Publications by authors named "Gerald Wertheim"

63 Publications

NTRK Fusions Identified in Pediatric Tumors: The Frequency, Fusion Partners, and Clinical Outcome.

JCO Precis Oncol 2021 14;1. Epub 2021 Jan 14.

Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

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 fusions were found exclusively in CNS tumors and fusions were highly enriched in papillary thyroid carcinomas, fusions were identified in all tumor categories. The most canonical fusion was observed in 10 patients with diverse types of tumors. Several novel NTRK fusions were observed in rare tumor types, including in ganglioglioma and 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 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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http://dx.doi.org/10.1200/PO.20.00250DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8140782PMC
January 2021

Constrained chromatin accessibility in PU.1-mutated agammaglobulinemia patients.

J Exp Med 2021 Jul 5;218(7). Epub 2021 May 5.

Department of Genetics, University of Alabama at Birmingham, Birmingham, AL.

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.
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http://dx.doi.org/10.1084/jem.20201750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8105723PMC
July 2021

Absolute lymphocyte count proliferation kinetics after CAR T-cell infusion impact response and relapse.

Blood Adv 2021 04;5(8):2128-2136

Division of Hematopathology, The Children's Hospital of Philadelphia, Philadelphia, PA.

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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http://dx.doi.org/10.1182/bloodadvances.2020004038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095130PMC
April 2021

Clinical impact of genomic characterization of 15 patients with acute megakaryoblastic leukemia-related malignancies.

Cold Spring Harb Mol Case Stud 2021 Apr 8;7(2). Epub 2021 Apr 8.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.

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 or a - fusion, and the remaining harbored a 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 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.
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http://dx.doi.org/10.1101/mcs.a005975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040732PMC
April 2021

Characterization of Plasmacytoid Dendritic Cells, Microbial Sequences, and Identification of a Candidate Public T-Cell Clone in Kikuchi-Fujimoto Disease.

Pediatr Dev Pathol 2021 May-Jun;24(3):193-205. Epub 2021 Feb 2.

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania.

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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http://dx.doi.org/10.1177/1093526620987961DOI Listing
February 2021

Active learning for fellows: The hematopathology "unknown case".

Pediatr Blood Cancer 2021 Apr 23;68(4):e28895. Epub 2021 Jan 23.

Division of Hematology/Oncology, Pediatric Hematology/Oncology, Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.

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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http://dx.doi.org/10.1002/pbc.28895DOI Listing
April 2021

Risk-Adapted Preemptive Tocilizumab to Prevent Severe Cytokine Release Syndrome After CTL019 for Pediatric B-Cell Acute Lymphoblastic Leukemia: A Prospective Clinical Trial.

J Clin Oncol 2021 Mar 8;39(8):920-930. Epub 2021 Jan 8.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

Purpose: To 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.

Methods: Children 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.

Results: The 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 ( < .001), but persistence was not different ( = .73). Event-free and overall survival were worse in the HTBC ( = .004, < .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 ( = .18).

Conclusion: Risk-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.
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http://dx.doi.org/10.1200/JCO.20.02477DOI Listing
March 2021

Eosinophilia/Hypereosinophilia in the Setting of Reactive and Idiopathic Causes, Well-Defined Myeloid or Lymphoid Leukemias, or Germline Disorders.

Am J Clin Pathol 2021 02;155(2):179-210

Division of Hematopathology, Mayo Clinic, Rochester, MN.

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.
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http://dx.doi.org/10.1093/ajcp/aqaa244DOI Listing
February 2021

A Novel KMT2A-ARHGEF12 Fusion Gene Identified in a High-Grade B-cell Lymphoma.

Cancer Genet 2020 08 7;246-247:41-43. Epub 2020 Aug 7.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA 19104, U.S.A; Department of Pediatrics, Children's Hospital of Philadelphia, Perelman School of Medicine, University of Pennsylvania, 3401 Civic Center Blvd, Philadelphia, PA 19104, U.S.A. Electronic address:

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http://dx.doi.org/10.1016/j.cancergen.2020.08.003DOI Listing
August 2020

Evolution of histomorphologic, cytogenetic, and genetic abnormalities in an untreated patient with MIRAGE syndrome.

