119 results on '"Gerald Wertheim"'
Search Results
2. CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy
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Vinodh Pillai, Kavitha Muralidharan, Wenzhao Meng, Asen Bagashev, Derek A. Oldridge, Jaclyn Rosenthal, John Van Arnam, Jos J. Melenhorst, Diwakar Mohan, Amanda M. DiNofia, Minjie Luo, Sindhu Cherian, Jonathan R. Fromm, Gerald Wertheim, Andrei Thomas-Tikhonenko, Michele Paessler, Carl H. June, Eline T. Luning Prak, Vijay G. Bhoj, Stephan A. Grupp, Shannon L. Maude, and Susan R. Rheingold
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Specialties of internal medicine ,RC581-951 - Abstract
Abstract: Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19– subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)– deep remission, whereas 67 patients had a recurrence after achieving a MRD– deep remission: 28 patients with CD19+ leukemia and 39 patients with CD19– leukemia. Return of CD19+ leukemia was associated with loss of CAR T-cell function, whereas CD19– leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19– events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD– remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.
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- 2019
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3. Poor outcome with hematopoietic stem cell transplantation for bone marrow failure and MDS with severe MIRAGE syndrome phenotype
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Jay Sarthy, Ji Zha, Daria Babushok, Archana Shenoy, Jian-Meng Fan, Gerald Wertheim, Adam Himebauch, Ashley Munchel, Agne Taraseviciute, Samuel Yang, Hirohito Shima, Satoshi Narumi, Soheil Meshinchi, and Timothy S. Olson
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Specialties of internal medicine ,RC581-951 - Published
- 2018
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4. Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells
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Maria Krevvata, Xiaochuan Shan, Chenghui Zhou, Cedric Dos Santos, Georges Habineza Ndikuyeze, Anthony Secreto, Joshua Glover, Winifred Trotman, Gisela Brake-Silla, Selene Nunez-Cruz, Gerald Wertheim, Hyun-Jeong Ra, Elizabeth Griffiths, Charalampos Papachristou, Gwenn Danet-Desnoyers, and Martin Carroll
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Diseases of the blood and blood-forming organs ,RC633-647.5 - Abstract
Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (
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- 2018
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5. Clinical Impact of Genomic Information in Pediatric Leukemia
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Emilie Lalonde, Gerald Wertheim, and Marilyn M. Li
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genomic profiling ,pediatric leukemia ,diagnosis ,prognosis ,therapy ,Pediatrics ,RJ1-570 - Abstract
Pediatric leukemia remains a significant contributor to childhood lethality rates. However, recent development of new technologies including next-generation sequencing (NGS) has increased our understanding of the biological and genetic underpinnings of leukemia, resulting in novel diagnostic and treatment paradigms. The most prevalent pediatric leukemias include B-cell acute lymphoblastic leukemia (B-ALL) and acute myeloid leukemia (AML). These leukemias are highly heterogeneous, both clinically and genetically. There are multiple genetic subgroups defined by the World Health Organization, each with distinct clinical management. Clinical laboratories have started adopting genomic testing strategies to include high-throughput sequencing assays which, together with conventional cytogenetic techniques, enable optimal patient care. This review summarizes genetic and genomic techniques used in clinical laboratories to support management of pediatric leukemia, highlighting technical, biological, and clinical advances. We illustrate clinical utilities of comprehensive genomic evaluation of leukemia genomes through clinical case examples, which includes the interrogations of hundreds of genes and multiple mutation mechanisms using NGS technologies. Finally, we provide a future perspective on clinical genomics and precision medicine.
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- 2017
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6. Iris and orbital myeloid sarcoma in a pediatric patient
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Vivian L. Qin, Lana Verkuil, Tatiana Zanganeh, Gerald Wertheim, and Stefanie L. Davidson
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Ophthalmology ,Pediatrics, Perinatology and Child Health - Published
- 2023
7. Supplementary Methods, Tables 1 - 19, Figures 1 - 5 from Identification of Predictive Biomarkers for Cytokine Release Syndrome after Chimeric Antigen Receptor T-cell Therapy for Acute Lymphoblastic Leukemia
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Stephan A. Grupp, David L. Porter, Carl H. June, Bruce L. Levine, Gerald Wertheim, Farzana Nazimuddin, Jason C. White, Stefan Rose-John, Zhaohui Zheng, Susan R. Rheingold, Colleen Callahan, Richard Aplenc, Robert A. Berg, Julie C. Fitzgerald, Scott L. Weiss, David M. Barrett, Jeffrey Finklestein, Fang Chen, Vanessa E. Gonzalez, Edward Pequignot, Noelle Frey, Shannon L. Maude, J. Joseph Melenhorst, Pamela A. Shaw, Simon F. Lacey, and David T. Teachey
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Supplementary methods, statistical methods, results, tables, and figures. Supplementary Table 1. CRS grading system. Supplementary Table 2. Samples collected and time points on the trials. Supplementary Table 3. HLH diagnostic criteria. Supplementary Table 4. Timing of severe CRS relative to CRS onset and time from infusion. Supplementary Table 5. Patient demographics and baseline characteristics. Supplementary Table 6. Co-morbid infections. Supplementary Table 7. Median (range) for baseline cytokine values comparing normal subjects and total study population, children on the trials, and adults on the trials. Supplementary Table 8. Median (range) baseline cytokine values comparing baseline disease burden. Supplementary Table 9. Median (range) one-month peak cytokine values in children and adults comparing CRS grades (0-3 vs 4-5). Supplementary Table 10. Time points of target cytokine assessment listed as day since CTL019 infusion, with the acceptable windows provided by the study protocols. Supplementary Table 11. Median (range) day 1-3 peak cytokine values for those with severe (grade 4-5) and without severe (grade 0-3) CRS for total study population, and split into adults and children. Supplementary Table 12. Summary of all predictive models generated by discovery cohort and validated. Supplementary Table 13. Clinical details on the validation cohort. Supplementary Table 14. Therapies used for CRS. Supplementary Table 15. Tocilizumab use by grade and age, as well as, clinical effects of tocilizumab on CRS. Supplementary Table 16. Lee CRS grading scale. Supplementary Table 17. Davila CRS grading scale. Supplementary Table 18. Reclassification of CRS severity by dividing grade 3 into 3a and 3b. Supplementary Table 19. Summary of top predictive models with alternate determination of CRS severity. Supplementary Figure 1. CRP and ferritin are poor early predictors of severe CRS. Supplementary Figure 2. Early increases in IFNγ and sgp130 after CTL019 infusion are associated with development of severe CRS. Supplementary Figure 3. Severity of CRS correlates moderately with CAR T cell expansion but early expansion of CAR T cells does not predict CRS severity. Supplementary Figure 4. Cytokine profiles predict CRS. Supplementary Figure 5. Alternate CRS prediction models.
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- 2023
8. A novel cryopreservation and biobanking strategy to study lymphoid tissue stromal cells in human disease
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Joshua D Brandstadter, Angelina De Martin, Mechthild Lütge, Antonio Ferreira, Brian T Gaudette, Yves Stanossek, Shumei Wang, Michael V Gonzalez, Edward Camiolo, Gerald Wertheim, Bridget Austin, David Allman, Megan S Lim, David C Fajgenbaum, Jon C Aster, Burkhard Ludewig, and Ivan Maillard
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Article - Abstract
Non-hematopoietic lymph node stromal cells (LNSCs) regulate lymphocyte trafficking, survival, and function for key roles in host defense, autoimmunity, alloimmunity, and lymphoproliferative disorders. However, study of LNSCs in human diseases is complicated by a dependence on viable lymphoid tissues, which are most often excised prior to establishment of a specific diagnosis. Here, we demonstrate that cryopreservation can be used to bank lymphoid tissue for the study of LNSCs in human disease. Using human tonsils, lymphoid tissue fragments were cryopreserved for subsequent enzymatic digestion and recovery of viable non-hematopoietic cells. Flow cytometry and single-cell transcriptomics identified comparable proportions of LNSC cell types in fresh and cryopreserved tissue. Moreover, cryopreservation had little effect on transcriptional profiles, which showed significant overlap between tonsils and lymph nodes. The presence and spatial distribution of transcriptionally defined cell types was confirmed by in situ analyses. Our broadly applicable approach promises to greatly enable research into the roles of LNSC in human disease.
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- 2023
9. NTRK Fusions Identified in Pediatric Tumors: The Frequency, Fusion Partners, and Clinical Outcome
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Adam C. Resnick, Mariarita Santi, Zubair W Baloch, Minjie Luo, Rebecca L. Linn, Portia A. Kreiger, Fumin Lin, Stephen P. Hunger, Andrew J. Bauer, Marilyn M. Li, Pierre Russo, Elizabeth Fox, Chelsea Kotch, Phillip B. Storm, Jennifer Pogoriler, Vinodh Pillai, Lea F. Surrey, Xiaonan Zhao, and Gerald Wertheim
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0301 basic medicine ,Cancer Research ,business.industry ,ORIGINAL REPORTS ,Bioinformatics ,03 medical and health sciences ,Cancer Genomics ,030104 developmental biology ,0302 clinical medicine ,Text mining ,Oncology ,030220 oncology & carcinogenesis ,Tyrosine Receptor Kinase ,Medicine ,business - Abstract
PURPOSE Neurotrophic tyrosine receptor kinase (NTRK) fusions have been described as oncogenic drivers in a variety of tumors. However, little is known about the overall frequency of NTRK fusion in unselected pediatric tumors. Here, we assessed the frequency, fusion partners, and clinical course in pediatric patients with NTRK fusion–positive tumors. PATIENTS AND METHODS We studied 1,347 consecutive pediatric tumors from 1,217 patients who underwent tumor genomic profiling using custom-designed DNA and RNA next-generation sequencing panels. NTRK fusions identified were orthogonally confirmed. RESULTS AND DISCUSSION NTRK fusions were identified in 29 tumors from 27 patients with a positive yield of 2.22% for all patients and 3.08% for solid tumors. Although NTRK2 fusions were found exclusively in CNS tumors and NTRK1 fusions were highly enriched in papillary thyroid carcinomas, NTRK3 fusions were identified in all tumor categories. The most canonical fusion was ETV6-NTRK3 observed in 10 patients with diverse types of tumors. Several novel NTRK fusions were observed in rare tumor types, including KCTD16-NTRK1 in ganglioglioma and IRF2BP2-NTRK3 in papillary thyroid carcinomas. The detection of an NTRK fusion confirmed the morphologic diagnosis including five cases where the final tumor diagnosis was largely based on the discovery of an NTRK fusion. In one patient, the diagnosis was changed because of the identification of an ETV6-NTRK3 fusion. One patient with infantile fibrosarcoma was treated with larotrectinib and achieved complete pathologic remission. CONCLUSION NTRK fusions are more frequently seen in pediatric tumors than in adult tumors and involve a broader panel of fusion partners and a wider range of tumors than previously recognized. These results highlight the importance of screening for NTRK fusions as part of the tumor genomic profiling for patients with pediatric cancer.
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- 2021
10. Pharmacologic Inhibition of DYRK1A Results in MYC Hyperactivation and ERK Hyperphosphorylation rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition
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Christian Hurtz, V. S. S. Abhinav Ayyadevara, Gerald Wertheim, John A Chukinas, Joseph P Loftus, Sung June Lee, Anil Kumar, Rahul S Bhansali, Srividya Swaminathan, Huimin Geng, Thomas Milne, Xianxin Hua, Kathrin M Bernt, Thierry Besson, Junwei Shi, John D. Crispino, Martin Carroll, and Sarah K Tasian
- Abstract
KMT2A-rearranged (KMT2A-R) B cell acute lymphoblastic leukemia (ALL) is a high-risk disease in children and adults that is often chemotherapy resistant. To identify non-cytotoxic approaches to therapy, we performed a domain-specific kinome-wide CRISPR screen in KMT2A-R cell lines and patient derived xenograft samples (PDX) and identified dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) as a potential target. Pharmacologic inhibition of the KMT2A-fusion transcriptional co-regulator Menin released the KMT2A-fusion complex from the DYRK1A promoter thereby lowering DYRK1A expression levels confirming DYRK1A as a direct target of the KMT2A fusion oncogene. Direct pharmacologic inhibition of DYRK1A decreased cell proliferation of KMT2A-R ALL, thereby confirming the requirement of DYRK1A in this ALL subtype. To further understand the biologic function of DYRK1A in KMT2A-R ALL, we leveraged pharmacologic DYRK1A inhibitors in KMT2A-R PDX and cell line models. DYRK1A inhibition consistently led to upregulation of MYC protein levels, and hyperphosphorylation of ERK, which we confirmed via in vivo treatment experiments. Furthermore, DYRK1A inhibition decreased ALL burden in mice. Our results further demonstrate that DYRK1A inhibition induces the proapoptotic factor BIM, but ERK hyperphosphorylation is the driving event that induces cell cycle arrest. In contrast, combined treatment of KMT2A-R ALL cells in vitro and in vivo with DYRK1A inhibitors and the BCL2 inhibitor, venetoclax, synergistically decreases cell survival and reduced the leukemic burden in mice. Taken together these results demonstrate a unique function of DYRK1A specially in KMT2A-R ALL. Synergistic inhibition of DRYK1A and BCL2 may provide a low-toxic approach to treat this high risk ALL subtype.
