood circulating exosomes contain distinguishable fractions of free and cell-surface-associated vesicles. We evaluated the influence of protein cargoes from exosomes from plasma, and exosomes from the total blood of healthy females (HFs) and breast cancer patients (BCPs), on cell motility. We conducted a mass spectrometric analysis of exosomal contents isolated from samples using ultrafiltration and ultracentrifugation approaches and verified their nature using transmission electron microscopy, nanoparticle tracking analysis and flow cytometry. We observed that malignant neoplasm-associated proteins in exosomes from BCP total blood were detected more often than in plasma (66% vs. 59%). FunRich analysis to assess Gene Ontology (GO) enrichment revealed that proteins with catalytic activities, transporter functions and protein metabolism activities were increased in exosomes from BCP blood. Finally, GO analysis revealed that proteomic profiles of exosomes from HF total blood were enriched with proteins inhibiting cell migration and invasion, which explains the low stimulating activity of exosomes from HF total blood on SKBR-3 cancer cell migration velocity. This allows exosomes to act as intermediaries providing intercellular communications through horizontal transfer of RNA and functionally active proteins, potentially affecting the development of both primary neoplasms and distant metastases.
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Chen, Justin Anthony, Yanli Hou, Krishna M. Roskin, Daniel A. Arber, Charles D. Bangs, Linda B. Baughn, Athena M. Cherry, et al. "Lymphoid blast transformation in an MPN with BCR-JAK2 treated with ruxolitinib: putative mechanisms of resistance." Blood Advances 5, no. 17 (September 10, 2021): 3492–96. http://dx.doi.org/10.1182/bloodadvances.2020004174.
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Abstract The basis for acquired resistance to JAK inhibition in patients with JAK2-driven hematologic malignancies is not well understood. We report a patient with a myeloproliferative neoplasm (MPN) with a BCR activator of RhoGEF and GTPase (BCR)–JAK2 fusion with initial hematologic response to ruxolitinib who rapidly developed B-lymphoid blast transformation. We analyzed pre-ruxolitinib and blast transformation samples using genome sequencing, DNA mate-pair sequencing (MPseq), RNA sequencing (RNA-seq), and chromosomal microarray to characterize possible mechanisms of resistance. No resistance mutations in the BCR-JAK2 fusion gene or transcript were identified, and fusion transcript expression levels remained stable. However, at the time of blast transformation, MPseq detected a new IKZF1 copy-number loss, which is predicted to result in loss of normal IKZF1 protein translation. RNA-seq revealed significant upregulation of genes negatively regulated by IKZF1, including IL7R and CRLF2. Disease progression was also characterized by adaptation to an activated B-cell receptor (BCR)–like signaling phenotype, with marked upregulation of genes such as CD79A, CD79B, IGLL1, VPREB1, BLNK, ZAP70, RAG1, and RAG2. In summary, IKZF1 deletion and a switch from cytokine dependence to activated BCR-like signaling phenotype represent putative mechanisms of ruxolitinib resistance in this case, recapitulating preclinical data on resistance to JAK inhibition in CRLF2-rearranged Philadelphia chromosome-like acute lymphoblastic leukemia.
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Nagata, Yasunobu, Masashi Sanada, Ayana Kon, Kenichi Yoshida, Yuichi Shiraishi, Aiko Sato-Otsubo, Hiraku Mori, et al. "Mutational Spectrum Analysis of Interesting Correlation and Interrelationship Between RNA Splicing Pathway and Commonly Targeted Genes in Myelodysplastic Syndrome." Blood 118, no. 21 (November 18, 2011): 273. http://dx.doi.org/10.1182/blood.v118.21.273.273.
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Abstract Abstract 273 Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms showing a frequent transition to acute myeloid leukemia. Although they are discriminated from de novo AML by the presence of a preleukemic period and dysplastic cell morphology, the difference in molecular genetics between both neoplasms has not been fully elucidated because of the similar spectrum of gene mutations. In this regards, the recent discovery of frequent pathway mutations (45∼90%) involving the RNA splicing machinery in MDS and related myeloid neoplasm with their rare mutation rate in de novo AML provided a novel insight into the distinct molecular pathogenesis of both neoplasms. Thus far, eight components of the RNA splicing machinery have been identified as the targets of gene mutations, among which U2AF35, SF3B1, SRSF2 and ZRSR2 show the highest mutation rates in MDS and CMML. Meanwhile, the frequency of mutations shows a substantial variation among disease subtypes, although the genetic/biological basis for these differences has not been clarified; SF3B1 mutations explain >90% of the spliceosome gene mutations in RARS and RCMD-RS, while mutations of U2AF35 and ZRSR2 are rare in these categories (< 5%) but common in CMML (16%) and MDS without increased ring sideroblasts (20%). On the other hand, SRSF2 mutations are most frequent in CMML (30%), compared with other subtypes (<10 %) (p<0.001) (Yoshida K, et al, unpublished data). So to obtain an insight into the genetic basis for these difference, we extensively explored spectrums of gene mutations in a set of 161 samples with MDS and related myeloid neoplasms, in which mutations of 10 genes thus far identified as major targets in MDS were examined and their frequencies were compared with regard to the species of mutated components of the splicing machinery. The mutation status of the 161 specimens was determined using the target exon enrichment followed by massively parallel sequencing. In total, 86 mutations were identified in 81(50%) in the 8 components of the splicing machinery. The mutations among 4 genes, U2AF35 (N = 20), SRSF2 (N = 31), SF3B1 (N = 15) and ZRSR2 (N = 10), explained most of the mutations with a much lower mutational rate for SF3A1 (N = 3), PRPF40B (N = 3), U2AF65 (N = 3) and SF1 (N = 1). Conspicuously, higher frequency 4 components of the splicing machinery were mutated in 76 out of the 161 cases (47.2%) in a mutually exclusive manner. On the other hand, 172 mutations of the 10 common targets were identified among 117, including 41 TET2 (25%), 32 RUNX1 (20%), 26 ASXL1 (16%), 24 RAS (NRAS/KRAS) (15%), 22 TP53 (14%), 17 IDH1/2 (10%), 10 CBL (6%) and 10 EZH2 (6%) mutations. We examined the difference between the major spliceosome mutations in terms of the number of the accompanying mutations in the 10 common gene targets. The possible bias from the difference in disease subtypes was compensated by multiple regressions. The SRSF2 mutations are more frequently associated with accompanying gene mutations with a significantly higher number of those mutations (N=29; OR 6.2; 95%CI 1.1–35) compared with that of the U2AF35 mutations (N=14) (p=0.038). Commonly involving the E/A splicing complexes, these splicing pathway mutations lead to compromised 3' splice site recognition. However, individual mutations may still have different impacts on cell functions, which could contribute to the determination of discrete disease phenotypes. It was demonstrated that SRSF2 was involved in the regulation of DNA stability and that depletion of SRSF2 can lead to DNA hypermutability, which may explain the higher number of accompanying gene mutation in SRSF2-mutated cases than cases with other spliceosome gene mutations. In conclusion, it may help to disclosing the genetic basis of MDS and related myeloid neoplasms that highly paralleled resequencing was confirmed SRSF2 mutated case significantly overlapped common mutations. Disclosures: No relevant conflicts of interest to declare.