Cancer Genet 2020 07 14;245:42-48. Epub 2020 Jun 14.

Department of Pathology and Laboratory Medicine, Children's Hospital of Philadelphia, Abramson Research Center, Room 716D, 3615 Civic Center Blvd., Philadelphia, PA 19104, United States; Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, United States. Electronic address:

Gain of function variants in SAMD9 cause MIRAGE syndrome, a rare Mendelian disorder that results in myeloid dysplastic syndrome (MDS), poor immune response, restricted growth, adrenal insufficiency, ambiguous genitalia, feeding difficulties and most often significantly reduced lifespan. In this study, we describe histomorphologic and genetic changes occurring in serial bone marrow measurements in a patient with MIRAGE syndrome and untreated MDS of 9 years. Histomorphological analysis during childhood showed progressive hypocellularity with erythroid and megakaryocytic dysplasia and cytogenetic testing demonstrated monosomy 7. Serial leukemia gene panel testing performed over a seven year period revealed multiple pre-leukemic clones arising at age 7 years followed by sequential mutational events in ETV6 and RUNX1 driving acute myeloid leukemia (AML) at age 9. Comprehensive genotype-phenotype analysis with 28 previously reported patients found the presence of MDS did not impact overall survival, but in silico variant pathogenicity prediction scores for SAMD9 distinguished patients with poor prognosis. Overall, our analysis shows progression of MDS to AML can be monitored by following mutation evolution in leukemia related genes in patients with MIRAGE syndrome, and specific SAMD9 mutations likely influence disease severity and overall survival.
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http://dx.doi.org/10.1016/j.cancergen.2020.06.002DOI Listing
July 2020

Targeting Bim via a lncRNA Morrbid Regulates the Survival of Preleukemic and Leukemic Cells.

Cell Rep 2020 06;31(12):107816

Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN, USA. Electronic address:

Inhibition of anti-apoptotic proteins BCL-2 and MCL-1 to release pro-apoptotic protein BIM and reactivate cell death could potentially be an efficient strategy for the treatment of leukemia. Here, we show that a lncRNA, MORRBID, a selective transcriptional repressor of BIM, is overexpressed in human acute myeloid leukemia (AML), which is associated with poor overall survival. In both human and animal models, MORRBID hyperactivation correlates with two recurrent AML drivers, TET2 and FLT3. Mice with individual mutations of Tet2 or Flt3 develop features of chronic myelomonocytic leukemia (CMML) and myeloproliferative neoplasm (MPN), respectively, and combined presence results in AML. We observe increased levels of Morrbid in murine models of CMML, MPN, and AML. Functionally, loss of Morrbid in these models induces increased expression of Bim and cell death in immature and mature myeloid cells, which results in reduced infiltration of leukemic cells in tissues and prolongs the survival of AML mice.
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http://dx.doi.org/10.1016/j.celrep.2020.107816DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371151PMC
June 2020

Runx1 negatively regulates inflammatory cytokine production by neutrophils in response to Toll-like receptor signaling.

Blood Adv 2020 03;4(6):1145-1158

Department of Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

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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http://dx.doi.org/10.1182/bloodadvances.2019000785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7094023PMC
March 2020

Increased mTOR activation in idiopathic multicentric Castleman disease.

Blood 2020 05;135(19):1673-1684

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA.