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- 2022
11. High Expression of TIM 3 and Galectin 9 on Immunohistochemistry Staining of Tumor Specimen at Diagnosis in Pediatric Patients with Ewing Sarcoma
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David M. Barrett, Gerald Wertheim, and Stephanie J. Si
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Pathology ,medicine.medical_specialty ,business.industry ,medicine ,Immunohistochemistry ,Sarcoma ,medicine.disease ,business ,Staining ,Galectin - Abstract
Significant progress has been made in the advancement of immune system modulation for cancer treatment in recent years. In particular, immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy have demonstrated remarkable clinical benefit in relapsed/refractory cancers. However, our understanding of the immuno-oncologic landscape in pediatric solid tumors remains limited and is a barrier to continued progress. We examined the immunohistochemical expression of checkpoint receptors PD-1, TIM-3, LAG-3 and their respective ligands in various pediatric cancers at diagnosis and found high expression of TIM-3/Galectin-9 in the infiltrating cells of Ewing sarcoma. Location of checkpoint receptor/ligand expressions is important, as some staining patterns were only seen along tumor borders. Finally, peripheral T cell function varied significantly among different tumors supporting a complex relationship between the tumor microenvironment and the global immune system.
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- 2022
12. Absolute lymphocyte count proliferation kinetics after CAR T-cell infusion impact response and relapse
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Vinodh Pillai, Xiaoming Xu, Stephan A. Grupp, Sophia Faude, Vijay Bhoj, Shannon L. Maude, Kavitha Muralidharan, Michele Paessler, Susan R. Rheingold, Jane Wei, and Gerald Wertheim
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0301 basic medicine ,medicine.medical_specialty ,Immunobiology and Immunotherapy ,Lymphocytosis ,T-Lymphocytes ,Lymphocyte ,Receptors, Antigen, T-Cell ,Immunotherapy, Adoptive ,CD19 ,03 medical and health sciences ,0302 clinical medicine ,Recurrence ,Internal medicine ,Infusion Procedure ,medicine ,Humans ,Lymphocyte Count ,Cell Proliferation ,Atypical Lymphocyte ,Hematology ,biology ,business.industry ,medicine.disease ,Minimal residual disease ,Kinetics ,Cytokine release syndrome ,030104 developmental biology ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Immunology ,biology.protein ,medicine.symptom ,business - Abstract
CD19-directed chimeric antigen receptor (CAR) T cells show characteristic proliferation kinetics after infusion that correlate with response. Clearance of circulating disease, B-cell aplasia (BCA), and cytokine release syndrome (CRS) are used to observe CAR T-cell function, given the lack of commercial CAR T-cell measurement assays. We investigated the utility of common hematology laboratory parameters in 166 patients with B-cell acute lymphoblastic leukemia (B-ALL) who were treated with CAR T-cell therapy targeting CD19. CAR T-cell infusion was followed by disappearance of circulating blasts in 86% of patients at a median of 6 days. After a lag phase, there was a rapid expansion in absolute lymphocyte count (ALC) in the second week that coincided with the appearance of atypical lymphocytes. The expansion phase was followed by a contraction phase with a concomitant decrease in atypical lymphocytes. In vitro CAR T-cell studies showed similar kinetics and morphological changes. Peak ALC and overall expansion was greater in sustained responders compared with that in nonresponders. Patients with early loss of BCA and those with eventual CD19+ minimal residual disease/relapse showed lower overall lymphocyte expansion compared with the controls. Pleomorphic lymphocytosis was noted in the cerebrospinal fluid at post-CAR time points. We conclude that lymphocyte counts and differential can also be used to evaluate CAR T-cell expansion after infusion, along with BCA and CRS. This is the first report to characterize the morphology of CAR T cells and determine the utility of lymphocyte kinetics.
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- 2021
13. A Novel Cryopreservation and Biobanking Strategy to Study Lymphoid Tissue Stromal Cells in Lymphoproliferative Disorders
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Joshua D Brandstadter, Angelina De Martin, Mechthild Lϋetge, Antonio Ferreira, Brian T. Gaudette, Michael Gonzalez, Yves Stanossek, Edward Camiolo, Gerald Wertheim, David Allman, David C Fajgenbaum, Jon C. Aster, Burkhard Ludewig, and Ivan Maillard
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
14. CD22-Targeted CAR-Modified T-Cells Safely Induce Remissions in Children and Young Adults with Relapsed, CD19-Negative B-ALL after Treatment with CD19-Targeted CAR T-Cells
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Regina M. Myers, Amanda M. DiNofia, Yimei Li, Caroline Diorio, Richard Aplenc, Diane Baniewicz, Jennifer L Brogdon, Colleen Callahan, Boris Engels, Joseph A. Fraietta, Vanessa Gonzalez, Emma Iannone, Allison Barz Leahy, Hongyan Liu, Susan E. McClory, Susan R. Rheingold, Laura Shinehouse, Gerald Wertheim, Lisa Wray, Noelle V. Frey, Shannon L. Maude, and Stephan A. Grupp
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
15. Targeting CD38 in T-ALL Upregulates the Targetable Polyamine Metabolism Pathway
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Caroline Diorio, Tiffaney L. Vincent, Tori Fuller, Rawan Shraim, Tina Glisovic-Aplenc, Theresa Ryan, Kimberly Veliz, Haley Newman, Gerald Wertheim, Hamid Bassiri, Annette Vu, Michael Hogarty, Carl H June, Stephan A. Grupp, Sarah K Tasian, Richard Aplenc, Saar Gill, and David T. Teachey
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Immunology ,Cell Biology ,Hematology ,Biochemistry - Published
- 2022
16. Characterization of Plasmacytoid Dendritic Cells, Microbial Sequences, and Identification of a Candidate Public T-Cell Clone in Kikuchi-Fujimoto Disease
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Wenzhao Meng, Geraldine S. Pinkus, Nya D Nelson, Aaron M. Rosenfeld, Gerald Wertheim, Michele Paessler, Jason Nomburg, Eline T. Luning Prak, Susan Bullman, Vinodh Pillai, Matthew Meyerson, Chandra Sekhar Pedamallu, and Jason L. Hornick
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Male ,0301 basic medicine ,Adolescent ,T-Lymphocytes ,Interleukin-3 Receptor alpha Subunit ,Disease ,Biology ,Pathology and Forensic Medicine ,Diagnosis, Differential ,03 medical and health sciences ,0302 clinical medicine ,T cell clone ,Humans ,Child ,Histiocytic Necrotizing Lymphadenitis ,Immune repertoire ,Kikuchi-Fujimoto Disease ,Dendritic Cells ,General Medicine ,Complementarity Determining Regions ,Clone Cells ,030104 developmental biology ,Child, Preschool ,030220 oncology & carcinogenesis ,Pediatrics, Perinatology and Child Health ,Immunology ,Etiology ,Female ,Identification (biology) ,Interleukin-3 receptor ,Biomarkers - Abstract
Objectives Kikuchi-Fujimoto disease (KFD) is a self-limited lymphadenitis of unclear etiology. We aimed to further characterize this disease in pediatric patients, including evaluation of the CD123 immunohistochemical (IHC) staining and investigation of potential immunologic and infectious causes. Methods Seventeen KFD cases and 12 controls were retrospectively identified, and the histologic and clinical features were evaluated. CD123 IHC staining was quantified by digital image analysis. Next generation sequencing was employed for comparative microbial analysis via RNAseq (5 KFD cases) and to evaluate the immune repertoire (9 KFD cases). Results In cases of lymphadenitis with necrosis, >0.85% CD123+ cells by IHC was found to be six times more likely in cases with a final diagnosis of KFD (sensitivity 75%, specificity 87.5%). RNAseq based comparative microbial analysis did not detect novel or known pathogen sequences in KFD. A shared complementarity determining region 3 (CDR3) sequence and use of the same T-cell receptor beta variable region family was identified in KFD LNs but not controls, and was not identified in available databases. Conclusions: Digital quantification of CD123 IHC can distinguish KFD from other necrotizing lymphadenitides. The presence of a unique shared CDR3 sequence suggests that a shared antigen underlies KFD pathogenesis.
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- 2021
17. Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL
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Katherine D. Cummins, Gerald Wertheim, Anne Lehman, Janis K. Burkhardt, Asen Bagashev, Martin Carroll, Sarah K. Tasian, Daniel M. Blumenthal, Alexander E. Perl, Bryan Manning, Yong Li, Joseph P. Loftus, and Christian Hurtz
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0301 basic medicine ,MAPK/ERK pathway ,B-cell receptor ,Receptors, Antigen, B-Cell ,Biology ,Dexamethasone ,Cell Line ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,Animals ,Humans ,Protein Kinase Inhibitors ,PI3K/AKT/mTOR pathway ,breakpoint cluster region ,General Medicine ,Neoplasm Proteins ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cancer research ,Signal transduction ,Cytokine receptor ,Tyrosine kinase ,Signal Transduction ,Research Article ,Proto-oncogene tyrosine-protein kinase Src - Abstract
Children and adults with Philadelphia chromosome–like B cell acute lymphoblastic leukemia (Ph-like B-ALL) experience high relapse rates despite best-available conventional chemotherapy. Ph-like ALL is driven by genetic alterations that activate constitutive cytokine receptor and kinase signaling, and early-phase trials are investigating the potential of the addition of tyrosine kinase inhibitors (TKIs) to chemotherapy to improve clinical outcomes. However, preclinical studies have shown that JAK or PI3K pathway inhibition is insufficient to eradicate the most common cytokine receptor–like factor 2–rearranged (CRLF2-rearranged) Ph-like ALL subset. We thus sought to define additional essential signaling pathways required in Ph-like leukemogenesis for improved therapeutic targeting. Herein, we describe an adaptive signaling plasticity of CRLF2-rearranged Ph-like ALL following selective TKI pressure, which occurs in the absence of genetic mutations. Interestingly, we observed that Ph-like ALL cells have activated SRC, ERK, and PI3K signaling consistent with activated B cell receptor (BCR) signaling, although they do not express cell surface μ-heavy chain (μHC). Combinatorial targeting of JAK/STAT, PI3K, and “BCR-like” signaling with multiple TKIs and/or dexamethasone prevented this signaling plasticity and induced complete cell death, demonstrating a more optimal and clinically pragmatic therapeutic strategy for CRLF2-rearranged Ph-like ALL.
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- 2020
18. Runx1 negatively regulates inflammatory cytokine production by neutrophils in response to Toll-like receptor signaling
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Bing He, Martha S. Jordan, Dana C. Bellissimo, Qin Zhu, G. Scott Worthen, Sumedha Bagga, Nancy A. Speck, D. Gary Gilliland, Chia hui Chen, Gerald Wertheim, Chung Tsai Lee, and Kai Tan
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Toll-like receptor ,Innate immune system ,Neutrophils ,Chemistry ,medicine.medical_treatment ,Toll-Like Receptors ,NF-kappa B ,Hematology ,Proinflammatory cytokine ,Cell biology ,Phagocytes, Granulocytes, and Myelopoiesis ,Cytokine ,Downregulation and upregulation ,hemic and lymphatic diseases ,Core Binding Factor Alpha 2 Subunit ,embryonic structures ,medicine ,TLR4 ,Tumor necrosis factor alpha ,Signal transduction ,Signal Transduction - Abstract
RUNX1 is frequently mutated in myeloid and lymphoid malignancies. It has been shown to negatively regulate Toll-like receptor 4 (TLR4) signaling through nuclear factor κB (NF-κB) in lung epithelial cells. Here we show that RUNX1 regulates TLR1/2 and TLR4 signaling and inflammatory cytokine production by neutrophils. Hematopoietic-specific RUNX1 loss increased the production of proinflammatory mediators, including tumor necrosis factor-α (TNF-α), by bone marrow neutrophils in response to TLR1/2 and TLR4 agonists. Hematopoietic RUNX1 loss also resulted in profound damage to the lung parenchyma following inhalation of the TLR4 ligand lipopolysaccharide (LPS). However, neutrophils with neutrophil-specific RUNX1 loss lacked the inflammatory phenotype caused by pan-hematopoietic RUNX1 loss, indicating that dysregulated TLR4 signaling is not due to loss of RUNX1 in neutrophils per se. Rather, single-cell RNA sequencing indicates the dysregulation originates in a neutrophil precursor. Enhanced inflammatory cytokine production by neutrophils following pan-hematopoietic RUNX1 loss correlated with increased degradation of the inhibitor of NF-κB signaling, and RUNX1-deficient neutrophils displayed broad transcriptional upregulation of many of the core components of the TLR4 signaling pathway. Hence, early, pan-hematopoietic RUNX1 loss de-represses an innate immune signaling transcriptional program that is maintained in terminally differentiated neutrophils, resulting in their hyperinflammatory state. We hypothesize that inflammatory cytokine production by neutrophils may contribute to leukemia associated with inherited RUNX1 mutations.