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Giannini, Riccardo, Clara Ugolini, Anello Marcello Poma, Maria Urpì, Cristina Niccoli, Rossella Elisei, Massimo Chiarugi, Paolo Vitti, Paolo Miccoli, and Fulvio Basolo. "Identification of Two Distinct Molecular Subtypes of Non-Invasive Follicular Neoplasm with Papillary-Like Nuclear Features by Digital RNA Counting." Thyroid 27, no. 10 (October 2017): 1267–76. http://dx.doi.org/10.1089/thy.2016.0605.
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Craver, Brianna, Quy Nguyen, Gajalakshmi Ramanathan, and Angela G. Fleischman. "Single-Cell RNA-Seq to Assess Differential Responses to Tnfα in Human Hematopoietic Stem and Progenitor Cells in Myeloproliferative Neoplasm." Blood 134, Supplement_1 (November 13, 2019): 2518. http://dx.doi.org/10.1182/blood-2019-123480.
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Somatic mutations in hematopoietic stem and progenitor cells (HSPCs) leading to constitutive activation of thrombopoietin receptor signaling result in myeloproliferative neoplasms (MPN). The most common mutation found in MPN patients occurs in the Janus kinase 2 gene (JAK2V617F). We have previously found that JAK2V617F hematopoietic progenitors are resistant to tumor necrosis factor alpha (TNFα), however this mechanism is not well defined. We hypothesize that resistance to TNFα in JAK2V617F hematopoietic stem and progenitors is a driver of the competitive advantage over non-malignant clones. Here, we used droplet-based single-cell RNA-sequencing to investigate transcriptional profiling in primary human HSPCs. First, we harvested white blood cells from fresh bone marrow aspirates from one MPN patient (Polycythemia Vera with 71% JAK2V617F allele burden) as well as one unaffected individual then sorted Lin-/CD34+/CD38- hematopoietic progenitors. Immediately following sorting, half of the cells were stimulated with TNFα for 4 hours while the other half of cells were used as unstimulated controls. We then utilized the 10X Chromium platform to generate single-cell droplets for the 8,129 total cells from the unaffected individual and 33,299 total cells from the MPN patient. We ran alignment using the CellRanger pipeline then performed analysis using the Seurat package in R. Expression profiles of untreated HSPCs in both normal and MPN cells revealed high expression of genes involved in important pathways for hematopoiesis (Polycomb repressive complexes, chromatin regulation, the ubiquitin proteasome system etc.). Expression of CD34 was confirmed in both MPN and non-MPN cells, though CD34 expression was reduced following TNFα stimulation. Expression of stem (i.e. THY1, ITGA6) and progenitor (i.e. PTPRC) genes were detected within both individuals, which highlights the heterogeneity within Lineage-/CD34+/CD38- cells. Following stimulation with TNFα, we observed expression of genes in canonical pathways downstream of TNF including NF-κB, Mitogen-Activated Protein Kinase (MAPK), and Transforming Growth Factor Beta (TGFβ). Indeed, we observed a baseline level of expression of TGFβ-related genes in both normal and MPN cells. Upon inflammatory stimulation, normal HSPCs upregulated SMAD expression which are involved in the TGFβ pathway. Strikingly, we did not observe an increase in SMAD expression in MPN cells following TNF. This suggests a dampened response via the TGFβ pathway to TNF in MPN cells. Additionally, we found that TNF-stimulated HPSCs from the unaffected individual expressed canonical genes of the TNF pathway that encode for chemokines, cytokines, transcription factors and negative feedback regulators. In normal TNF-stimulated cells, we identified highly expressed genes involved in the caspase cascade, suggesting a robust apoptotic response in normal HPSCs. However, there was a lower expression of caspases in stimulated MPN cells, suggesting a dampened apoptotic response to TNF. One observation that was unique to TNF-stimulated cells from the MPN individual was the expression of glycoproteins involved in angiogenesis and platelet aggregation. Taken together, these data serve as a proof of principle for transcriptional profiling of primary human hematopoietic stem and progenitor cells and that this cell population rapidly and robustly alter their gene expression program upon TNFα stimulation. In conclusion, we show that HSPCs from an MPN patient exhibit a dampened response to TNF compared to normal HSPCs. Specifically, we observed a lower expression of genes involved with apoptosis and TGFβ signaling in MPN cells compared to normal cells following TNF stimulation. The finding of a dampened apoptotic response to TNF is consistent with the hypothesis that JAK2V617F cells gain a selective advantage over normal cells under inflammatory stress. To our knowledge, this is the first report of single-cell RNA-seq analysis on primary human HSPCs following FACS and inflammatory stimulation. Disclosures Fleischman: incyte: Speakers Bureau.
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Hershberger, Courtney E., Devlin C. Moyer, Wencke Walter, Stephan Hutter, Claudia Haferlach, Torsten Haferlach, Jaroslaw P. Maciejewski, and Richard A. Padgett. "The Biological and Clinical Implications of the Alternative Splicing Landscape of 1,258 Myeloid Neoplasm Cases." Blood 134, Supplement_1 (November 13, 2019): 769. http://dx.doi.org/10.1182/blood-2019-128278.