Idiopathic multicentric Castleman disease (iMCD) is a rare and poorly understood hematologic disorder characterized by lymphadenopathy, systemic inflammation, cytopenias, and life-threatening multiorgan dysfunction. Interleukin-6 (IL-6) inhibition effectively treats approximately one-third of patients. Limited options exist for nonresponders, because the etiology, dysregulated cell types, and signaling pathways are unknown. We previously reported 3 anti-IL-6 nonresponders with increased mTOR activation who responded to mTOR inhibition with sirolimus. We investigated mTOR signaling in tissue and serum proteomes from iMCD patients and controls. mTOR activation was increased in the interfollicular space of iMCD lymph nodes (N = 26) compared with control lymph nodes by immunohistochemistry (IHC) for pS6, p4EBP1, and p70S6K, known effectors and readouts of mTORC1 activation. IHC for pS6 also revealed increased mTOR activation in iMCD compared with Hodgkin lymphoma, systemic lupus erythematosus, and reactive lymph nodes, suggesting that the mTOR activation in iMCD is not just a product of lymphoproliferation/inflammatory lymphadenopathy. Further, the degree of mTOR activation in iMCD was comparable to autoimmune lymphoproliferative syndrome, a disease driven by mTOR hyperactivation that responds to sirolimus treatment. Gene set enrichment analysis of serum proteomic data from iMCD patients (n = 88) and controls (n = 42) showed significantly enriched mTORC1 signaling. Finally, functional studies revealed increased baseline mTOR pathway activation in peripheral monocytes and T cells from iMCD remission samples compared with healthy controls. IL-6 stimulation augmented mTOR activation in iMCD patients, which was abrogated with JAK1/2 inhibition. These findings support mTOR activation as a novel therapeutic target for iMCD, which is being investigated through a trial of sirolimus (NCT03933904).
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http://dx.doi.org/10.1182/blood.2019002792DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205815PMC
May 2020

Oncogene-independent BCR-like signaling adaptation confers drug resistance in Ph-like ALL.

J Clin Invest 2020 07;130(7):3637-3653

Division of Oncology, and.

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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http://dx.doi.org/10.1172/JCI134424DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324172PMC
July 2020

Significance of minimal residual disease in pediatric mixed phenotype acute leukemia: a multicenter cohort study.

Leukemia 2020 07 14;34(7):1741-1750. Epub 2020 Feb 14.

Children's Center for Cancer and Blood Diseases, Children's Hospital Los Angeles, Los Angeles, CA, USA.

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 (<0.01%) remission in most patients (70%). EOI MRD positivity was predictive of 5-year EFS (HR = 6.00, p < 0.001) and OS (HR = 9.57, p = 0.003). Patients who cleared MRD by EOC had worse survival compared with those EOI MRD negative. In contrast to adults with MPAL, ALL therapy without transplantation was adequate to treat most pediatric patients. Earlier MRD clearance was associated with better treatment success and survival. Prospective trials are now necessary to validate and refine MRD thresholds within the pediatric MPAL population and to identify salvage strategies for those with poor predicted survival.
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http://dx.doi.org/10.1038/s41375-020-0741-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7332384PMC
July 2020

Tatton-Brown-Rahman syndrome: Six individuals with novel features.

Am J Med Genet A 2020 04 21;182(4):673-680. Epub 2020 Jan 21.

Department of Genetics, Children's Hospital of Eastern Ontario, Ottawa, Ontario, Canada.

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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http://dx.doi.org/10.1002/ajmg.a.61475DOI Listing
April 2020

CAR T-cell therapy is effective for CD19-dim B-lymphoblastic leukemia but is impacted by prior blinatumomab therapy.

Blood Adv 2019 11;3(22):3539-3549

Division of Oncology, Cancer Immunotherapy Program, Children's Hospital of Philadelphia, Philadelphia, PA; and.

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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http://dx.doi.org/10.1182/bloodadvances.2019000692DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6880911PMC
November 2019

Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in IL-6-blockade-refractory idiopathic multicentric Castleman disease.

J Clin Invest 2019 08 13;129(10):4451-4463. Epub 2019 Aug 13.

Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

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.

Methods: We analyzed tissues and blood samples from three IL-6-blockade refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in three iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor, sirolimus.

Results: Studies of three IL-6-blockade refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus significantly attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all three patients with durable and ongoing remissions of 66, 19, and 19 months.

Conclusion: This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically-targetable pathogenic process in IL-6-blockade refractory iMCD. Prospective evaluation of sirolimus in treatment-refractory iMCD is planned (NCT03933904).

Funding: Castleman's Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and National Heart Lung and Blood Institute.
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http://dx.doi.org/10.1172/JCI126091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6763254PMC
August 2019

Trib1 regulates eosinophil lineage commitment and identity by restraining the neutrophil program.

Blood 2019 05 27;133(22):2413-2426. Epub 2019 Mar 27.

Department of Pathology and Laboratory Medicine and.