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- 2020
19. Significance of minimal residual disease in pediatric mixed phenotype acute leukemia: a multicenter cohort study
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Jyotinder N. Punia, Maurice R.G. O'Gorman, Karen R. Rabin, Richard Sposto, Gerald Wertheim, Viviane C. Cahen, Terri Guinipero, William G. Woods, Reuven J. Schore, Dragos C. Luca, Etan Orgel, Matthew J. Oberley, Jemily Malvar, Alix E. Seif, and Sunil S. Raikar
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Male ,0301 basic medicine ,Oncology ,Cancer Research ,medicine.medical_specialty ,Neoplasm, Residual ,medicine.medical_treatment ,Population ,Article ,Cohort Studies ,03 medical and health sciences ,0302 clinical medicine ,hemic and lymphatic diseases ,Internal medicine ,Humans ,Medicine ,Child ,education ,Survival rate ,Chemotherapy ,education.field_of_study ,Leukemia ,Mixed phenotype acute leukemia ,business.industry ,Hematopoietic Stem Cell Transplantation ,Induction Chemotherapy ,Hematology ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,Prognosis ,Minimal residual disease ,United States ,Survival Rate ,body regions ,Transplantation ,Phenotype ,030104 developmental biology ,030220 oncology & carcinogenesis ,Cohort ,Female ,business ,Follow-Up Studies ,Cohort study - Abstract
The rarity of mixed phenotype acute leukemia (MPAL) has precluded adequate data to incorporate minimal residual disease (MRD) monitoring into therapy. Fluidity in MPAL classification systems further complicates understanding its biology and outcomes; this includes uncertainty surrounding the impact of shifting diagnostic requirements even between iterations of the World Health Organization (WHO) classification. Our primary objective was to address these knowledge gaps. To do so, we analyzed clinicopathologic features, therapy, MRD, and survival in a centrally-reviewed, multicenter cohort of MPAL uniformly diagnosed by the WHO classification and treated with acute lymphoblastic leukemia (ALL) regimens. ALL induction therapy achieved an EOI MRD negative (
- Published
- 2020
20. Tatton‐Brown‐Rahman syndrome: Six individuals with novel features
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Elaine H. Zackai, Julien L. Marcadier, Jennifer M. Kalish, Melissa T. Carter, Alana Strong, Anne Reilly, Gerald Wertheim, Lea F. Surrey, John M. Maris, Gail E. Graham, and Tugce B. Balci
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Adult ,Male ,Pediatrics ,medicine.medical_specialty ,Central sleep apnea ,Adolescent ,Context (language use) ,DNA Methyltransferase 3A ,Young Adult ,Orthostatic vital signs ,Intellectual Disability ,Intellectual disability ,Genetics ,Humans ,Medicine ,Abnormalities, Multiple ,DNA (Cytosine-5-)-Methyltransferases ,Child ,Preschool ,Growth Disorders ,Genetics (clinical) ,Ganglioneuroblastoma ,business.industry ,Lymphoblastic lymphoma ,Infant ,Dysautonomia ,Congenital diaphragmatic hernia ,Syndrome ,medicine.disease ,Phenotype ,Clathrin Heavy Chains ,Child, Preschool ,Mutation ,Female ,Abnormalities ,medicine.symptom ,business ,Multiple - Abstract
Tatton-Brown Rahman syndrome (TBRS) is an overgrowth-intellectual disability syndrome caused by heterozygous variants in DNMT3A. Seventy-eight individuals have been reported with a consistent phenotype of somatic overgrowth, mild to moderate intellectual disability, and similar dysmorphisms. We present six individuals with TBRS, including the youngest individual thus far reported, first individual to be diagnosed with tumor testing and two individuals with variants at the Arg882 domain, bringing the total number of reported cases to 82. Patients reported herein have additional clinical features not previously reported in TBRS. One patient had congenital diaphragmatic hernia. One patient carrying the recurrent p.Arg882His DNMT3A variant, who was previously reported as having a phenotype due to a truncating variant in the CLTC gene, developed a ganglioneuroblastoma at 18 months and T-cell lymphoblastic lymphoma at 6 years of age. Four patients manifested symptoms suggestive of autonomic dysfunction, including central sleep apnea, postural orthostatic hypotension, and episodic vasomotor instability in the extremities. We discuss the molecular and clinical findings in our patients with TBRS in context of existing literature.
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- 2020
21. Pediatric Somatic Tumor Sequencing Identifies Underlying Cancer Predisposition
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Suzanne P. MacFarland, Gerald Wertheim, Stephen P. Hunger, Garrett M. Brodeur, Minjie Luo, Kristin Zelley, Daniel Gallo, Pichai Raman, Lea F. Surrey, and Marilyn M. Li
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0301 basic medicine ,Oncology ,Cancer Research ,medicine.medical_specialty ,business.industry ,Cancer predisposition ,Somatic cell ,MEDLINE ,Cancer ,medicine.disease ,Article ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,030220 oncology & carcinogenesis ,Internal medicine ,Medicine ,Treatment decision making ,business - Abstract
PURPOSE The diagnosis of cancer predisposition in pediatric patients with cancer is vital for treatment decisions, surveillance, and management of at-risk family members. Somatic tumor testing can identify potential underlying constitutional variants that confer increased cancer risk. Here, we report the characteristics of constitutional variants identified through tumor testing. MATERIALS AND METHODS Data were abstracted from medical record review of 1,023 patients who received in-house somatic tumor testing over a 28-month period. Patients were identified for testing using referral criteria developed as a collaboration between genomic diagnostics, pathology, and oncology. Characteristics of patients who underwent constitutional testing, including family history and variant loss of heterozygosity, were tracked. RESULTS From 1,023 patients who underwent somatic tumor sequencing in a 28-month period, 210 variants were identified in 141 patients (13.8%) that were concerning for cancer predisposition syndromes requiring intervention. A total of 73 variants in 41 patients have undergone clinical confirmatory testing thus far. Of these, 26 variants were confirmed to be constitutionally present (35.6%). Among patients tested, 23 (56.1%) of 41 total patients were diagnosed with a cancer predisposition syndrome. CONCLUSION Our data demonstrate that more than one third of variants in tumor somatic sequencing that were concerning for underlying cancer predisposition were constitutionally confirmed. Overall, somatic tumor testing identified potential cancer predisposition syndromes in pediatric patients, and some would not have been identified on the basis of clinical history alone.
- Published
- 2019
22. A novel
- Author
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Feng, Xu, Erfan, Aref-Eshghi, Jinhua, Wu, Jeffrey, Schubert, Gerald, Wertheim, Tricia, Bhatti, Jennifer, Pogoriler, Maha, Patel, Kajia, Cao, Ariel, Long, Zhiqian, Fan, Elizabeth H, Denenberg, Elizabeth A, Fanning, Donna M, Wilmoth, Minjie, Luo, Laura K, Conlin, Aleksandra S, Dain, Kristin, Zelley, Sarah, Baldino, Naomi, Balamuth, Suzanne, MacFarland, Marilyn M, Li, and Yiming, Zhong
- Subjects
Adult ,Li-Fraumeni Syndrome ,Gene Duplication ,Humans ,Breast Neoplasms ,Female ,Genetic Predisposition to Disease ,Tumor Suppressor Protein p53 ,Child ,Adrenal Cortex Neoplasms ,Germ-Line Mutation - Abstract
Li-Fraumeni syndrome (LFS) is one of the most common cancer predisposition syndromes that affects both children and adults. Individuals with LFS are at an increased risk of developing various types of cancer over their lifetime including soft tissue sarcomas, osteosarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Heterozygous germline pathogenic variants in the tumor suppressor gene
- Published
- 2021
23. Molecular evolution of classic Hodgkin lymphoma revealed through whole genome sequencing of Hodgkin and Reed Sternberg cells
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Francesco Maura, Bachisio Ziccheddu, Jenny Z. Xiang, Bhavneet Bhinder, Federico Abascal, Kylee H. Maclachlan, Kenneth Wha Eng, Manik Uppal, Feng He, Wei Zhang, Qi Gao, Venkata Yellapantula, Sunita Park, Matthew Oberley, Elizabeth Ruckdeschel, Megan S. Lim, Gerald Wertheim, Matthew Barth, Terzah M. Horton, Christopher Forlenza, Yanming Zhang, Ola Landgren, Craig H Moskowitz, Ethel Cesarman, Marcin Imielinski, Olivier Elemento, Mikhail Roshal, and Lisa Giulino-Roth
- Subjects
macromolecular substances - Abstract
The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells within a classic Hodgkin lymphoma (cHL) biopsy limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Here we leveraged this method to report the first whole genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events prior to the clinical diagnosis of cHL. We identified alterations in driver genes not previously described in cHL, a high activity of the APOBEC mutational signature, and the presence complex structural variants including chromothripsis. We found that the high ploidy observed in cHL is often acquired through multiple, independent large chromosomal gain events including whole genome duplication. The first of these likely occurs several years prior to the diagnosis of cHL, and the last gains typically occur very close to the time of diagnosis. Evolutionary timing analyses revealed that driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID driven mutagenesis usually preceded large chromosomal gains. The study provides the first temporal reconstruction of cHL pathogenesis and suggests a relatively long time course between the first pathogenic event and the clinical diagnosis.
- Published
- 2021
24. Transcriptome and unique cytokine microenvironment of Castleman disease
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Michele Paessler, Anna Wing, Aaron M. Rosenfeld, David T. Teachey, Eline T. Luning Prak, Gerald Wertheim, Dale Frank, Vinodh Pillai, Jason Xu, Kai Tan, Adam Bagg, Megan S. Lim, David C. Fajgenbaum, Wenzhao Meng, and Elizabeth Y. Li
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Vascular Endothelial Growth Factor A ,Pathology ,medicine.medical_specialty ,medicine.medical_treatment ,T cell ,Biology ,Article ,Pathology and Forensic Medicine ,Plasma cell differentiation ,medicine ,Humans ,CXCL13 ,Lymph node ,Clusterin ,Follicular dendritic cells ,Interleukin-6 ,Castleman Disease ,Germinal center ,eye diseases ,ADAM Proteins ,medicine.anatomical_structure ,Cytokine ,biology.protein ,Cancer research ,Cytokines ,Transcriptome - Abstract
Castleman disease (CD) represents a group of rare, heterogeneous and poorly understood disorders that share characteristic histopathological features. Unicentric CD (UCD) typically involves a single enlarged lymph node whereas multicentric CD (MCD) involves multiple lymph node stations. To understand the cellular basis of CD, we undertook a multi-platform analysis using targeted RNA sequencing, RNA in-situ hybridization (ISH), and adaptive immune receptor rearrangements (AIRR) profiling of archived tissue from 26 UCD, 14 MCD, and 31 non-CD reactive controls. UCD showed differential expression and upregulation of follicular dendritic cell markers (CXCL13, clusterin), angiogenesis factors (LPL, DLL4), extracellular matrix remodeling factors (TGFβ, SKIL, LOXL1, IL-1β, ADAM33, CLEC4A), complement components (C3, CR2) and germinal center activation markers (ZDHHC2 and BLK) compared to controls. MCD showed upregulation of IL-6 (IL-6ST, OSMR and LIFR), IL-2, plasma cell differentiation (XBP1), FDC marker (CXCL13, clusterin), fibroblastic reticular cell cytokine (CCL21), angiogenesis factor (VEGF), and mTORC1 pathway genes compared to UCD and controls. ISH studies demonstrated that VEGF was increased in the follicular dendritic cell-predominant atretic follicles and the interfollicular macrophages of MCD compared to UCD and controls. IL-6 expression was higher along interfollicular vasculature-associated cells of MCD. Immune repertoire analysis revealed oligoclonal expansions of T-cell populations in MCD cases (2/6) and UCD cases (1/9) that are consistent with antigen-driven T cell activation. The findings highlight the unique genes, pathways and cell types involved in UCD and MCD. We identify potential novel targets in CD that may be harnessed for therapeutics.