Full textAbstract:
NGS has led to the discovery of somatic mutations in splicing factors (SF), a group of functionally related genes previously not implicated in leukemogenesis. At least one genetic aberration in the most commonly affected 7 splicing factor genes is present in the majority of patients with MDS and related diseases (MDS/MPN and AML). The most popular and plausible hypothesis is that individual splicing mutations are associated with mis-splicing of key pathogenic genes in leukemia. However, searches for the essential mis-spliced gene or pathway in engineered cell lines and murine models have not been successful despite identification of many downstream gene targets. We have designed a strategy that overcomes pitfalls and advances results of previous attempts to identify the most essential targets. First, we have collected an expansive dataset (RNA-Seq and WGS of 1258 patient samples and 63 healthy controls) which allowed us to overcome sample size limitations and exclude cases with low tumor burdens, decreasing the analytic noise. In addition to studying the common mutant SRSF2 (n=208), SF3B1 (n=282), and U2AF1 (n=69) cases, we have also studied LOH lesions (fs, ns, deletions) in the less frequently affected splicing factors LUC7L2, DDX41, PRPF8, and ZRSR2 (n=211) (Fig.1A). Unsupervised hierarchical clustering segregated patient splicing signatures by disease type, SF mutation, and SF expression. To detect significantly dysregulated alternative splicing (AS) events, samples from each disease subtype, with mutations in SF3B1 (various), SRSF2P95, U2AF1S34, or U2AF1Q157, were compared to patients without SF mutations and also healthy controls. The disease cohorts were also stratified by LUC7L2, DDX41, PRPF8, and ZRSR2 expression levels, and the lower expression groups were compared to both the higher expression groups and healthy controls. Meta-analysis revealed over 17,000 splicing variations that were significantly dysregulated in at least one of 64 comparisons (PSI≥5%, q≤.05). Statistically significant AS events in each group were overlaid to identify commonly dysregulated AS events when compared to both the disease control and the healthy bone marrow controls (Fig.1B). We characterized AS events that were unique to the myeloid neoplasm subtypes as well as specific to genetic aberrations in SFs. We also identified genes and transcripts mis-spliced in multiple groups, suggesting a convergence of splicing factor mutations on a common target gene. The vast majority of our analysis identified alterations in isoform balance, however some splice sites that were activated only in the MDS and AML cohorts but never utilized in healthy controls. Examples of these tumor-specific splicing events are found in CERS2, which was found in a majority of patient samples, and in FMNL1, which was overwhelmingly mis-spliced in SF3B1 mutant patient samples (data not shown). We have highlighted the 52 AS events that were changed most often in comparisons against disease controls and/or healthy controls. Examples of targeted exons and introns included those in ubiquitination factors, transcription factors, DNA repair factors, and oncogenes. We classified significantly changed exons by the functional domains of the translated protein. The cohorts were then stratified according to the inclusion level of the exon or intron. The inclusion groups were compared to distinguish differences both in gene expression and in dysregulation of downstream pathways. Furthermore, the exons and introns were examined for any correlation with survival in the myeloid neoplasm subtypes. Integration of these datasets provided insights into the functional impact of AS in myeloid neoplasms, e.g., TDP1 exon 12, or exon 10b of NCOR1 inclusion both is positively correlated with expression of MYC targets and negatively associated with survival in AML patients (Fig.1C-D) In sum, we have identified strong isoform candidates for the practical study of AS driven pathogenesis, utilizing both RNA-seq and the integration of publicly available exon annotation and pathway databases. Notably, our analyses have unveiled hundreds of splicing changes dysregulated at a statistically significant level, thus warranting further assessments. This assemblage of splicing patterns found in myeloid neoplasms patients' samples is the largest in existence and should greatly advance the study of pathogenic AS. Disclosures Walter: MLL Munich Leukemia Laboratory: Employment. Hutter:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Maciejewski:Alexion: Consultancy; Novartis: Consultancy.
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Pang, Xiaocong, Ying Zhao, Jinhua Wang, Wan Li, Qian Xiang, Zhuo Zhang, Shiliang Wu, Ailin Liu, Guanhua Du, and Yimin Cui. "Competing Endogenous RNA and Coexpression Network Analysis for Identification of Potential Biomarkers and Therapeutics in association with Metastasis Risk and Progression of Prostate Cancer." Oxidative Medicine and Cellular Longevity 2019 (August 5, 2019): 1–21. http://dx.doi.org/10.1155/2019/8265958.
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Prostate cancer (PCa) is the most frequently diagnosed malignant neoplasm in men. Despite the high incidence, the underlying pathogenic mechanisms of PCa are still largely unknown, which limits the therapeutic options and leads to poor prognosis. Herein, based on the expression profiles from The Cancer Genome Atlas (TCGA) database, we investigated the interactions between long noncoding RNA (lncRNA) and mRNA by constructing a competing endogenous RNA network. Several competing endogenous RNAs could participate in the tumorigenesis of PCa. Six lncRNA signatures were identified as potential candidates associated with stage progression by the Kolmogorov-Smirnov test. In addition, 32 signatures from the coexpression network had potential diagnostic value for PCa lymphatic metastasis using machine learning algorithms. By targeting the coexpression network, the antifungal compound econazole was screened out for PCa treatment. Econazole could induce growth restraint, arrest the cell cycle, lead to apoptosis, inhibit migration, invasion, and adhesion in PC3 and DU145 cell lines, and inhibit the growth of prostate xenografts in nude mice. This systematic characterization of lncRNAs, microRNAs, and mRNAs in the risk of metastasis and progression of PCa will aid in the identification of candidate prognostic biomarkers and potential therapeutic drugs.
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Ortega, Miguel A., Oscar Fraile-Martínez, Luis G. Guijarro, Carlos Casanova, Santiago Coca, Melchor Álvarez-Mon, Julia Buján, Natalio García-Honduvilla, and Ángel Asúnsolo. "The Regulatory Role of Mitochondrial MicroRNAs (MitomiRs) in Breast Cancer: Translational Implications Present and Future." Cancers 12, no. 9 (August 28, 2020): 2443. http://dx.doi.org/10.3390/cancers12092443.
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Breast cancer is the most prevalent and incident female neoplasm worldwide. Although survival rates have considerably improved, it is still the leading cause of cancer-related mortality in women. MicroRNAs are small non-coding RNA molecules that regulate the posttranscriptional expression of a wide variety of genes. Although it is usually located in the cytoplasm, several studies have detected a regulatory role of microRNAs in other cell compartments such as the nucleus or mitochondrion, known as “mitomiRs”. MitomiRs are essential modulators of mitochondrion tasks and their abnormal expression has been linked to the aetiology of several human diseases related to mitochondrial dysfunction, including breast cancer. This review aims to examine basic knowledge of the role of mitomiRs in breast cancer and discusses their prospects as biomarkers or therapeutic targets.
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Kameda-Smith, Michelle, Helen Zhu, EnChing Luo, Chitra Venugopal, Agata Xella, Kevin Brown, Raymond Fox, et al. "MBRS-01. DISSECTING REGULATORS OF THE ABERRANT POST-TRANSCRIPTIONAL LANDSCAPE IN MYC-AMPLIFIED GROUP 3 MEDULLOBLASTOMA." Neuro-Oncology 22, Supplement_3 (December 1, 2020): iii399. http://dx.doi.org/10.1093/neuonc/noaa222.522.
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Abstract Medulloblastoma (MB) is the most common solid malignant pediatric brain neoplasm, with Group 3 (G3) MB representing the most aggressive subgroup. MYC amplification is an independent poor prognostic factor in G3 MB, however, therapeutic targeting of the MYC pathway remains limited and alternative therapies for G3 MB are urgently needed. Here we show that an RNA-binding protein, Musashi-1 (MSI1) is an essential mediator of G3 MB in both MYC-overexpressing mouse models and patient-derived xenografts. Unbiased integrative multi-omics analysis of MSI1 function in human G3 MB suggests a paradigm shift beyond traditional gene-based profiling of oncogenes. Here we identify MSI1 as an oncogene in G3 MB driving stem cell self-renewal through stabilization of HIPK1 mRNA, a downstream context-specific therapeutic target for drug discovery.