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. expression markedly increases with eosinophil-lineage commitment in eosinophil progenitors (EoPs), downstream of the granulocyte/macrophage progenitor (GMP). Using hematopoietic- and eosinophil-lineage-specific 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.
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http://dx.doi.org/10.1182/blood.2018872218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6543518PMC
May 2019

Pediatric Somatic Tumor Sequencing Identifies Underlying Cancer Predisposition.

JCO Precis Oncol 2019 16;3. Epub 2019 Dec 16.

Division of Oncology, Department of Pediatrics, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA.

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 inhouse 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.
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http://dx.doi.org/10.1200/po.19.00062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7416724PMC
December 2019

Heterogeneity of surface CD19 and CD22 expression in B lymphoblastic leukemia.

Am J Hematol 2018 11 9;93(11):E352-E355. Epub 2018 Sep 9.

Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania.

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http://dx.doi.org/10.1002/ajh.25235DOI Listing
November 2018

Cytokines increase engraftment of human acute myeloid leukemia cells in immunocompromised mice but not engraftment of human myelodysplastic syndrome cells.

Haematologica 2018 06 15;103(6):959-971. Epub 2018 Mar 15.

Division of Hematology and Oncology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA

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 (<2%), did not increase over time, and was only transiently affected by the use of NSG-S mice. Co-injection of mesenchymal stem cells did not enhance human myelodysplastic syndrome cell engraftment. Overall, we conclude that engraftment of acute myeloid leukemia samples is more robust compared to that of myelodysplastic syndrome samples and unlike those, acute myeloid leukemia cells respond positively to human cytokines, whereas myelodysplastic syndrome cells demonstrate a general unresponsiveness to them.
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http://dx.doi.org/10.3324/haematol.2017.183202DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6058784PMC
June 2018

Clinical Impact of Genomic Information in Pediatric Leukemia.

Front Pediatr 2017 14;5:263. Epub 2017 Dec 14.

Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, United States.

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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http://dx.doi.org/10.3389/fped.2017.00263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5735078PMC
December 2017

A PERK-miR-211 axis suppresses circadian regulators and protein synthesis to promote cancer cell survival.

Nat Cell Biol 2018 01 11;20(1):104-115. Epub 2017 Dec 11.

Department of Biochemistry and Molecular Biology and Hollings Cancer Center, Medical University of South Carolina, Charleston, SC, USA.

The unfolded protein response (UPR) is a stress-activated signalling pathway that regulates cell proliferation, metabolism and survival. The circadian clock coordinates metabolism and signal transduction with light/dark cycles. We explore how UPR signalling interfaces with the circadian clock. UPR activation induces a 10 h phase shift in circadian oscillations through induction of miR-211, a PERK-inducible microRNA that transiently suppresses both Bmal1 and Clock, core circadian regulators. Molecular investigation reveals that miR-211 directly regulates Bmal1 and Clock via distinct mechanisms. Suppression of Bmal1 and Clock has the anticipated impact on expression of select circadian genes, but we also find that repression of Bmal1 is essential for UPR-dependent inhibition of protein synthesis and cell adaptation to stresses that disrupt endoplasmic reticulum homeostasis. Our data demonstrate that c-Myc-dependent activation of the UPR inhibits Bmal1 in Burkitt's lymphoma, thereby suppressing both circadian oscillation and ongoing protein synthesis to facilitate tumour progression.
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http://dx.doi.org/10.1038/s41556-017-0006-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5741512PMC
January 2018

MAFB enhances oncogenic Notch signaling in T cell acute lymphoblastic leukemia.

Sci Signal 2017 11 14;10(505). Epub 2017 Nov 14.

Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104, USA.