- Published
- 2021
25. 134. Novel fusions in aggressive infant sarcomas: Expanding the scope of 'CIC-rearranged' sarcoma without CIC rearrangement
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Feng Xu, Jenny Ruiz, Jeffery Schubert, Jinhua Wu, Jiani Chen, Kajia Cao, Zhiqian Fan, Ariel Long, Yiming Zhong, Portia Kreiger, Lea Surrey, Gerald Wertheim, Kristina Cole, Mariarita Santi, Marilyn Li, and Phillip Storm
- Subjects
Cancer Research ,Genetics ,Molecular Biology - Published
- 2022
26. Infant Acute Leukemia
- Author
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Gerald Wertheim
- Subjects
Adult ,Pediatrics ,medicine.medical_specialty ,Acute leukemia ,Leukemia ,business.industry ,Biochemistry (medical) ,Clinical Biochemistry ,Infant ,Aggressive disease ,medicine.disease ,Young age ,Acute megakaryoblastic leukemia ,Leukemia, Megakaryoblastic, Acute ,hemic and lymphatic diseases ,Acute Disease ,medicine ,Humans ,Down Syndrome ,business ,Child - Abstract
Infant acute leukemia is a rare but aggressive disease. Although infant acute leukemia is cytologically and histologically similar to acute leukemia seen in older children and adults, it displays unique and characteristic clinical and genetic characteristics. The features, as well as the extremely young age of the patients, present multiple challenges for treatment. This review focuses on the unique pathology of acute leukemia of infancy, including the genetic characteristics that are specific for these diseases.
- Published
- 2021
27. Humanized CD19-Targeted Chimeric Antigen Receptor (CAR) T Cells in CAR-Naive and CAR-Exposed Children and Young Adults With Relapsed or Refractory Acute Lymphoblastic Leukemia
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Pamela A Shaw, Don L. Siegel, Andrew D. Fesnak, Christina Fasano, Kelly D. Getz, Richard Aplenc, Jennifer Brogdon, Amanda M. DiNofia, Allison Barz Leahy, Yimei Li, Megan M. Davis, Diane Baniewicz, Hongyan Liu, Lisa Wray, Carl H. June, David T. Teachey, Elizabeth O. Hexner, Stephan A. Grupp, Edward Pequignot, Bruce L. Levine, Colleen Callahan, Simon F. Lacey, Shannon L. Maude, Gerald Wertheim, Anne Chew, Chelsie Bartoszek, Regina M. Myers, Susan R. Rheingold, and David M. Barrett
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Adult ,Male ,Cancer Research ,Adolescent ,Lymphoblastic Leukemia ,Antigens, CD19 ,Receptors, Antigen, T-Cell ,Pilot Projects ,CD19 ,Young Adult ,Refractory ,Medicine ,Humans ,Young adult ,Child ,Receptors, Chimeric Antigen ,biology ,business.industry ,ORIGINAL REPORTS ,Precursor Cell Lymphoblastic Leukemia-Lymphoma ,Chimeric antigen receptor ,Oncology ,Child, Preschool ,Immunology ,biology.protein ,Female ,Car t cells ,business - Abstract
PURPOSE CD19-targeted chimeric antigen receptor (CAR)–modified T cells demonstrate unprecedented responses in B-cell acute lymphoblastic leukemia (B-ALL); however, relapse remains a substantial challenge. Short CAR T-cell persistence contributes to this risk; therefore, strategies to improve persistence are needed. METHODS We conducted a pilot clinical trial of a humanized CD19 CAR T-cell product (huCART19) in children and young adults with relapsed or refractory B-ALL (n = 72) or B-lymphoblastic lymphoma (n = 2), treated in two cohorts: with (retreatment, n = 33) or without (CAR-naive, n = 41) prior CAR exposure. Patients were monitored for toxicity, response, and persistence of huCART19. RESULTS Seventy-four patients 1-29 years of age received huCART19. Cytokine release syndrome developed in 62 (84%) patients and was grade 4 in five (6.8%). Neurologic toxicities were reported in 29 (39%), three (4%) grade 3 or 4, and fully resolved in all cases. The overall response rate at 1 month after infusion was 98% (100% in B-ALL) in the CAR-naive cohort and 64% in the retreatment cohort. At 6 months, the probability of losing huCART19 persistence was 27% (95% CI, 14 to 41) for CAR-naive and 48% (95% CI, 30 to 64) for retreatment patients, whereas the incidence of B-cell recovery was 15% (95% CI, 6 to 28) and 58% (95% CI, 33 to 77), respectively. Relapse-free survival at 12 and 24 months, respectively, was 84% (95% CI, 72 to 97) and 74% (95% CI, 60 to 90) in CAR-naive and 74% (95% CI, 56 to 97) and 58% (95% CI, 37 to 90) in retreatment cohorts. CONCLUSION HuCART19 achieved durable remissions with long-term persistence in children and young adults with relapsed or refractory B-ALL, including after failure of prior CAR T-cell therapy.
- Published
- 2021
28. Constrained chromatin accessibility in PU.1-mutated agammaglobulinemia patients
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Natasha L. Rudy, Anna C.E. Hurst, Alexander Marson, Steven H. Kroft, James Garifallou, Sarah K. Nicholas, Piyush Pillarisetti, Gerald Wertheim, Di Sun, Andrew D. Wells, Titus J. Boggon, Gregory M.K. Poon, Kelly Maurer, Brian Nolan, Caroline Khanna, Francis Wright, Jennifer M. Puck, Struan F.A. Grant, Suela Xhani, Amy Rymaszewski, Ivan K. Chinn, Viktoria Zakharova, Samuel Yoon, James W. Verbsky, Neil Romberg, David N. Nguyen, Linda T. Vo, Kathleen E. Sullivan, Chun Su, Anna Shcherbina, Alix E. Seif, T. Prescott Atkinson, Amy L. Stiegler, Hakon Hakonarson, Anna Mukhina, Amanda V. Albrecht, Timothy S. Olson, Peixin Amy Chen, John M. Routes, Benjamin Demaree, Joud Hajjar, James R. Lupski, Adam R. Abate, Michael Gonzalez, and Carole Le Coz
- Subjects
0301 basic medicine ,Mutation ,Euchromatin ,Immunology ,Biology ,medicine.disease_cause ,Cell biology ,Chromatin ,03 medical and health sciences ,Haematopoiesis ,030104 developmental biology ,0302 clinical medicine ,medicine.anatomical_structure ,medicine ,Immunology and Allergy ,Lymphopoiesis ,Stem cell ,Progenitor cell ,030217 neurology & neurosurgery ,B cell - Abstract
The pioneer transcription factor (TF) PU.1 controls hematopoietic cell fate by decompacting stem cell heterochromatin and allowing nonpioneer TFs to enter otherwise inaccessible genomic sites. PU.1 deficiency fatally arrests lymphopoiesis and myelopoiesis in mice, but human congenital PU.1 disorders have not previously been described. We studied six unrelated agammaglobulinemic patients, each harboring a heterozygous mutation (four de novo, two unphased) of SPI1, the gene encoding PU.1. Affected patients lacked circulating B cells and possessed few conventional dendritic cells. Introducing disease-similar SPI1 mutations into human hematopoietic stem and progenitor cells impaired early in vitro B cell and myeloid cell differentiation. Patient SPI1 mutations encoded destabilized PU.1 proteins unable to nuclear localize or bind target DNA. In PU.1-haploinsufficient pro–B cell lines, euchromatin was less accessible to nonpioneer TFs critical for B cell development, and gene expression patterns associated with the pro– to pre–B cell transition were undermined. Our findings molecularly describe a novel form of agammaglobulinemia and underscore PU.1’s critical, dose-dependent role as a hematopoietic euchromatin gatekeeper.
- Published
- 2021
29. A Novel KMT2A-ARHGEF12 Fusion Gene Identified in a High-Grade B-cell Lymphoma
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Gerald Wertheim, Adam J. Wolpaw, Marilyn M. Li, Minjie Luo, Fumin Lin, Anne F. Reilly, Elizabeth Margolskee, and Hou-Sung Jung
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Cancer Research ,Oncogene Proteins ,High grade B-cell lymphoma ,Biology ,medicine.disease ,Lymphoma ,Fusion gene ,KMT2A ,Genetics ,medicine ,biology.protein ,Cancer research ,B-cell lymphoma ,Molecular Biology - Published
- 2020
30. Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in IL-6 blockade–refractory idiopathic multicentric Castleman disease
- Author
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Amrit Singh, Anthony P. Schwarer, Helen L. Partridge, Frits van Rhee, Daniel J. Arenas, David C. Fajgenbaum, Katie L. Stone, Mariko Okumura, Jason R. Ruth, Nelson Hamerschlak, Michael R. Betts, Thomas S. Uldrick, Gerald Wertheim, Christopher S. Nabel, Taku Kambayashi, Michael B. Jordan, Sheila K Pierson, Fabio Freire José, Alberto Sada Japp, Arthur H. Rubenstein, Adam D. Cohen, Ruth-Anne Langan, and Vera P. Krymskaya
- Subjects
0301 basic medicine ,medicine.medical_specialty ,Hematology ,biology ,business.industry ,General Medicine ,Systemic inflammation ,medicine.disease ,Anasarca ,Organomegaly ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,hemic and lymphatic diseases ,030220 oncology & carcinogenesis ,Internal medicine ,medicine ,Cancer research ,biology.protein ,medicine.symptom ,business ,Myelofibrosis ,Interleukin 6 ,Protein kinase B ,PI3K/AKT/mTOR pathway - Abstract
BACKGROUND Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6 (IL-6) blockade–refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6 blockade–refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype.
- Published
- 2019
31. Trib1 regulates eosinophil lineage commitment and identity by restraining the neutrophil program
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Sarah J. Stein, Kelly S. Rome, Gerald Wertheim, Ethan A. Mack, Warren S. Pear, Lanwei Xu, and Rossana C. N. Melo
- Subjects
Neutrophils ,Immunology ,Population ,Mice, Transgenic ,Protein Serine-Threonine Kinases ,Biology ,Granulocyte ,Biochemistry ,Mice ,Phagocytes, Granulocytes, and Myelopoiesis ,Eosinophil migration ,Granulocyte-Macrophage Progenitor Cells ,Enhancer binding ,medicine ,Animals ,education ,Interleukin 5 ,Myelopoiesis ,education.field_of_study ,Eosinophil differentiation ,Intracellular Signaling Peptides and Proteins ,Cell Biology ,Hematology ,Eosinophil ,Cell biology ,Eosinophils ,medicine.anatomical_structure ,Gene Expression Regulation - Abstract
Eosinophils and neutrophils are critical for host defense, yet gaps in understanding how granulocytes differentiate from hematopoietic stem cells (HSCs) into mature effectors remain. The pseudokinase tribbles homolog 1 (Trib1) is an important regulator of granulocytes; knockout mice lack eosinophils and have increased neutrophils. However, how Trib1 regulates cellular identity and function during eosinophilopoiesis is not understood. Trib1 expression markedly increases with eosinophil-lineage commitment in eosinophil progenitors (EoPs), downstream of the granulocyte/macrophage progenitor (GMP). Using hematopoietic- and eosinophil-lineage–specific Trib1 deletion, we found that Trib1 regulates both granulocyte precursor lineage commitment and mature eosinophil identity. Conditional Trib1 deletion in HSCs reduced the size of the EoP pool and increased neutrophils, whereas deletion following eosinophil lineage commitment blunted the decrease in EoPs without increasing neutrophils. In both modes of deletion, Trib1-deficient mice expanded a stable population of Ly6G+ eosinophils with neutrophilic characteristics and functions, and had increased CCAAT/enhancer binding protein α (C/EBPα) p42. Using an ex vivo differentiation assay, we found that interleukin 5 (IL-5) supports the generation of Ly6G+ eosinophils from Trib1-deficient cells, but is not sufficient to restore normal eosinophil differentiation and development. Furthermore, we demonstrated that Trib1 loss blunted eosinophil migration and altered chemokine receptor expression, both in vivo and ex vivo. Finally, we showed that Trib1 controls eosinophil identity by modulating C/EBPα. Together, our findings provide new insights into early events in myelopoiesis, whereby Trib1 functions at 2 distinct stages to guide eosinophil lineage commitment from the GMP and suppress the neutrophil program, promoting eosinophil terminal identity and maintaining lineage fidelity.