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Umar, Liya Agustin. "Effect of Non-Coding Region RNA Gene XIST (X-Inactive Specific Transcript) on Human Breast Cancer." Biomedical Journal of Indonesia 7, no. 2 (March 22, 2021): 285–92. http://dx.doi.org/10.32539/bji.v7i2.290.
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X chromosome is a sex chromosome found in both women and men. The inactivation of the X chromosome is linked to a non-coding region of RNA known as the XIST gene (X-inactive specific transcript). This gene is located in the X inactivation center (XIC X-inactive center). The XIST gene is a region that belongs to the RNA group, non-coding transcripts (NCT), also known as microRNA. Breast cancer (Breast Cancer) is a type of cancer that commonly affects women, but men can develop breast cancer, but the chances are small, about 1 in 1000. Breast cancer is a cancerous neoplasm that is malignant, and occurs in the mammary gland. The presence of a specific XIST gene on the X chromosome and the prevalence of breast cancer, which is mostly in women, raises the idea that there is an influence of this gene on breast cancer (breast cancer) in the epigenetic process. The XIST gene related to microRNA has an opportunity to be looked at because certain microRNAs have a greater or lesser level (concentration) in cancer cells than normal cells. This is a new opportunity to continue to be developed as a consideration for a new treatment method involving gene therapy.
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You, Xiaolan, Yuanjie Wang, Jian Wu, Qinghong Liu, Dehu Chen, Dong Tang, and Daorong Wang. "Aberrant Cytokeratin 20 mRNA Expression in Peripheral Blood and Lymph Nodes Indicates Micrometastasis and Poor Prognosis in Patients With Gastric Carcinoma." Technology in Cancer Research & Treatment 18 (January 1, 2019): 153303381983285. http://dx.doi.org/10.1177/1533033819832856.
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Several studies suggest that peripheral blood and lymph node micrometastases may be a causative factor for gastric cancer recurrence. Cytokeratin 20 shows enriched expression in intestinal epithelial cells. This study aimed to evaluate the clinical utility of monitoring cytokeratin 20 levels in peripheral blood and lymph nodes of patients with gastric cancer for detecting micrometastasis and predicting prognosis. We detected messenger RNA levels of cytokeratin 20 in gastric cancer cell lines and in the peripheral blood of 125 patients (85 patients with gastric cancer and 40 patients with benign neoplasm) by fluorescence quantitative real-time polymerase chain reaction both before and after radical resection. In all, 1586 lymph node samples from 85 patients with gastric cancer were evaluated for cytokeratin 20 expression using real-time polymerase chain reaction, as well as by immunohistochemistry staining with anti-pan-keratin and anti-cytokeratin 20 antibodies. All patients underwent follow-up until cancer-related death or for more than 3 years after tumor resection. We found that elevated cytokeratin 20 expression in peripheral blood as detected by quantitative real-time polymerase chain reaction closely correlates with poor clinicopathological characteristics. Detecting cytokeratin 20 messenger RNA in the lymph nodes by quantitative real-time polymerase chain reaction enabled more accurate determination of the clinicopathological staging of gastric cancer, best treatment approach, and prognosis. Our findings show that patients with increased cytokeratin 20 messenger RNA expression in the peripheral blood or lymph nodes have a shorter time to recurrence and poorer overall survival.
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Togami, Katsuhiro, Vikas Madan, Jia Li, Alexandra-Chloe Villani, Siranush Sarkizova, Mahmoud Ghandi, Kevin Buczkowski, et al. "Blastic Plasmacytoid Dendritic Cell Neoplasm (BPDCN) Harbors Frequent Splicesosome Mutations That Cause Aberrant RNA Splicing Affecting Genes Critical in pDC Differentiation and Function." Blood 128, no. 22 (December 2, 2016): 738. http://dx.doi.org/10.1182/blood.v128.22.738.738.
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Abstract Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive malignancy thought to result from transformation of plasmacytoid dendritic cells (pDCs). Clinical outcomes are poor and pathogenesis is unclear. To better understand BPDCN genomics and disease mechanisms, we performed whole exome- (12 BPDCNs), targeted DNA- (additional 12 BPDCNs), bulk whole transcriptome RNA- (12 BPDCNs and 6 BPDCN patient-derived xenografts [PDXs]), and single cell RNA-sequencing (scRNA-seq) compared to normal DCs. We observed RNA splicing factor mutations in 16/24 cases (7 ZRSR2, 6 SRSF2, 1 each SF3B1, U2AF1, SF3A2, SF3B4). Additional recurrent alterations were in genes known to be mutated in other blood cancers: TET2, ASXL1, TP53, GNB1, NRAS, IDH2, ETV6, DNMT3A, and RUNX1. From exome sequencing we also discovered recurrent mutations in CRIPAK (6/12 cases), NEFH (4/12), HNF1A (2/12), PAX3 (2/12), and SSC5D (2/12) that may be unique to BPDCN. ZRSR2 is notable among the recurrently mutated splicing factors in hematologic malignancies in that all mutations are loss-of-function (e.g., nonsense, frameshift). Of note, BPDCN is very male predominant, ZRSR2 is located on chrX and all mutations are in males. ZRSR2 plays a critical role in "minor" or U12-type intron splicing (only 0.3% of all introns). Thus, we hypothesized that mis-splicing, possibly of U12 genes, contributes to BPDCN pathogenesis. Using RNA-seq, we measured aberrant splicing in BPDCN. Intron retention was the most frequent abnormality in ZRSR2 mutant BPDCNs and PDXs compared to non-mutant cases. ZRSR2 mutant intron retention predominantly affected U12 introns (patients: 29.4% of retained introns, P<0.0001; PDX: 94%, P<0.0001). To test if ZRSR2 loss directly causes U12 intron retention in otherwise isogenic cells, we performed ZRSR2 knockdown using doxycycline-inducible shRNAs in the BPDCN cell line, CAL1, which has no known splicing factor mutation. RNA-seq was performed 0, 2, and 7 days after addition of doxycycline in 3 independent clones each of control or ZRSR2 knockdown. Consistent with what we observed in primary BPDCN, intron retention events were higher in ZRSR2 compared to control shRNA cells after 7 days of doxycycline (mean 885.7 vs 122.7 events, P=0.041). Aberrant intron retention after ZRSR2 knockdown largely involved U12 introns (30/732 U12 vs 37/207,344 U2 introns, P<0.0001). SRSF2 and SF3B1 mutations in BPDCN were at hotspots seen in other cancers: SRSF2 P95H/L/R and SF3B1 K666N, mutants that induce specific types of aberrant splicing (Kim, Ca Cell 2015; Darman, Cell Rep 2015). Mutant BPDCNs demonstrated the same aberrations: SRSF2, exon inclusion/exclusion based on CCNG/GGNG exonic splicing enhancer motifs; SF3B1, aberrant 3' splice site recognition. We hypothesized that aberrant splicing may affect RNAs important for pDC development or function. To further define genes uniquely important in BPDCN, we performed scRNA-seq on 4 BPDCNs and on DCs from healthy donors. By principal component analysis, BPDCNs were more similar to pDCs than to conventional DCs (cDCs) or other HLA-DR+ cells. However, several critical genes for pDC function had markedly lower expression in BPDCN including the transcription factors IRF4 and IRF7. Next we determined which genes were commonly mis-spliced in splicing factor mutant BPDCNs. Strikingly, this list included genes already known to be important in driving DC biology or identified in our scRNA-seq as being differentially expressed between BPDCN and healthy pDCs, including IRF7, IRF8, IKZF1, FLT3, and DERL3. To determine if splicing factor mutations affect DC function, we stimulated ZRSR2 knockdown or control CAL1 cells with Toll-like receptor (TLR) 7, 8, and 9 agonists (R848 or CpG oligo). ZRSR2 knockdown inhibited upregulation of the CD80 costimulatory molecule and aggregation of CAL1 cells, suggesting impairment in activation. Using mouse conditional knock-in bone marrow in ex vivo multipotent progenitor assays, DC differentiation induced by FLT3 ligand was biased toward pDCs and away from cDCs in SRSF2 P95H mutant compared to wild-type cells. However, cDC and monocyte differentiation in the presence of GM-CSF was not affected. In conclusion, splicing factors are frequently mutated in BPDCN and lead to specific splicing defects. Splicing factor mutations may promote BPDCN by affecting pathways important in DC maturation or activation, which could contribute to transformation. Disclosures Seiler: H3 Biomedicine: Employment. Buonamici:H3 Biomedicine: Employment. Lane:Stemline Therapeutics: Research Funding; N-of-1: Consultancy.