Activating mutations in the gene encoding the cell-cell contact signaling protein Notch1 are common in human T cell acute lymphoblastic leukemias (T-ALLs). However, expressing mutant alleles in mice fails to efficiently induce the development of leukemia. We performed a gain-of-function screen to identify proteins that enhanced signaling by leukemia-associated Notch1 mutants. The transcription factors MAFB and ETS2 emerged as candidates that individually enhanced Notch1 signaling, and when coexpressed, they synergistically increased signaling to an extent similar to that induced by core components of the Notch transcriptional complex. In mouse models of T-ALL, MAFB enhanced leukemogenesis by the naturally occurring Notch1 mutants, decreased disease latency, and increased disease penetrance. Decreasing MAFB abundance in mouse and human T-ALL cells reduced the expression of Notch1 target genes, including and , and sustained MAFB knockdown impaired T-ALL growth in a competitive setting. MAFB bound to ETS2 and interacted with the acetyltransferases PCAF and P300, highlighting its importance in recruiting coactivators that enhance Notch1 signaling. Together, these data identify a mechanism for enhancing the oncogenic potential of weak Notch1 mutants in leukemia models, and they reveal the MAFB-ETS2 transcriptional axis as a potential therapeutic target in T-ALL.
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http://dx.doi.org/10.1126/scisignal.aam6846DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5885022PMC
November 2017

Flow Cytometry in Pediatric Hematopoietic Malignancies.

Clin Lab Med 2017 12 28;37(4):879-893. Epub 2017 Sep 28.

Department of Pathology and Laboratory Medicine, Division of Hematopathology, Children's Hospital of Philadelphia, Philadelphia, PA, USA; Department of Pathology and Laboratory Medicine, Hospital of University of Pennsylvania, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

Utility of flow cytometry in the evaluation of pediatric hematopoietic neoplasms and the differences from adult hematopoietic neoplasms are discussed in this review. Distinction of hematogones from B-lymphoblasts, detection of residual/relapsed disease after novel targeted therapies, and evaluation of pediatric myeloid neoplasms are discussed.
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http://dx.doi.org/10.1016/j.cll.2017.07.009DOI Listing
December 2017

Severe Mucha-Habermann-Like Ulceronecrotic Skin Disease in T-Cell Acute Lymphoblastic Leukemia Responsive to Basiliximab and Stem Cell Transplant.

Pediatr Dermatol 2017 Sep;34(5):e265-e270

Division of Oncology, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

A 5-year-old girl with T-cell acute lymphoblastic leukemia (T-ALL) developed a progressive eruption of crusted papules and ulcerative plaques involving 80% of her body surface area with histopathology consistent with febrile ulceronecrotic Mucha-Habermann disease (FUMHD), although multiple specimens also contained clonal leukemic cells. Her skin disease was refractory to many classic treatments for FUMHD, including methotrexate, and became so severe that concern about superinfection prevented intensification of chemotherapy for her malignancy. The addition of basiliximab promoted gradual improvement of the skin, allowing for chemotherapy intensification and subsequent bone marrow transplantation, after which the eruption resolved completely. This report describes a severe case of FUMHD-like eruption associated with clonal leukemic cells that improved with basiliximab, suggesting anti-CD25 therapy as a novel treatment for ulceronecrotic skin disease in the setting of high interleukin-2 levels.
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http://dx.doi.org/10.1111/pde.13235DOI Listing
September 2017

Microsphere-Based Assessment of DNA Methylation for AML Prognosis.

Methods Mol Biol 2017 ;1633:125-136

Department of Pathology & Laboratory Medicine, Weill Cornell Medical College, 525 E. 68th St., F-703, New York, NY, 10065, USA.

Epigenetic dysregulation, including aberrant methylation of cytosine residues in DNA, is a hallmark of cancer and clearly results in oncogenic cellular alterations such as transcriptional attenuation of tumor suppressors and genomic instability. A number of studies have examined DNA methylation alterations in patients with acute myeloid leukemia (AML) and have shown that analysis of multilocus methylation patterns can identify biologically distinct AML subclasses and can predict patient prognosis. In order to utilize the prognostic capability of methylation analysis in a clinical setting, we have developed a microsphere-based HpaII tiny fragment enrichment by ligation-mediated PCR (xMELP) assay to interrogate the methylation state of genomic multiple loci along with a random forest-based classification algorithm that correlates DNA methylation status with patient prognosis. These tools can be easily implemented in a clinical molecular pathology laboratory and can be utilized for more accurate risk stratification of AML patients.
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http://dx.doi.org/10.1007/978-1-4939-7142-8_8DOI Listing
May 2018