- Published
- 2019
32. Risk-Adapted Preemptive Tocilizumab to Prevent Severe Cytokine Release Syndrome After CTL019 for Pediatric B-Cell Acute Lymphoblastic Leukemia: A Prospective Clinical Trial
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Regina McGuire, Julie C. Fitzgerald, Diane Baniewicz, Lisa Wray, Colleen Callahan, Allison Barz Leahy, Whitney L. Gladney, Stephan Kadauke, Regina M. Myers, Joseph G Dolan, Laura S Motley, Hongyan Liu, Yimei Li, David M. Barrett, Gerald Wertheim, Simon F. Lacey, David T. Teachey, Stephan A. Grupp, Richard Aplenc, Shannon L. Maude, and Amanda M. DiNofia
- Subjects
Oncology ,Adult ,Male ,Cancer Research ,medicine.medical_specialty ,Adolescent ,medicine.medical_treatment ,Antigens, CD19 ,Salvage therapy ,Pilot Projects ,Antibodies, Monoclonal, Humanized ,Immunotherapy, Adoptive ,chemistry.chemical_compound ,Young Adult ,Tocilizumab ,Risk Factors ,Internal medicine ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,medicine ,Humans ,Prospective Studies ,Prospective cohort study ,Child ,Retrospective Studies ,Salvage Therapy ,business.industry ,Infant ,Retrospective cohort study ,Immunotherapy ,ORIGINAL REPORTS ,medicine.disease ,Prognosis ,Clinical trial ,Survival Rate ,Cytokine release syndrome ,chemistry ,Drug Resistance, Neoplasm ,Child, Preschool ,Monoclonal ,Female ,Neoplasm Recurrence, Local ,business ,Cytokine Release Syndrome ,Follow-Up Studies - Abstract
PURPOSETo prospectively evaluate the effectiveness of risk-adapted preemptive tocilizumab (PT) administration in preventing severe cytokine release syndrome (CRS) after CTL019, a CD19 chimeric antigen receptor T-cell therapy.METHODSChildren and young adults with CD19-positive relapsed or refractory B-cell acute lymphoblastic leukemia were assigned to high- (≥ 40%) or low- (< 40%) tumor burden cohorts (HTBC or LTBC) based on a bone marrow aspirate or biopsy before infusion. HTBC patients received a single dose of tocilizumab (8-12 mg/kg) after development of high, persistent fevers. LTBC patients received standard CRS management. The primary end point was the frequency of grade 4 CRS (Penn scale), with an observed rate of ≤ 5 of 15 patients in the HTBC pre-defined as clinically meaningful. In post hoc analyses, the HTBC was compared with a historical cohort of high-tumor burden patients from the initial phase I CTL019 trial.RESULTSThe primary end point was met. Seventy patients were infused with CTL019, 15 in the HTBC and 55 in the LTBC. All HTBC patients received the PT intervention. The incidence of grade 4 CRS was 27% (95% CI, 8 to 55) in the HTBC and 3.6% (95% CI, 0.4 to 13) in the LTBC. The best overall response rate was 87% in the HTBC and 100% in the LTBC. Initial CTL019 expansion was greater in the HTBC than the LTBC ( P < .001), but persistence was not different ( P = .73). Event-free and overall survival were worse in the HTBC ( P = .004, P < .001, respectively). In the post hoc analysis, grade 4 CRS was observed in 27% versus 50% of patients in the PT and prior phase I cohorts, respectively ( P = .18).CONCLUSIONRisk-adapted PT administration resulted in a decrease in the expected incidence of grade 4 CRS, meeting the study end point, without adversely impacting the antitumor efficacy or safety of CTL019.
- Published
- 2021
33. Eosinophilia/Hypereosinophilia in the Setting of Reactive and Idiopathic Causes, Well-Defined Myeloid or Lymphoid Leukemias, or Germline Disorders
- Author
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Hans-Peter Horny, Rebecca L. King, Megan O. Nakashima, Gerald Wertheim, Sa A. Wang, Katalin Kelemen, Lisa M. Rimsza, Kaaren K. Reichard, Leonie Saft, Fiona E. Craig, Tracy I. George, Leticia Quintanilla-Martinez, and Attilio Orazi
- Subjects
0301 basic medicine ,Male ,Myeloid ,Fusion Proteins, bcr-abl ,Hypereosinophilia ,Leukemia, Myeloid, Accelerated Phase ,Precursor T-Cell Lymphoblastic Leukemia-Lymphoma ,Diagnosis, Differential ,03 medical and health sciences ,0302 clinical medicine ,hemic and lymphatic diseases ,Leukemia, Myelogenous, Chronic, BCR-ABL Positive ,Eosinophilia ,Hypereosinophilic Syndrome ,medicine ,Humans ,Genetic Predisposition to Disease ,Pathology, Molecular ,Myeloproliferative neoplasm ,Chronic eosinophilic leukemia ,Acute leukemia ,Leukemia ,business.industry ,Histological Techniques ,Myeloid leukemia ,General Medicine ,medicine.disease ,Leukemia, Lymphoid ,030104 developmental biology ,medicine.anatomical_structure ,Germ Cells ,Hematologic Neoplasms ,Myelodysplastic Syndromes ,Immunology ,Female ,medicine.symptom ,business ,030215 immunology - Abstract
Objectives To report the findings of the 2019 Society for Hematopathology/European Association for Haematopathology Workshop within the categories of reactive eosinophilia, hypereosinophilic syndrome (HES), germline disorders with eosinophilia (GDE), and myeloid and lymphoid neoplasms associated with eosinophilia (excluding entities covered by other studies in this series). Methods The workshop panel reviewed 109 cases, assigned consensus diagnosis, and created diagnosis-specific sessions. Results The most frequent diagnosis was reactive eosinophilia (35), followed by acute leukemia (24). Myeloproliferative neoplasms (MPNs) received 17 submissions, including chronic eosinophilic leukemia, not otherwise specified (CEL, NOS). Myelodysplastic syndrome (MDS), MDS/MPN, and therapy-related myeloid neoplasms received 11, while GDE and HES received 12 and 11 submissions, respectively. Conclusions Hypereosinophilia and HES are defined by specific clinical and laboratory criteria. Eosinophilia is commonly reactive. An acute leukemic onset with eosinophilia may suggest core-binding factor acute myeloid leukemia, blast phase of chronic myeloid leukemia, BCR-ABL1–positive leukemia, or t(5;14) B-lymphoblastic leukemia. Eosinophilia is rare in MDS but common in MDS/MPN. CEL, NOS is a clinically aggressive MPN with eosinophilia as the dominant feature. Bone marrow morphology and cytogenetic and/or molecular clonality may distinguish CEL from HES. Molecular testing helps to better subclassify myeloid neoplasms with eosinophilia and to identify patients for targeted treatments.
- Published
- 2020
34. Clinical impact of genomic characterization of 15 patients with acute megakaryoblastic leukemia-related malignancies
- Author
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Kajia Cao, Fumin Lin, Amrom E. Obstfeld, Lea F. Surrey, Xiaonan Zhao, Marilyn M. Li, Stefan Rentas, Richard Aplenc, Minjie Luo, Emilie Lalonde, and Gerald Wertheim
- Subjects
Oncology ,Male ,Research Report ,medicine.medical_specialty ,Down syndrome ,Somatic cell ,Karyotype ,Kruppel-Like Transcription Factors ,Genome ,Chromosomes ,Leukemoid Reaction ,Acute megakaryoblastic leukemia ,Leukemia, Megakaryoblastic, Acute ,Internal medicine ,Neoplasms ,Medicine ,Humans ,Clinical significance ,GATA1 Transcription Factor ,Genetic Predisposition to Disease ,Child ,Leukemia ,business.industry ,Infant, Newborn ,Myeloid leukemia ,High-Throughput Nucleotide Sequencing ,Infant ,GATA1 ,General Medicine ,Genomics ,Histone-Lysine N-Methyltransferase ,medicine.disease ,acute megakaryocytic leukemia ,Neoplasm Proteins ,DNA-Binding Proteins ,Repressor Proteins ,Child, Preschool ,Female ,Down Syndrome ,business ,Trisomy ,Retinoblastoma-Binding Protein 2 ,Myeloid-Lymphoid Leukemia Protein - Abstract
Acute megakaryoblastic leukemia (AMKL) is a rare subtype of acute myeloid leukemia but is approximately 500 times more likely to develop in children with Down syndrome (DS) through transformation of transient abnormal myelopoiesis (TAM). This study investigates the clinical significance of genomic heterogeneity of AMKL in children with and without DS and in children with TAM. Genomic evaluation of nine patients with DS-related TAM or AMKL, and six patients with non-DS AMKL, included conventional cytogenetics and a comprehensive next-generation sequencing panel for single-nucleotide variants/indels and copy-number variants in 118 genes and fusions involving 110 genes. Recurrent gene fusions were found in all patients with non-DS, including two individuals with complex genomes and either a NUP98–KDM5A or a KMT2A–MLLT6 fusion, and the remaining harbored a CBFA2T3–GLIS2 fusion, which arose from both typical and atypical cytogenetic mechanisms. These fusions guided treatment protocols and resulted in a change in diagnosis in two patients. The nine patients with DS had constitutional trisomy 21 and somatic GATA1 mutations, and those with DS-AMKL had two to four additional clinically significant somatic mutations. Comprehensive genomic characterization provides critical information for diagnosis, risk stratification, and treatment decisions for patients with AMKL. Continued genetic and clinical characterization of these rare cancers will aid in improving patient management.
- Published
- 2020
35. A Novel TP53 Tandem Duplication in a Child with Li-Fraumeni Syndrome
- Author
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Feng Xu, Erfan Aref-Eshghi, Jinhua Wu, Jeffrey Schubert, Gerald Wertheim, Tricia Bhatti, Jennifer Pogoriler, Maha Patel, Kajia Cao, Ariel Long, Zhiqian Fan, Elizabeth Denenberg, Elizabeth Fanning, Donna Wilmoth, Minjie Luo, Laura Conlin, Aleksandra Sarah Dain, Sarah Baldino, Kristin Zelley, Naomi J Balamuth, Suzanne Macfarland, Marilyn M Li, and Yiming Zhong
- Subjects
General Medicine - Abstract
Li-Fraumeni syndrome (LFS) is one of the most common cancer predisposition syndromes that affects both children and adults. Individuals with LFS are at an increased risk of developing various types of cancer over their lifetime including soft tissue sarcomas, osteosarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Heterozygous germline pathogenic variants in the tumor suppressor gene TP53 are the known causal genetic defect for LFS. Single nucleotide variants (SNVs) including missense substitutions that occur in the highly conserved DNA binding domain of the protein are the most common alterations, followed by nonsense and splice site variants. Gross copy number changes in TP53 are rare and account for less than 1% of all variants. Using next-generation sequencing (NGS) panels, we identified a paternally inherited germline intragenic duplication of TP53 in a child with metastatic osteosarcoma who later developed acute myeloid leukemia (AML). Transcriptome sequencing (RNA-Seq) demonstrated the duplication was tandem, encompassing exons 2-6 and 28 nucleotides of the untranslated region (UTR) upstream of the start codon in exon 2. The inclusion of the 28 nucleotides is expected to result in a frameshift with a stop codon 18 codons downstream of the exon 6, leading to a loss-of-function allele. This case highlights the significance of simultaneous identification of both significant copy number variants as well as SNVs/indels using NGS panels.
- Published
- 2022
36. eP288: Genomic characterization of a TP53 tandem duplication in a pediatric patient with Li-Fraumeni syndrome
- Author
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Feng Xu, Erfan Aref-Eshghi, Jinhua Wu, Jeffrey Schubert, Maha Patel, Zhiqian Fan, Kajia Cao, Ariel Long, Elizabeth Denenberg, Elizabeth Fanning, Donna Wilmoth, Gerald Wertheim, Minjie Luo, Laura Conlin, Tricia Bhatti, Aleksandra Dain, Kristin Zelley, Naomi Balamuth, Suzanne MacFarland, Marilyn Li, and Yiming Zhong
- Subjects
Genetics (clinical) - Published
- 2022
37. 28. Identification of TP53 germline variants in pediatric patients undergoing tumor testing
- Author
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Minjie Luo, Suzanne Macfarland, Kristin Zelley, Fumin Lin, Daniel Gallo, Jinhua Wu, Jeffery Schubert, Elizabeth Denenberg, Elizabeth Fanning, Jiani Chen, Hou-sung Jung, Laura Conlin, Gerald Wertheim, Yiming Zhong, Lea Surrey, Garrett Brodeur, and Marilyn Li
- Subjects
Cancer Research ,Genetics ,Molecular Biology - Published
- 2022
38. 27. KMT2A-ARHGEF12 fusion in Burkitt-like Lymphoma
- Author
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Minjie Luo, Anne F. Reilly, Hou-Sung Jung, Adam J. Wolpaw, Fumin Lin, Marilyn M. Li, and Gerald Wertheim
- Subjects
Burkitt-like lymphoma ,Cancer Research ,KMT2A ,biology ,Genetics ,biology.protein ,Cancer research ,Molecular Biology - Published
- 2021
39. Clinical impact of genomic characterization of 15 patients with acute megakaryoblastic leukemia related malignancies
- Author
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Emilie Lalonde, Stefan Rentas, Gerald Wertheim, Lea Surrey, Fumin Lin, Xiaonan Zhao, Amrom Obstfeld, Richard Aplenc, Minjie Luo, and Marilyn Li
- Published
- 2020
40. Active learning for fellows: The hematopathology 'unknown case'
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Rahela Aziz-Bose, Leslie S. Kersun, and Gerald Wertheim
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Adult ,medicine.medical_specialty ,education ,Medical Oncology ,Pediatrics ,03 medical and health sciences ,0302 clinical medicine ,Humans ,Medicine ,Set (psychology) ,Parallels ,Adult Learning ,Curriculum ,Medical education ,business.industry ,Learning environment ,Problem-Based Learning ,Hematology ,Oncology ,Education, Medical, Graduate ,030220 oncology & carcinogenesis ,Pediatrics, Perinatology and Child Health ,Active learning ,Pediatric hematology ,business ,Hematopathology ,030215 immunology - Abstract
The pediatric hematology/oncology fellowship program at the Children's Hospital of Philadelphia set out to create a case-based learning curriculum for common hematologic malignancies that would apply principles of adult learning theory and improve fellows' retention of information in a supportive, goal-oriented learning environment. A framework we employed in developing this curriculum is that of "flow theory," which parallels many of the tenets of adult learning theory. After implementing this curriculum, which we call "the unknown case," the percentage of fellows correctly identifying a common hematopathologic diagnosis improved from 50% to 85%.