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Hao, Yafei, Changzhou Li, Teng Zhao, and Hansheng Liu. "Long Non-Coding RNA GATA3-AS1 Promotes 5-Fluorouracil Resistance of Ovarian Cancer via Mediating miR-6771-3p/SOX4 Target Axis." Journal of Biomaterials and Tissue Engineering 11, no. 6 (June 1, 2021): 1099–107. http://dx.doi.org/10.1166/jbt.2021.2666.
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Ovarian cancer (OC) ranks as the 5th highest cause of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) exert significant effects on chemotherapy resistance. The effects of lncRNA GATA3-AS1 on the 5-Fluorouracil (5-FU) resistance in ovarian carcinoma were explored in the current study. The results showed that GATA3-AS1 was highly expressed in OC tissues and 5-FU resistant OC cells. Moreover, GATA3-AS1 knockdown reduced 50% inhibitory concentration (IC50) of 5-Fluorouracil and promoted cell apoptosis, while GATA3-AS1-overexpression showed the opposite effect. In vivo experiment and murine xenograft assay indicated GATA3-AS1 knockdown inhibited neoplasm growth, promoted cell apoptosis, and altered the expression level of apoptosis-associated proteins. GATA3-AS1 promoted SOX4 expression, a well-known transcription factor regulating apoptosis, via targeting miR-6771-3p. In summary, our findings suggested GATA3-AS1 was associated with OC 5-Fluorouracil viaregulating miR-6771-3p/SOX4, providing novel insights into OC chemoresistance.
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Jour, George, Jonathan Serrano, Christian Koelsche, David T. W. Jones, Andreas von Deimling, Jeffrey Allen, and Matija Snuderl. "Primary CNS Alveolar Rhabdomyosarcoma: Importance of Epigenetic and Transcriptomic Assays for Accurate Diagnosis." Journal of Neuropathology & Experimental Neurology 78, no. 11 (August 14, 2019): 1073–75. http://dx.doi.org/10.1093/jnen/nlz083.
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Abstract We present the case of a 22-year-old woman who developed increasing headaches, nausea, and vomiting. Imaging identified a 3 × 3 cm heterogeneously enhancing cystic mass in the posterior III ventricular/pineal region. Pathology review of the initial lesion revealed a highly malignant spindle cell neoplasm composed of round to mostly oval elongated cells with relatively small amounts of cytoplasm arranged in sheets and fascicles with focal storiform pattern. Whole genome methylation analysis through unsupervised clustering with data generated from other primary intracranial tumors and peripheral sarcomas was performed at the German Cancer Research Center (DKFZ) and classified the tumor with the group of alveolar rhabdomyosarcomas (ARMS). Further RNA sequencing revealed an in frame PAX3 (EX 7)-NCOA2 (EX12) fusion confirming the diagnosis. This is the first evidence of occurrence of PAX3-NCOA2 in primary CNS ARMS.
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Shenoy, Archana, Lea Surrey, Payal Jain, Jessica Foster, Joshua Straka, Adam Resnick, Angela Waanders, et al. "Sclerosing Epithelioid Fibrosarcoma of the Bone With Rare EWSR1-CREB3L3 Translocation Driving Upregulation of the PI3K/mTOR Signaling Pathway." Pediatric and Developmental Pathology 22, no. 6 (July 23, 2019): 594–98. http://dx.doi.org/10.1177/1093526619864230.
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Sclerosing epithelioid fibrosarcoma (SEF) is an uncommon neoplasm that rarely presents in bone. It is characterized by epithelioid cells arranged in nests and single-file cords within a sclerotic stromal background which may mimic neoplastic bone. SEF harbors an EWSR1 translocation, which may complicate its distinction from Ewing sarcoma in cases with histomorphologic overlap. We present a diagnostically challenging case of SEF in the mandible of a 16-year-old girl. Our experience highlights the lack of specificity of traditional morphology and EWSR1 break-apart fluorescent in situ hybridization. Open-ended RNA-based fusion gene testing coupled with MUC4 immunohistochemistry aided the eventual diagnosis in this case. Herein, we report the third case of SEF with EWSR1-CREB3L3 translocation and show that this fusion leads to aberrant upregulation of the phosphoinositide 3-kinase/mammalian target of rapamycin signaling pathway in heterologous cell models.
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Shi, Guanfang, Kiron Nair, Preethi Ramachandran, Chi Chen, Ching Wong, Gardith Joseph, Vladimir Gotlieb, Maksim Liaukovich, and Jen-Chin Wang. "TLR, RAGE, and HMGB1 in the Inflammatory Response in Ph(-) Myeloproliferative Neoplasm." Blood 136, Supplement 1 (November 5, 2020): 31–32. http://dx.doi.org/10.1182/blood-2020-134988.