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- 2020
41. CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy
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Asen Bagashev, Michele Paessler, Andrei Thomas-Tikhonenko, Carl H. June, Wenzhao Meng, John S. Van Arnam, Eline T. Luning Prak, Minjie Luo, Sindhu Cherian, Derek A. Oldridge, Amanda M. DiNofia, Vinodh Pillai, Jaclyn Rosenthal, Stephan A. Grupp, J. Joseph Melenhorst, Vijay Bhoj, Gerald Wertheim, Susan R. Rheingold, Diwakar Mohan, Shannon L. Maude, Jonathan R. Fromm, and Kavitha Muralidharan
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Oncology ,Adult ,Cytotoxicity, Immunologic ,Male ,medicine.medical_specialty ,Neoplasm, Residual ,Immunobiology and Immunotherapy ,Adolescent ,medicine.medical_treatment ,T-Lymphocytes ,Antigens, CD19 ,Receptors, Antigen, T-Cell ,chemical and pharmacologic phenomena ,Immunotherapy, Adoptive ,Immunophenotyping ,Young Adult ,Antineoplastic Agents, Immunological ,Antigen ,immune system diseases ,Recurrence ,Internal medicine ,hemic and lymphatic diseases ,Precursor B-Cell Lymphoblastic Leukemia-Lymphoma ,Antibodies, Bispecific ,Medicine ,Humans ,Child ,business.industry ,Infant ,hemic and immune systems ,Hematology ,Immunotherapy ,medicine.disease ,Minimal residual disease ,Combined Modality Therapy ,Chimeric antigen receptor ,Leukemia ,Prior Therapy ,Treatment Outcome ,Child, Preschool ,Blinatumomab ,Female ,business ,medicine.drug - Abstract
Tisagenlecleucel, a chimeric antigen receptor (CAR) T-cell product targeting CD19 is approved for relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, the impact of pretreatment variables, such as CD19 expression level, on leukemic blasts, the presence of CD19(–) subpopulations, and especially prior CD19-targeted therapy, on the response to CAR T-cell therapy has not been determined. We analyzed 166 patients treated with CAR T-cell therapy at our institution. Eleven patients did not achieve a minimal residual disease (MRD)(–) deep remission, whereas 67 patients had a recurrence after achieving a MRD(–) deep remission: 28 patients with CD19(+) leukemia and 39 patients with CD19(–) leukemia. Return of CD19(+) leukemia was associated with loss of CAR T-cell function, whereas CD19(–) leukemia was associated with continued CAR T-cell function. There were no significant differences in efficacy of CAR T cells in CD19-dim B-ALL, compared with CD19-normal or -bright B-ALL. Consistent with this, CAR T cells recognized and lysed cells with very low levels of CD19 expression in vitro. The presence of dim CD19 or rare CD19(–) events by flow cytometry did not predict nonresponse or recurrence after CAR T-cell therapy. However, prior therapy with the CD19-directed, bispecific T-cell engager blinatumomab was associated with a significantly higher rate of failure to achieve MRD(–) remission or subsequent loss of remission with antigen escape. Finally, immunophenotypic heterogeneity and lineage plasticity were independent of underlying clonotype and cytogenetic abnormalities.
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- 2019
42. DYRK1A Is Regulated By Oncogenic KMT2A and Required for Survival of KMT2A-Rearranged Acute Lymphoblastic Leukemia
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Gerald Wertheim, Anne Lehman, John D. Crispino, Thierry Besson, Christian Hurtz, Martin Carroll, Rahul S. Bhansali, Sarah K. Tasian, Grace R. Jeschke, University of Pennsylvania [Philadelphia], Children’s Hospital of Philadelphia (CHOP ), University of Illinois College of Medicine, University of Illinois System, The University of Chicago Medicine [Chicago], Chimie Organique et Bioorganique : Réactivité et Analyse (COBRA), Institut Normand de Chimie Moléculaire Médicinale et Macromoléculaire (INC3M), Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Normandie Université (NU)-Institut national des sciences appliquées Rouen Normandie (INSA Rouen Normandie), Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Université Le Havre Normandie (ULH), Normandie Université (NU)-Université de Rouen Normandie (UNIROUEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Université de Caen Normandie (UNICAEN), Normandie Université (NU)-Institut de Chimie du CNRS (INC)-École Nationale Supérieure d'Ingénieurs de Caen (ENSICAEN), Normandie Université (NU)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie Organique Fine (IRCOF), Université de Rouen Normandie (UNIROUEN), and Institut National des Sciences Appliquées (INSA)-Normandie Université (NU)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)
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Cell signaling ,Immunology ,achilles tendon ,cell lines ,acute lymphocytic leukemia ,Biochemistry ,burkitt's lymphoma ,chemotherapy regimen ,03 medical and health sciences ,0302 clinical medicine ,Acute lymphocytic leukemia ,b-lymphocytes ,medicine ,Transcriptional regulation ,[CHIM]Chemical Sciences ,030304 developmental biology ,0303 health sciences ,acute megakaryocytic leukemias ,Oncogene ,biology ,business.industry ,Cell Biology ,Hematology ,DOT1L ,medicine.disease ,Fusion protein ,3. Good health ,binding (molecular function) ,Leukemia ,KMT2A ,biological products ,Cancer research ,biology.protein ,adverse effects ,business ,030215 immunology - Abstract
Background: Research efforts have focused upon uncovering critical leukemia-associated genetic alterations that may be amenable to therapeutic targeting with new drugs. Targeting the oncogenic BCR-ABL1 fusion protein in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (B-ALL) with tyrosine kinase inhibitors to shut down constitutive signaling activation and induce leukemia cell cytotoxicity has remarkably improved patients' survival and has established a precision medicine paradigm for kinase-driven leukemias. However, multiple subtypes of B-ALL are driven through non-tyrosine fusion proteins, including the high-risk KMT2A-rearranged (KMT2A-R) subtype common in infants with B-ALL, leaving many patients with insufficient treatment options. Objectives: KMT2A-R B-ALL is associated with chemoresistance, relapse, and poor survival with a frequency of 75% in infants and 10% in older children/adults with B-ALL. Current intensive multiagent chemotherapy regimens induce significant side effects yet fail to cure the majority of patients, demonstrating continued need for novel therapeutic approaches. The goals of our study were to i) identify signaling molecules required for KMT2A-R B-ALL cell survival, ii) select ALL-associated targets that are not essential in normal tissues, and iii) develop new treatment strategies that may benefit patients with KMT2A-R ALL. Results: We performed a genome-wide kinome CRISPR screen using the pediatric KMT2A-R cell line SEM and identified DYRK1A among other signaling molecules as required for leukemia cell survival. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as a critical oncogene in a murine Down syndrome (DS) model of megakaryoblastic leukemia. In normal hematopoiesis, DYRK1A controls the transition from proliferation to quiescence during lymphoid development. Deletion of DYRK1A results in increased numbers of B cells in S-G2-M phase, yet also significantly reduces cell proliferation. Meta-analysis of ChIP-Seq data from two KMT2A-AFF1 cell lines (SEM and RS4;11) and a human KMT2A-Aff1-FLAG-transduced ALL model demonstrates that both N-terminal (KMT2AN) and C-terminal (AFF1C) and the FLAG-tagged KMT2A-Aff1 fusion directly bind to the DYRK1A promoter. Gene expression and RT-PCR analyses of SEM cells treated with inhibitors against two important KMT2A fusion complex proteins, DOT1L (histone methyltransferase) and menin (tumor suppressor), demonstrate that only menin inhibition induced DYRK1A downregulation. Interestingly, deletion of germline KMT2A in murine B-cells did not decrease DYRK1A expression. Taken together, these results suggest direct transcriptional regulation through the KMT2A fusion complex. Surprisingly, RNA and protein expression of DYRK1A was reduced in KMT2A-R ALL compared to other B-ALL subtypes. We then identified MYC as a potential negative regulator of DYRK1A that could explain the lower RNA and protein expression levels observed. A gain-of-function experiment showed marked downregulation of DYRK1A when MYC was ectopically expressed in murine B-cells, while loss of MYC resulted in DYRK1A upregulation. Parallel analysis of publicly available gene expression data from children with high-risk B-ALL (NCI TARGET database) showed significantly higher MYC RNA expression levels in KMT2A-R ALL as compared to other ALL subtypes, further validating our findings that MYC acts as a negative regulator of DYRK1A. Finally, to assess pharmacologic inhibition, we treated multiple KMT2A-rearranged ALL cell lines with the novel DYRK1A inhibitor EHT 1610 and identified sensitivity to DYRK1A inhibition. We then queried the Achilles database and identified that DYRK1A is not a common essential gene in normal tissues, suggesting minimal potential for on-target/off-tumor effects of DYRK1A inhibition. Conclusions: We identified a novel mechanism in KMT2A-R ALL in which DYRK1A is positively regulated by the KMT2A fusion protein and negatively regulated by MYC. Genetic deletion and pharmacologic inhibition of DYRK1A resulted in significant growth disadvantage of KMT2A-R ALL cells. While further studies are needed, we predict that combining DYRK1A inhibitors with chemotherapy could decrease relapse risk and improve long-term survival of patients with KMT2A-R B-ALL. Disclosures Crispino: MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy; Scholar Rock: Research Funding; Forma Therapeutics: Research Funding. Tasian:Incyte Corportation: Research Funding; Gilead Sciences: Research Funding; Aleta Biotherapeutics: Membership on an entity's Board of Directors or advisory committees. Carroll:Astellas Pharmaceuticals: Research Funding; Incyte: Research Funding; Janssen Pharmaceuticals: Consultancy.
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- 2019
43. Clinical efficacy of ruxolitinib and chemotherapy in a child with Philadelphia chromosome-like acute lymphoblastic leukemia with GOLGA5-JAK2 fusion and induction failure
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Richard C. Harvey, I-Ming Chen, Zhaohui Gu, Gerald Wertheim, Tracey Jubelirer, Yang Y. Ding, Yong Li, Charles G. Mullighan, Fengqi Chang, Marilyn M. Li, Cheryl L. Willman, Sarah K. Tasian, Fumin Lin, Stephen P. Hunger, and Julie W. Stern
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0301 basic medicine ,Oncology ,medicine.medical_specialty ,Chemotherapy ,Ruxolitinib ,business.industry ,medicine.medical_treatment ,Lymphoblastic Leukemia ,Treatment outcome ,Karyotype ,Hematology ,Philadelphia chromosome ,medicine.disease ,03 medical and health sciences ,Remission induction ,030104 developmental biology ,0302 clinical medicine ,hemic and lymphatic diseases ,030220 oncology & carcinogenesis ,Internal medicine ,Medicine ,Clinical efficacy ,business ,medicine.drug - Abstract
Patients with Philadelphia chromosome-like B-cell lymphoblastic leukemia (Ph-like or BCR-ABL1 -like BALL) experience high relapse rates and are difficult to cure with conventional chemotherapy.[1][1],[2][2] The Ph-like ALL subtype comprises 15-25% of B-ALL in older children and adolescents/young
- Published
- 2018
44. Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells
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Anthony Secreto, Charalampos Papachristou, Gerald Wertheim, Elizabeth A. Griffiths, Georges Habineza Ndikuyeze, Maria Krevvata, Joshua Glover, Cedric Dos Santos, Chenghui Zhou, Hyun-Jeong Ra, Selene Nunez-Cruz, Xiaochuan Shan, Winifred Trotman, Martin Carroll, Gwenn Danet-Desnoyers, and Gisela Brake-Silla
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0301 basic medicine ,Myeloid ,business.industry ,Myelodysplastic syndromes ,Mesenchymal stem cell ,Myeloid leukemia ,Hematology ,medicine.disease ,3. Good health ,Transplantation ,03 medical and health sciences ,Leukemia ,Haematopoiesis ,030104 developmental biology ,medicine.anatomical_structure ,hemic and lymphatic diseases ,medicine ,Cancer research ,Bone marrow ,business - Abstract
Patient-derived xenotransplantation models of human myeloid diseases including acute myeloid leukemia, myelodysplastic syndromes and myeloproliferative neoplasms are essential for studying the biology of the diseases in pre-clinical studies. However, few studies have used these models for comparative purposes. Previous work has shown that acute myeloid leukemia blasts respond to human hematopoietic cytokines whereas myelodysplastic syndrome cells do not. We compared the engraftment of acute myeloid leukemia cells and myelodysplastic syndrome cells in NSG mice to that in NSG-S mice, which have transgene expression of human cytokines. We observed that only 50% of all primary acute myeloid leukemia samples (n=77) transplanted in NSG mice provided useful levels of engraftment (>0.5% human blasts in bone marrow). In contrast, 82% of primary acute myeloid leukemia samples engrafted in NSG-S mice with higher leukemic burden and shortened survival. Additionally, all of 5 injected samples from patients with myelodysplastic syndrome showed persistent engraftment on week 6; however, engraftment was mostly low (
- Published
- 2018
45. Poor outcome with hematopoietic stem cell transplantation for bone marrow failure and MDS with severe MIRAGE syndrome phenotype
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Gerald Wertheim, Satoshi Narumi, Jay F. Sarthy, Timothy S. Olson, Adam S. Himebauch, Ashley Munchel, Agne Taraseviciute, Daria V. Babushok, Soheil Meshinchi, Samuel Yang, Archana Shenoy, Hirohito Shima, Jian-Meng Fan, and Ji Zha
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Male ,0301 basic medicine ,Oncology ,medicine.medical_specialty ,medicine.medical_treatment ,Hematopoietic stem cell transplantation ,03 medical and health sciences ,0302 clinical medicine ,Internal medicine ,medicine ,Congenital Bone Marrow Failure Syndromes ,Humans ,Bone Marrow Diseases ,business.industry ,Myelodysplastic syndromes ,Hematopoietic Stem Cell Transplantation ,Intracellular Signaling Peptides and Proteins ,Bone marrow failure ,Infant ,Proteins ,Syndrome ,Hematology ,Prognosis ,medicine.disease ,MIRAGE SYNDROME ,Thrombocytopenia ,Pancytopenia ,Phenotype ,Treatment Outcome ,030104 developmental biology ,Myelodysplastic Syndromes ,030220 oncology & carcinogenesis ,Mutation ,Congenital amegakaryocytic thrombocytopenia ,Female ,Exceptional Case Report ,business - Abstract
Key Points Success of hematopoietic stem cell transplantation for MIRAGE syndrome may be limited by syndrome-specific comorbidities. SAMD9 mutations associated with MIRAGE syndrome are a newly described cause of congenital amegakaryocytic thrombocytopenia.
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- 2018
46. FLT3 Inhibition Downregulates EZH2 in AML and Promotes Myeloid Differentiation
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Hongbo Xie, Simone S. Riedel, Daniel Tsai, Martin Carroll, Sara E. Meyer, Gerald Wertheim, Bryan Manning, Benjamin A. Garcia, Katarzyna Kulej, Pamela J. Sung, Kathrin M. Bernt, and Simone Sidoli
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Myeloid ,medicine.anatomical_structure ,hemic and lymphatic diseases ,Immunology ,EZH2 ,medicine ,Cancer research ,Cell Biology ,Hematology ,Biology ,Biochemistry - Abstract
Internal tandem duplication mutations in the Fms-like tyrosine kinase 3 (FLT3-ITD) are frequently recurring in AML and confer a poor prognosis. FLT3 inhibitors (FLT3i) such as gilteritinib are efficacious in relapsed AML. Clinical responses to FLT3i include myeloid differentiation of the FLT3-ITD clone in about 50% of patients. How FLT3i induce this response in a subset of patients is unknown. The FLT3i-induced differentiation response seen in clinical trials has not previously been demonstrated in animal models. We modeled FLT3i-induced differentiation in murine Flt3 ITD/ITDDnmt3a -/- AML model (Meyer et al., Cancer Discovery, 2016). Treatment with FLT3i in vitro accelerated differentiation of cKIT+ leukemic splenocytes as assessed by colony morphology in serial re-plating assays. To characterize the differentiation response in vivo, we transplanted CD45.2+ leukemic splenocytes from moribund mice into sub-lethally irradiated healthy congenic CD45.1+ mice. After confirmation of engraftment at 2 weeks post-irradiation, mice were treated with vehicle or gilteritinib for 4 weeks. Animals treated with gilteritinib demonstrated increased neutrophil and decreased stem/progenitor cell populations, recapitulating the clinically observed increase in granulocytic differentiation of the FLT3-ITD clone. We next sought to understand the molecular mechanism of FLT3i-induced differentiation. We used a proteomic-based screen in a human AML cell line treated with FLT3i to identify novel targets of FLT3-ITD that could be potential mediators of the differentiation response. We identified downregulation of Enhancer of Zeste Homolog 2 (EZH2), the catalytic component of the Polycomb Repressive Complex 2 (PRC2). EZH2 and PRC2 were previously shown to be required for leukemic maintenance in mouse models of MLL-AF9 AML. We treated murine Flt3 ITD/ITDDnmt3a -/- cKIT+ leukemic splenocytes with FLT3i or the EZH1/2 inhibitor (EZH1/2i). Both promoted myeloid differentiation to similar degrees as assessed by colony morphology in this model. We hypothesized that FLT3-ITD regulates EZH2 to maintain leukemia cells in a stem/progenitor cell state. We, therefore, characterized the effect of FLT3i on PRC2 in more detail. We confirmed that FLT3i decreases EZH2 protein levels in FLT3-ITD cell lines and primary human AML within 24 hours of treatment as suggested by our proteomic data (Figure 1A-B). We found that the mechanism of EZH2 downregulation is complex with both transcriptional effects and a decrease in EZH2 protein half-life. ChIP-Seq for H3K27me3 demonstrated decreased peaks at the transcription start sites of PRC2 target genes (Figure 1C). RNA-Seq gene expression profiles of FLT3i- and EZH1/2i-treated human AML cells overlapped at 253 differentially expressed genes (Figure 1D). Critically, both FLT3i and EZH1/2i expression profiles enriched in differentiated myeloid cell gene signatures. Overall, we found that EZH2 is a novel, unexpected, and clinically relevant target of FLT3-ITD. Our data suggest that reduced EZH2 activity following FLT3 inhibition promotes myeloid differentiation of FLT3-ITD leukemic cells, providing a mechanistic explanation for the FLT3i-induced differentiation response seen in patients. These data demonstrate that FLT3-ITD has at least two functions in leukemogenesis, the well described activation of signaling pathways, and second, a previously undefined, regulation of PRC2 to maintain a myeloid stem cell state. Our results may lead to improved approaches to therapy for FLT3 mutated AML. Figure 1 Figure 1. Disclosures Bernt: Syndax: Research Funding; Merck: Other: Spouse is an employee of Merck.. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.
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- 2021
47. Standardization in the Diagnosis of Mixed Phenotype Acute Leukemia (MPAL): Semiquantitative, Universally Applicable Flow Cytometric Criteria for Immunophenotypic Lineage Assignment and Isolated MPO
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Brent L. Wood, Sunil S. Raikar, William G. Woods, Alexandra E. Kovach, Maurice R.G. O'Gorman, Gerald Wertheim, Karen R. Rabin, Alix E. Seif, Terri Guinipero, Jyotinder N. Punia, Viviane Cahen, Reuven J. Schore, Etan Orgel, Matthew J. Oberley, and Dragos C. Luca
- Subjects
Mixed phenotype acute leukemia ,Lineage (genetic) ,Immunology ,Cell Biology ,Hematology ,Biology ,Biochemistry - Abstract
Mixed phenotype acute leukemia (MPAL) is a rare group of acute leukemias defined by immunophenotypic expression of more than one hematopoietic cell lineage. The World Health Organization (WHO) diagnostic immunophenotypic criteria for MPAL rely on the intensity of lineage-defining antigen expression, predominantly a qualitative assessment, and are often difficult to apply to a phenotypically heterogeneous leukemia. Cases of MPAL defined by isolated myeloperoxidase (isoMPO) expression on otherwise typical acute lymphoblastic leukemia (ALL) are variably diagnosed as MPAL or ALL based on the incompletely defined criteria for assigning MPO expression. We hypothesized that quantitative criteria for antigen intensity could be developed and applied in a uniform manner across flow cytometry instruments, reagents, and analysis software to enable a consistent approach to diagnosing MPAL and better defining isoMPO. We previously reported on a multicenter cohort identified by respective institutions as MPAL with subsequent central review according to 2008 WHO criteria (Oberley et al 2020). Of these, 100 had suitable dot plots for reevaluation (89: B/Myeloid, 10: T/Myeloid, 1: B/T). Antigen expression was concurrently reviewed by two hematopathologists to reach consensus (BLW, AEK). The cohort was divided a priori into randomly selected training and testing cases (n=30/n=70). Classification criteria were generated in the training cohort for WHO lineage-defining antigen expression (myeloid: cMPO, CD64, CD14; B: CD19, T: cCD3) from 10 cases and then refined through review of an additional 20 cases. Positive antigen expression was classified as maximal intensity approaching that of the mature normal counterpart population (NCP) (cMPO: neutrophils; CD64, CD14 and CD11c: monocytes; CD19: B cells; cCD3: T cells) either by 1) range of expression recapitulating maturation of the NCP, irrespective of the percentage on the leukemic population (Figure 1A); or 2) uniform expression above background on a discrete (sub)population (Figure 1B). To account for technical variation within and among laboratories, maximal antigen intensity on the leukemic population was measured in 0.5 log increments and normalized to the maximal intensity of the NCP. An intensity of ≥50% of the NCP above background was defined as sufficient for MPAL lineage assignment and Using this approach, 41/98 (42%) cases previously classified as MPAL remained classified as MPAL: 31 as B/Myeloid (25 by maturational MPO expression, 6 by maturational CD64 and/or CD14 expression); 9 as T/Myeloid (8 by maturational MPO expression, 1 with maturational CD64, CD14 and CD11c expression); and 1 as B/T. No cases in the cohort showed uniform expression ≥50% of the NCP. The remaining 57 showed isolated low-level MPO expression (maximal intensity Novel semiquantitative immunophenotypic criteria applied to a large cohort of acute leukemias originally diagnosed as MPAL was able to objectively identify a large subset as having dim, uniform MPO expression (isoMPO). Our approach emphasizes antigen expression recapitulating maturational expression similar to their non-leukemic cellular counterparts, normalizes absolute intensities to internal positive and negative control populations to minimize technical variability between observers and assays, and as per the 2017 WHO, does not require a specific percent threshold of positivity. While requiring validation, this is a critical first step toward establishing a reproducible delineation of these complex cases to practically implement the WHO classification to support treatment decisions and ongoing investigation into MPAL genomics and outcomes (available for this cohort by ASH). Figure 1 Figure 1. Disclosures Oberley: Caris LIfe Science: Current Employment. Orgel: Jazz Pharmaceuticals: Consultancy.
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- 2021
48. Pharmacologic Inhibition of DYRK1A Results in Hyperactivation and Hyperphosphorylation of MYC and ERK Rendering KMT2A-R ALL Cells Sensitive to BCL2 Inhibition
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Joseph P. Loftus, Anil Kumar, Sarah K. Tasian, John D. Crispino, Srividya Swaminathan, Christian Hurtz, Junwei Shi, Thierry Besson, Martin Carroll, Rahul S. Bhansali, Sung June Lee, Gerald Wertheim, and John Chukinas
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MAPK/ERK pathway ,biology ,Hyperactivation ,DYRK1A ,Chemistry ,Immunology ,Hyperphosphorylation ,Cell Biology ,Hematology ,Biochemistry ,Rendering (computer graphics) ,Cell biology ,KMT2A ,biology.protein - Abstract
Background: KMT2A-rearranged (R) ALL is a high-risk disease with a frequency of 75% in infants and 10% in children and adults with ALL and is associated with chemoresistance, relapse, and poor survival. Current intensive multiagent chemotherapy regimens induce significant side effects, yet fail to cure many patients, demonstrating continued need for novel therapeutic approaches. We performed a kinome-wide CRISPR screen and identified that DYRK1A is specifically required for the survival of KMT2A-R ALL cell. DYRK1A is a member of the dual-specificity tyrosine phosphorylation-regulated kinase family and has been reported as negatively regulator of cell proliferation. Results: We performed a kinome-wide CRISPR screen in human ALL cell lines and PDX models and identified DYRK1A as a novel target in KMT2A-R ALL. DYRK1A is a serine-threonine kinase with a proposed, but poorly defined role in cell cycle regulation. We performed a meta-analysis of multiple ChIP-Seq experiments and identified that oncogenic KMT2A fusions directly bind to the DYRK1A promoter. Our RT-PCR and Western blot analyses of KMT2A-R ALL cells treated with a menin inhibitor (MI-503) to disrupt the transcriptional activity of the KMT2A-R complex resulted in the downregulation of DYRK1A, indicating that DYRK1A is directly regulated by the KMT2A fusion complex. We further observed that pharmacologic inhibition of DYRK1A with EHT1610 induced potent leukemic cell growth inhibition in vitro and in vivo, demonstrating that DYRK1A could be a new therapeutic target in KMT2A-R ALL cells. To further elucidate the mechanism of DYRK1A function, we treated several KMT2A-R ALL cell lines in vitro with EHT1610, which surprisingly resulted in the upregulation of MYC and hyperphosphorylation of the RAS/MAPK target ERK. Given that ERK hyperactivation stops B cell proliferation during early B cell development to allow them to rearrange their B cell receptor, we hypothesized that cell cycle inhibition upon ERK hyperactivation remains as a conserved mechanism of cell cycle regulation in KMT2A-R ALL. Strikingly, combining DYRK1A inhibition with the MEK inhibitor trametinib antagonistically rescued KMT2A-R ALL cell proliferation, indicating that ERK hyperactivation is the main driver of DYRK1A inhibitor mediated cell cycle arrest. Given that DYRK1A inhibitor does not induce apoptosis and cells restart cell proliferation after EHT1610 withdrawal we concluded that a DYRK1A monotherapy may not be an ideal new treatment option. However, it has been reported that increased MYC activity induces the accumulation of BIM in Burkitt's Lymphoma. Given the increased expression of MYC following DYRK1A inhibition we performed a new Western blot analysis and validated increased expression of BIM in our KMT2A-R ALL cell lines after EHT1610 treatment. To test if targeting the interaction of BIM with BCL2 will induce an apoptotic effect when combined with EHT1610, we treated four KMT2A-R ALL cell lines with increasing concentrations of EHT1610 and the BCL2 inhibitor venetoclax. Strikingly, the combination of DYRK1A inhibition with BCL2 inhibition synergistically killed KMT2A-R ALL cells. Conclusion: Our results validate DYRK1A as an important molecule to regulate cell proliferation via inhibition of MYC and ERK. Targeting DYRK1A results in the accumulation of BIM, which renders the cells sensitive to BCL2 inhibition via venetoclax. While further in vivo studies are needed, we predict that combining DYRK1A inhibition with venetoclax may be a novel precision medicine strategy for the treatment of KMT2A-R ALL. Figure 1 Figure 1. Disclosures Crispino: Forma Therapeutics: Research Funding; Scholar Rock: Research Funding; MPN Research Foundation: Membership on an entity's Board of Directors or advisory committees; Sierra Oncology: Consultancy. Tasian: Aleta Biotherapeutics: Consultancy; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Incyte Corporation: Research Funding. Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy.