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Recent evidence of increased constitutional symptoms and inflammatory cytokines in Philadelphia chromosome negative (Ph (-)) MPN suggests that an inflammatory response is important in the pathogenesis of Ph (-) MPN. Toll-like receptors (TLR), Receptor for Advanced Glycation End products (RAGE) and High mobility group protein B1 (HMGB1) are the important pathways for the inflammatory response. All these three important pathway proteins were studied in MPN diseases in the current studies. Materials and Methods: TLR assay. TLR 2,3, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells (peripheral blood) which were incubated with fluorescence-conjugated anti-TLR-2,3, 4, 7, 9 antibodies and assayed by flow cytometry. HMGB1assay:HMGB1 ELISA kit from Immuno-Biological Laboratories, Inc. (IBL-America) were used. The plasma samples were diluted four times with the provided sample dilution buffer, and assayed in duplicate according to the manufacturer's suggestion. RAGE (RT-PCR) Assay: Total RNA was extracted from normal control or patient mononuclear cells. Predesigned primers for RAGE, and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of RAGE were compared with each internal control. Average of three was used to calculate the ratio of final patient to normal Results: Total of 97 patients with MPN were studied 1) TLR: TLR 3,7,9 was not significantly different from controls. But TLR 2 was significantly increased in both PV, as well as in the MPN group when PV, ET and MF were grouped together as MPN (Fig A). TLR 4 was not significantly increased in PV, ET, MF individually but was found to be significantly increased than the controls, when they are grouped together as MPN (Fig B). 2) RAGE: No significant difference was found between ET, PV, MF individually or when they were grouped together as MPN than the controls (Fig C). 3) HMGB1: No significant difference was seen between ET, PV, MF or when they were grouped as MPN (Fig D). Conclusion: Current study suggests that TLR pathway especially TLR2, and to a lesser extent TLR4 are the important pathways for inflammatory response with increased inflammatory cytokines in MPN, while HMGB1 and RAGE pathways were not different from controls. Figure Disclosures No relevant conflicts of interest to declare.
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Weber, Georg F. "Molecular Analysis of a Recurrent Sarcoma Identifies a Mutation in FAF1." Sarcoma 2015 (2015): 1–20. http://dx.doi.org/10.1155/2015/839182.
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A patient presented with a recurrent sarcoma (diagnosed as leiomyosarcoma) 12 years after the removal of an initial cancer (diagnosed as extracompartmental osteosarcoma) distally on the same limb. Following surgery, the sarcoma and unaffected muscle and bone were subjected to measurements of DNA exome sequence, RNA and protein expression, and transcription factor binding. The investigation provided corroboration of the diagnosis leiomyosarcoma, as the major upregulations in this tumor comprise muscle-specific gene products and calcium-regulating molecules (calcium is an important second messenger in smooth muscle cells). A likely culprit for the disease is the point mutation S181G in FAF1, which may cause a loss of apoptotic function consecutive to transforming DNA damage. The RNA levels of genes for drug transport and metabolism were extensively skewed in the tumor tissue as compared to muscle and bone. The results suggest that the tumor represents a recurrence of a dormant metastasis from an originally misdiagnosed neoplasm. A loss of FAF1 function could cause constitutive WNT pathway activity (consistent with the downstream inductions of IGF2BP1 and E2F1 in this cancer). While the study has informed on drug transport and drug metabolism pharmacogenetics, it has fallen short of identifying a suitable target for molecular therapy.
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Lawrence, Ben, Simon Schimmack, Bernhard Svejda, Ignat Drozdov, Daniele Alaimo, Barton Kenney, Mark Kidd, and Irvin M. Modlin. "Prognostication by multigene proliferative marker panel compared with Ki-67 in small-intestinal NENs." Journal of Clinical Oncology 30, no. 4_suppl (February 1, 2012): 242. http://dx.doi.org/10.1200/jco.2012.30.4_suppl.242.
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242 Background: Ki-67 is the major proliferative marker in clinical use to determine neuroendocrine neoplasm (NEN) prognosis. Ki-67 is unable to predict the outcome of SI-NENs, as the majority have a low (≤2) Ki-67%. Therefore, we aimed to identify a sensitive panel of proliferative markers using qRT-PCR to more accurately define the proliferation of these slow growing tumors. Methods: We identified genes with a mechanistic function in cell cycle progression that were over-expressed in RNA microarrays of SI-NENs (n=8) compared to adjacent normal tissue (n=4) (dCHIP, annotation databases). Timing of marker gene expression (qRT-PCR) in proliferating cell-cycle phases (S, G2, M) was determined in flow-sorted SI-NEN cell lines (KRJ-1, H-STS) after propidium iodide staining. RNA expression of candidate proliferative markers was then investigated using an in vivo model and two independent tumor datasets, and transcript level compared to Ki-67% protein expression (immunohistochemical staining). Results: Twenty genes with a mechanistic role in proliferation were identified and 17 confirmed to be expressed in proliferating cell cycle phases. Each tumor expressed a unique profile of the 17 proliferative markers. Both Ki-67 protein and Ki-67 RNA transcript levels failed to differentiate in vivo SI-NEN models or patient samples despite variable proliferative capacity (e.g., WDNETs versus WDNECs). Although most tumors showed low levels of Ki-67 expression, the tumors expressed high levels of select alternative proliferative markers. Hierarchical clustering provided a novel and clinically meaningful prognostic classification. Conclusions: Proliferation of individual SI-NENs is regulated by unique combinations of multiple genes with a mechanistic role in cell-cycle progression. Regulation of proliferation in SI-NENs is therefore complex and cannot accurately be defined by Ki-67 as a single marker. A panel of proliferative RNA markers has potential to significantly improve prognostication in patients with SI-NENs.
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Malcovati, Luca, Elli Papaemmanuil, Eva Hellström-Lindberg, Jacqueline Boultwood, David Bowen, Paresh Vyas, Cristiana Pascutto, et al. "Somatic Mutation of SF3B1, a Gene Encoding a Core Component of RNA Splicing Machinery, in Myelodysplasia with Ring Sideroblasts." Blood 118, no. 21 (November 18, 2011): 3. http://dx.doi.org/10.1182/blood.v118.21.3.3.