- Published
- 2021
49. Alisertib Synergistically Strengthens the Anti-Leukemia Activity of Venetoclax in TCF3-Hlf B-ALL
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Colin Wakefield, Martin Carroll, Sarah K. Tasian, Joseph P. Loftus, Yana Pikman, Kimberly Stegmaier, Asen Bagashev, Gerald Wertheim, and Christian Hurtz
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Leukemia ,chemistry.chemical_compound ,chemistry ,Venetoclax ,TCF3 ,Immunology ,Alisertib ,medicine ,Cancer research ,Cell Biology ,Hematology ,medicine.disease ,Biochemistry - Abstract
Background: Despite maximally-intensive chemotherapy and stem cell transplantation, survival of patients with the very rare t(17;19)/TCF3-HLF B-acute lymphoblastic leukemia (B-ALL) subtype remains effectively 0%. Prior studies have demonstrated association of the oncogenic TCF3-HLF fusion protein with multi-drug resistance via increased expression of ABC and P-glycoprotein drug efflux transporters, as well as via upregulation of pro-survival Ras and BCL-2 pathways. Preclinical studies and small clinical case series of targeted inhibitor addition to chemotherapy or antibody-based and cellular immunotherapies have aimed to improve outcomes for children with TCF3-HLF ALL. Unfortunately, targeting of these activated pathways with the BCL-2 inhibitor venetoclax or other small molecule inhibitors (SMIs) has been minimally or only transiently effective, suggesting more complex mechanism(s) of chemoresistance. In recent years, many patients with relapsed TCF3-HLF ALL have enrolled on clinical trials of CD19- or CD22-targeted immunotherapies. However, TCF3-HLF ALL frequently harbours deactivating mutations in PAX5, a major B-cell regulator and indispensable CD19 transcription factor, placing immunotherapy-treated patients at higher risk of CD19 antigen-loss relapse. New therapies remain needed to prevent relapse and attempt cure. Methods: We designed an unbiased kinome-wide CRISPR/Cas9 library to identify essential drivers in TCF3-HLF leukemogenesis. We screened the human TCF3-HLF ALL cell line HAL-01 and our TCF3-HLF ALL patient-derived xenograft (PDX) model ALL1807 (Hurtz JCI 2020, Schultz Genome Biol 2021), then validated identified targets using 49 SMIs targeting receptor tyrosine kinases (RTK), MEK signaling, and cell cycle pathways. We selected promising candidate inhibitor pairings with non-overlapping mechanisms of action and assessed for in vitro drug synergy via SynergyFinder analyses. Finally, we assessed the in vivo activity of targeted inhibitors in ALL1807 and two newly established TCF3-HLF ALL PDX models (CPCT-0002, CPCT-0003) created from primary pediatric specimens obtained via the LEAP Consortium (Pikman Cancer Disc 2021). Results: RNA-sequencing of HAL-01 and ALL1807 cells followed by functional protein association (STRING) analysis confirmed a network of significantly upregulated (>3-fold) plasma membrane and cytoplasm components of RTK pathways as well as BCL-2. The intersection of the results of the SMI drug library screen with the top 1% targets identified in CRIPSR/Cas9 screen determined p120-RasGAP and Aurora kinase A (AURKA) as therapeutic targets in TCF3-HLF ALL. In vitro treatment of HAL-01 or ALL1807 cells with the RasGAP inhibitor, pluripotin, or the AURKA inhibitor, alisertib, across a range of concentrations demonstrated robust anti-ALL activity. AURKA and RasGAP co-immunoprecipitated and this protein complex was disrupted with alisertib or pluripotin treatment. The AURKB inhibitor barisertib had minimal activity against TCF3-HLF ALL cells, confirming preferential dependency of these cells upon AURKA. Treatment of TCF3-HLF ALL cells with the BCL-2i venetoclax did not disrupt the AURKA/RasGAP complex, suggesting its different mechanism of action and potential for combinatorial drug therapy. Next, we found that alisertib and venetoclax synergistically killed TCF3-HLF ALL cells. Finally, we observed superior inhibition of in vivo leukemia with dual AURKA and BCL-2 inhibitor treatment of three TCF3-HLF ALL PDX models compared to single-agent alisertib or venetoclax (Figure 1). Conclusions: We identified AURKA as a critical new driver in TCF3-HLF ALL via orthogonal genetic and functional assays and confirmed prior observations of BCL-2 dependency in our models. We validated these key targets via in vitro and in vivo pharmacologic inhibition studies with drug synergy detected with combined alisertib and venetoclax in human TCF3-HLF ALL cell lines and PDX models. We posit that dual AURKA and BCL-2 inhibition is a clinically-pragmatic and potentially effective therapeutic strategy for patients with this rare, but highly fatal, leukemia subtype that merits formal clinical investigation. Figure 1 Figure 1. Disclosures Carroll: Incyte Pharmaceuticals: Research Funding; Janssen Pharmaceutical: Consultancy. Stegmaier: Auron Therapeutics, Kronos Bio, AstraZeneca, Novartis Institute of Biomedical Research: Consultancy, Research Funding. Tasian: Incyte Corporation: Research Funding; Gilead Sciences: Research Funding; Kura Oncology: Consultancy; Aleta Biotherapeutics: Consultancy.
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- 2021
50. Molecular Evolution of Classical Hodgkin Lymphoma Revealed Though Whole Genome Sequencing of Hodgkin and Reed-Sternberg Cells
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Sunita I. Park, Olivier Elemento, Gerald Wertheim, Manik Uppal, Kenneth Eng, Craig H. Moskowitz, Ethel Cesarman, Marcin Imielinski, Francesco Maura, Venkata Yellapantula, Wei Zhang, Matthew J. Barth, Kylee H Maclachlan, Terzah M. Horton, Elizabeth Ruckdeschel, Bhavneet Binder, Megan S. Lim, Mikhail Roshal, Feng He, Qi Gao, Federico Abascal, Ola Landgren, Bachisio Ziccheddu, Matthew J. Oberley, Jenny Xiang, and Lisa Giulino Roth
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Genetics ,Whole genome sequencing ,Reed–Sternberg cell ,Molecular evolution ,Immunology ,Classical Hodgkin lymphoma ,medicine ,Cell Biology ,Hematology ,Biology ,medicine.disease ,Biochemistry - Abstract
Introduction: Classical Hodgkin lymphoma (cHL) is characterized by a small fraction of Hodgkin and Reed-Sternberg (HRS) tumor cells (~1%) which are surrounded by an extensive immune infiltrate. The rare nature of HRS cells limits the ability to study the genomics of cHL using standard platforms. To circumvent this, our group has optimized fluorescence-activated cell sorting to isolate HRS cells and intratumor B- and T- cells and to perform whole exome sequencing (WES; Reichel, Blood 2015). To date, however, there have been no reports on whole genome sequencing (WGS) of cHL. Methods: We performed flow-sorting of HRS cells and WGS to define the genomic landscape of cHL including: i) mutational processes involved in pathogenesis, ii) large and focal copy number variants, iii) structural variants including complex events, iv) the sequence and evolution of molecular events in cHL. We interrogated WGS from 25 cases of cHL: 10 pediatric patients (age40). Intra-tumoral T-cells were used as germline control. An additional 36 cHL cases were evaluated by WES. Results: The average depth of coverage among the 25 WGS cases was 27.5x. After having identified and removed amplification-based palindromic sequencing artifacts, we observed a median of 5006 single base substitutions (SBS; range 1763-18436). Pediatric and AYA patients had a higher SBS burden compared to older adults (median 5279 vs. 2945, p=0.009). Five main SBS signatures were identified: SBS1 and SBS5 (aging), SBS2 and SBS13 (APOBEC), and SBS25 (chemotherapy, in a relapsed case). A dNdScv driver discovery analysis performed on the combined WES and WGS cases identified 24 driver genes including BCL7A and CISH which had not been previously reported as drivers in cHL. An investigation of copy number alterations (CNAs) confirmed high ploidy in cHL (median 2.95, range 1.66-5.33). Whole genome duplication was identified in 64% cases. We also observed clear evidence of complex events such as chromothripsis (n=4), double minutes (dm, n=2), breakage-fusion-bridge (bfb; n=4). Some of these events were responsible for the acquisition of distinct drivers. For example, we observed one dm and one bfb responsible for CD274 and REL gains, respectively (>10 copies). Leveraging the high prevalence of large chromosomal gains, we performed an investigation of the relative timing of acquisition of driver mutations. Clonal mutations within chromosomal gains can be defined as duplicated (VAF~66%; acquired before the gain) or non-duplicated (VAF~33%; acquired before or after the gain). Sixty-one percent (152/249) of driver genes were duplicated suggesting that they were acquired prior to large chromosomal gains. Next, we used the corrected ratio between duplicated and non-duplicated mutations within large chromosomal gains to estimate the molecular time of each duplicated segment (Rustad, Nat Comm 2020). In 11/22 genomes the final CNA profile was acquired through at least two temporally distinct events. To convert these relative estimations into absolute timing (i.e., the age at which events occurred), we leveraged the clock-like mutation signatures (SBS1, SBS5). We first confirmed that the SBS1 and SBS5 mutation rate were constant over time (R 2=0.84; p Conclusion: Here we report the first WGS in cHL. We identify novel drivers and genomic mechanisms involved in cHL pathogenesis. We found that mutations in driver genes are often acquired earlier then chromosomal gains, potentially preceding the cHL diagnosis by several years. In addition, we observed key differences in biology of cHL across age groups including accelerated mutagenesis and increased mutational burden among younger patients. Disclosures Maura: OncLive: Honoraria; Medscape: Consultancy, Honoraria. Oberley: Caris LIfe Science: Current Employment. Lim: EUSA Pharma: Honoraria. Landgren: Janssen: Other: IDMC; Celgene: Research Funding; Janssen: Honoraria; Amgen: Honoraria; Janssen: Research Funding; Amgen: Research Funding; Takeda: Other: IDMC; GSK: Honoraria. Moskowitz: Merck & Co., Inc.: Research Funding. Roshal: Celgene: Other: Provision of services; Auron Therapeutics: Other: Ownership / Equity interests; Provision of services; Physicians' Education Resource: Other: Provision of services. Elemento: Owkin: Consultancy, Other: Current equity holder; AstraZeneca: Research Funding; Champions Oncology: Consultancy; Volastra Therapeutics: Consultancy, Other: Current equity holder, Research Funding; One Three Biotech: Consultancy, Other: Current equity holder; Eli Lilly: Research Funding; Johnson and Johnson: Research Funding; Freenome: Consultancy, Other: Current equity holder in a privately-held company; Janssen: Research Funding. Roth: Janssen: Consultancy; Merck: Consultancy.
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- 2021
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