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Abstract Abstract 3 Myelodysplastic syndromes (MDS) are myeloid neoplasms characterized by dysplasia in one or more cell lines, ineffective hematopoiesis, and variable risk of progression to acute myeloid leukemia (AML). As any other neoplasm, MDS is expected to be driven by mutation, and its clonal evolution is likely a multistep process in which several genetic events occur. Somatic mutations of TET2 have been found in about 25% of MDS patients, while additional mutant genes (including ASXL1, ETV6, EZH2, IDH1, IDH2, RUNX1, and TP53) have been detected in smaller proportions of patients, particularly in those with poor prognosis. Refractory anemia with ring sideroblasts (RARS) is a phenotypically well-defined subtype of MDS, characterized by 15% or more ring sideroblasts (RS, erythroblasts with perinuclear iron-loaded mitochondria) in the bone marrow. We reasoned that the identification of recurrently mutated genes in RARS could provide novel insights into molecular pathogenesis of MDS, and used massively parallel sequencing technology to identify somatically acquired point mutations across all protein-coding exons in the genome in 8 patients with RARS. We identified 62 point mutations across the 8 patients, and the mutation spectrum showed a predominance of transitions, especially C>T/G>A mutations. Within 5/8 patients studied, the observed proportion of reads reporting a mutant allele showed significantly greater variability than expected by chance, indicating that the population of malignant cells was genetically heterogeneous. In 6/8 RARS patients, we identified recurrent somatic mutations (found in granulocytes but not in T-lymphocytes) in a gene that encodes a core component of the RNA splicing machinery, SF3B1. Based on the proportion of reads reporting the mutant allele, the mutations all appeared to be heterozygous and present in the dominant clone of cells. To characterize the spectrum and frequency of SF3B1 mutations in greater detail, both in myeloid malignancies and other cancers, we undertook targeted resequencing of the gene. In particular, we studied patients with MDS, myelodysplastic/myeloproliferative neoplasm (MDS/MPN) or AML evolving from MDS. Somatic mutations of SF3B1 were found in 150/533 (28.1%) patients with MDS, 16/83 (19.3%) patients with MDS/MPN, and 2/38 (5.3%) patients with AML. The gene was also mutated in 1–5% of diverse other tumor types. All mutations appeared to be heterozygous substitutions, and we observed no frameshift indels, splice site mutations or nonsense substitutions. The mutations clustered in exons 12–15 of the gene, and K700E accounted for 97/168 (57.7%) of the variants observed. SF3B1 mutations were less deleterious than expected by chance, implying that the mutated protein retains structural integrity with altered function. Gene expression profiling revealed SF3B1 mutations are associated with down-regulation of key gene networks, including core mitochondrial pathways. Close relationships were found between mutant SF3B1 and presence of RS (P<.001), and between mutant allele burden and percentage of RS (P=.002). Overall, 83/105 (79%) of patients with RARS, 30/54 (57.7%) of those with refractory cytopenia with multilineage dysplasia and RS, and 12/18 (66.7%) of those with RARS associated with marked thrombocytosis (RARS-T) carried a somatic mutation of SF3B1. On the other hand, 97% of patients carrying a mutant SF3B1 had RS, and the mutant gene had a positive predictive value for RS of 97.7% (95% CI, 93.5–99.5%). We then studied the prognostic significance of the genetic lesion. In multivariable analysis including established risk factors, SF3B1 mutations were independently associated with better overall survival (HR=0.18, P=.028) and lower risk of progression to AML (HR=0.32, P=.048). In conclusion, mutations in SF3B1 implicate abnormalities of mRNA splicing, a pathway not previously known as a target for mutation, in the pathogenesis of MDS. The close relationship between this molecular lesion and RS is consistent with a causal relationship, and makes SF3B1 the first gene to be strongly associated with a specific morphological feature in MDS. Finally, SF3B1 mutations are independent predictors of favorable clinical outcome, and their detection may improve risk assessment in MDS. The first two authors equally contributed to this paper, which is on behalf of the International Cancer Genome Consortium Chronic Myeloid Disorders Working Group. Disclosures: No relevant conflicts of interest to declare.
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Qin, Wei, Huina Lu, Jianfei Fu, and Aibin Liang. "Alteration of SOCS Is a Possible Pathogenetic Mechanism of Myeloproliferative Neoplasm." Blood 116, no. 21 (November 19, 2010): 4121. http://dx.doi.org/10.1182/blood.v116.21.4121.4121.
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Abstract Abstract 4121 The activation of the JAK/STAT pathway caused by the JAK2 gene mutations is an important pathogenetic mechanism of myeloproliferative neoplasm(MPN). Recently, many evidences suggest that there are factors besides the mutations of JAK2 gene participate in the pathogenesis of MPN. Suppressors of cytokine signaling (SOCS) proteins are potent inhibitors of JAK/STAT pathway, therefore we hypothesized that the down regulation of SOCS protein system may be a possible pathogenetic mechanism of MPN through the activation of the JAK/STAT pathway. In order to testify our hypothesis, we investigated mutated points, the expression and methylation status of the SOCS1, SOCS2 and SOCS3 gene in 100 MPN patients(44 polycythemia vera (PV), 38 essential thrombocythemia (ET) and 18 idiopathic myelofibrosis(MF)). We obtained some interesting results: (1) By using DNA sequence analysis, two mutations of SOCS3 were identified with in the coding region in 1 of PV patients and 1 of ET patients (2%), respectively, and both of these 2 patients are with JAK2V617F mutation.(wide type ACG, coding Threonine, alterering to mutant type AAG, coding Lysine). Furthermore, three types of nonsense mutations were identified in SOCS3:Firstly,38 (38%) mutations of SOCS3 were identified with in the coding region in 19 of PV patients,17 of ET patients and 2 of MF respectively, (wide type CCC, coding Proline, alterering to mutant type CCA, coding Proline); Secondly, 44 (44%) mutations of SOCS3 were identified with in the coding region in 21 of PV patients,18 of ET patients and 5 of MF respectively, (wide type GTA, coding Valine, alterering to mutant type GTG, coding Valine); At last, 35 (35%) mutations of SOCS3 were identified with in the coding region in 13 of PV patients,20 of ET patients and 2 of MF respectively, (wide type GAT, coding Aspartic acid, alterering to mutant type GAC, coding Aspartic acid).Five nonsense mutations were found in SOCS2: 2 of PV patients,3 of ET patients, (wide type AAT, coding Asparagine, alterering to mutant type AAC, coding Asparagine). On the contrary, the presence of JAK2V617F mutation did not affect the nonsense mutations of SOCS2 or SOCS3. (2) By using Methylation Specific PCR (MSP), SOCS1 hypermethylation was identified in 27 patients. Hypermethylation of the SOCS2 promoter was identified in 9 of 100 (9%) patients. Hypermethylation of the SOCS3 promoter was identified in 35 of 100 (35%) patients. There was no hypermethylation of the SOCS1, SOCS2 and SOCS3 gene in 173 normal controls. (3) By using semi-quantitative PCR, the RNA expression levels of SOCS1, SOCS2 and SOCS3 were also investigated. We observed hypermethylated patients had lower SOCS1 or SOCS3 mRNA levels than unmethylated MPN samples, also observed that among patients with unmethylated SOCS1 and SOCS3, mRNA expression was higher from patients carrying the JAK2V617F mutation as compared with JAK2 wild type patients. On the contrary, the presence of JAK2V617F mutation did not affect the expression of SOCS1 or SOCS3 mRNA in methylated patients. Moreover, SOCS3 transcript levels were highest in patients with polycythemia vera and other JAK2 V617F negative myeloproliferative neoplasm. (4) According to SOCS1, SOCS3 methylation was not significantly correlated with survival or other clinical variables. In conclusion, SOCS1 and SOCS3 hypermethylation can activate the JAK/STATsignaling pathway in alternative or together with JAK2 mutations. These alterations might represent a potential therapeutic target. Disclosures: No relevant conflicts of interest to declare.
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Cai, Zhigang, Reuben Kapur, Xiaoyu Lu, Chi Zhang, Sai Nelanuthala, Jonathan Kotzin, Adam Williams, et al. "Hyperglycemia Cooperates with Tet2 Heterozygosity to Induce Leukemia Driven By Pro-Inflammatory Cytokine Induced Lncrna Morrbid." Blood 136, Supplement 1 (November 5, 2020): 30. http://dx.doi.org/10.1182/blood-2020-141335.
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Diabetes mellitus (DM) is a risk factor for cancer development. However, the role of DM induced hyperglycemic stress (HG) in the development of blood cancer is poorly understood, largely due to lack of appropriate animal models. Epidemiologic studies show that individuals with DM are more likely to possess higher rate of mutations in genes found in pre-leukemic stem and progenitor cells (pre-LHSC/Ps) including in the epigenetic regulator TET2. TET2-mutant pre-LHSC/Ps require additional hits to evolve into a full-blown leukemia and/or aggressive myeloproliferative neoplasm (MPN). Cell intrinsic mutations have been shown to cooperate with Tet2 to promote leukemic transformation. However, the role of extrinsic factors is poorly understood. Utilizing a novel mouse model bearing haploinsufficiency of Tet2, to mimic the human pre-LHSC/P condition and HG stress, in the form of an Ins2Akita/+mutation, which induces HG and Type-1 DM, we show that the compound mutant mice develop a lethal form of MPN and/or acute myeloid leukemia (AML). RNA-seq revealed that this is in part due to upregulation of pro-inflammatory pathways, thereby generating a feed-forward loop, including the expression of an anti-apoptotic lncRNA Morrbid. Loss of Morrbid in the compound mutants rescues the lethality and mitigates the development of MPN/AML. Our results describe a novel mouse model for age-dependent AML/MPN and suggest that HG stress acts as an environmental driver for myeloid neoplasm, which could be effectively prevented by reducing the expression of inflammation-related lncRNA Morrbid. Disclosures No relevant conflicts of interest to declare.
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Morimatsu, Katsuya, Shinichi Aishima, Hidetaka Yamamoto, Akifumi Hayashi, Kohei Nakata, Yasunori Oda, Koji Shindo, Minoru Fujino, Masao Tanaka, and Yoshinao Oda. "Insulin-like growth factor II messenger RNA–binding protein-3 is a valuable diagnostic and prognostic marker of intraductal papillary mucinous neoplasm." Human Pathology 44, no. 9 (September 2013): 1714–21. http://dx.doi.org/10.1016/j.humpath.2012.12.020.
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Basu, Gargi D., Janine LoBello, and Audrey Ozols. "Employing RNA sequencing to enhance treatment options for cancer patients." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 3628. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.3628.
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3628 Background: Fusions and translocations account for 20% of cancer mortality globally. Maximizing their detection enhances the utility of precision medicine for various solid and hematologic cancers. Practice guidelines stress the importance of RNA sequencing. Novel assay techniques employing a comprehensive genomic profiling approach, including RNA sequencing, yield information beyond conventional DNA next generation sequencing (NGS) alone. Methods: Tumor samples (N = 1517) were assayed combining whole transcriptome (RNA) sequencing, whole exome (DNA) sequencing, and comparison of tumor sequence vs. paired normal DNA. Results were analyzed to determine the frequency of rare and common RNA fusion and variant detection. Findings were mapped to a knowledge-base of targeted treatment options. Results: Analysis detected 79 (5.2%) actionable fusions and 15 (1%) transcript variants across major solid and heme-based malignancies. Notably, we observed actionable transcript variants that are not detectable at the DNA level including: EGFRvIII, EGFRvIVa and EGFRvIVb in GBM; ARv7 in prostate, and METe14 in TNBC. Many fusion cases (42%, n = 33) had no other actionable molecular abnormalities. Novel fusions included: SLC12A/ROS1 in low-grade spindle cell neoplasm with myogenic differentiation, KANK1/NTRK2 in ganglioneuroblastoma, ETV6/NTRK3 in metastatic mammary analogue secretory carcinoma, FGFR1/SCT in germ cell tumor, ZNF33B/RET fusion in GBM, SH3BP4/ERBB4 and EML4/ALK in RCC, VTCN1/NRG1 in pancreatic cancer, and AGRN/NRG1 in cholangiocarcinoma. More common actionable fusion events included: EML4/ALK in NSCLC, KIAA1549/BRAF in pilocytic astrocytoma, FGFR2 and FGFR3 in cholangiocarcinoma and urothelial cancers and ESR1 in endocrine therapy-resistant breast cancers. The fusion events detected in heme-based malignancies included MLLT10 and MLLT4 in AML, BCR/ABL in leukemias, TCF3/PBX1 in B cell ALL, NPM1/ALK in ALCL, and novel fusion CIITA/CD274 in DLBCL. All RNA fusions and transcript variants found were matched to FDA-approved or investigational treatment options. Conclusions: Maximizing the rate of variant detection for targeted therapy relies on precise identification of common and rare fusion events. Without the addition of RNA sequencing, 15 transcript variants in our cohort would have been missed and 33 of the fusions may have gone undetected by conventional DNA NGS testing, resulting in zero targeted treatment options for this vulnerable population. Further use of comprehensive genomic profiling is vital to optimizing cancer care.
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Foy, Allister, and Mary Frances McMullin. "Somatic SF3B1 mutations in myelodysplastic syndrome with ring sideroblasts and chronic lymphocytic leukaemia." Journal of Clinical Pathology 72, no. 11 (August 31, 2019): 778–82. http://dx.doi.org/10.1136/jclinpath-2019-205895.
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SF3B1 is the largest subunit of the Spliceosome Factor 3b (SF3B) complex and part of the U2 small nuclear ribosomal protein. It functions as an important part of spliceosomal assembly, converting precursor messenger RNA (mRNA) to mRNA ready for ribosomal translation. Mutations of SF3B1 are commonly seen in myelodysplastic syndromes with ring sideroblasts (MDS-RS)and MDS/myeloproliferative neoplasm (MPN-RS-T). These mutations are typically heterozygous missense substitutions, of which, 55% involve K700E. MDS-RS and MDS/MPN-RS-T usually carry a more favourable prognosis than other subtypes of MDS. SF3B1 itself does not influence survival in these conditions, but does correlate with increased thrombotic risk. Mutated SF3B1 is present in 9%–15% of chronic lymphocytic leukaemia cases and on its own correlates with improved responsiveness to ibrutinib, but is associated with additional adverse genetic abnormalities including TP53 and ATM mutations, which traditionally confer adverse outcomes.