Publications by authors named "Elliot Stieglitz"

40 Publications

Inhibition of BTK and PI3Kδ impairs the development of human JMML stem and progenitor cells.

Mol Ther 2022 Apr 20. Epub 2022 Apr 20.

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

Juvenile myelomonocytic leukemia (JMML) is an aggressive myeloproliferative neoplasia that lacks effective targeted chemotherapies. Clinically, JMML manifests as monocytic leukocytosis, splenomegaly with consequential thrombocytopenia. Most commonly, patients have gain-of-function (GOF) oncogenic mutations in PTPN11 (SHP2), leading to Erk and Akt hyperactivation. Mechanism(s) involved in co-regulation of Erk and Akt in the context of GOF SHP2 are poorly understood. Here, we show that Bruton's tyrosine kinase (BTK) is hyperphosphorylated in GOF Shp2-bearing cells and utilizes B cell adaptor for PI3K to cooperate with p110δ, the catalytic subunit of PI3K. Dual inhibition of BTK and p110δ reduces the activation of both Erk and Akt. In vivo, individual targeting of BTK or p110δ in a mouse model of human JMML equally reduces monocytosis and splenomegaly; however, the combined treatment results in a more robust inhibition and uniquely rescues anemia and thrombocytopenia. RNA-seq analysis of drug-treated mice showed a profound reduction in the expression of genes associated with leukemic cell migration and inflammation, leading to correction in the infiltration of leukemic cells in the lung, liver, and spleen. Remarkably, in a patient derived xenograft model of JMML, leukemia-initiating stem and progenitor cells were potently inhibited in response to the dual drug treatment.
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http://dx.doi.org/10.1016/j.ymthe.2022.04.009DOI Listing
April 2022

Therapy-related myeloid neoplasms resembling juvenile myelomonocytic leukemia: a case series and review of the literature.

Pediatr Blood Cancer 2022 05 22;69(5):e29499. Epub 2021 Dec 22.

Department of Pediatrics, UCSF Benioff Children's Hospital San Francisco, University of California San Francisco, San Francisco, California, USA.

Therapy-related myeloid neoplasms (t-MN) are a distinct subgroup of myeloid malignancies with a poor prognosis that include cases of therapy-related myelodysplastic syndrome (t-MDS), therapy-related myeloproliferative neoplasms (t-MPN) and therapy-related acute myeloid leukemia (t-AML). Here, we report a series of patients with clinical features consistent with juvenile myelomonocytic leukemia (JMML), an overlap syndrome of MDS and myeloproliferative neoplasms that developed after treatment for another malignancy.
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http://dx.doi.org/10.1002/pbc.29499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8957526PMC
May 2022

Simple and robust methylation test for risk stratification of patients with juvenile myelomonocytic leukemia.

Blood Adv 2021 12;5(24):5507-5518

Department of Pediatrics, Nagoya University Graduate School of Medicine, Nagoya, Japan.

Juvenile myelomonocytic leukemia (JMML) is a rare myelodysplastic/myeloproliferative neoplasm that develops during infancy and early childhood. The array-based international consensus definition of DNA methylation has recently classified patients with JMML into the following 3 groups: high (HM), intermediate (IM), and low methylation (LM). To develop a simple and robust methylation clinical test, 137 patients with JMML were analyzed using the Digital Restriction Enzyme Analysis of Methylation (DREAM), which is a next-generation sequencing-based methylation analysis. Unsupervised consensus clustering of the discovery cohort (n = 99) using DREAM data identified HM (HM_DREAM; n = 35) and LM subgroups (LM_DREAM; n = 64). Of the 98 cases that could be compared with the international consensus classification, 90 HM (n = 30) and LM (n = 60) cases had 100% concordance with DREAM clustering results. Of the remaining 8 cases comprising the IM group, 4 were classified as belonging to the HM_DREAM group and 4 to the LM_DREAM group. A machine-learning classifier was successfully constructed using a support vector machine (SVM), which divided the validation cohort (n = 38) into HM (HM_SVM, n = 18) and LM (LM_SVM; n = 20) groups. Patients with the HM_SVM profile had a significantly poorer 5-year overall survival rate than those with the LM_SVM profile. In conclusion, we developed a robust methylation test using DREAM for patients with JMML. This simple and straightforward test can be easily incorporated into diagnosis to generate a methylation classification for patients so they can receive risk-adapted treatment in the context of future clinical trials.
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http://dx.doi.org/10.1182/bloodadvances.2021005080DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8714717PMC
December 2021

JMML tumor cells disrupt normal hematopoietic stem cells by imposing inflammatory stress through overproduction of IL-1β.

Blood Adv 2022 01;6(1):200-206

Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Winship Cancer Institute, Children's Healthcare of Atlanta, Emory University School of Medicine, Atlanta, GA.

Development of normal blood cells is often suppressed in juvenile myelomonocytic leukemia (JMML), a myeloproliferative neoplasm (MPN) of childhood, causing complications and impacting therapeutic outcomes. However, the mechanism underlying this phenomenon remains uncharacterized. To address this question, we induced the most common mutation identified in JMML (Ptpn11E76K) specifically in the myeloid lineage with hematopoietic stem cells (HSCs) spared. These mice uniformly developed a JMML-like MPN. Importantly, HSCs in the same bone marrow (BM) microenvironment were aberrantly activated and differentiated at the expense of self-renewal. As a result, HSCs lost quiescence and became exhausted. A similar result was observed in wild-type (WT) donor HSCs when co-transplanted with Ptpn11E76K/+ BM cells into WT mice. Co-culture testing demonstrated that JMML/MPN cells robustly accelerated differentiation in mouse and human normal hematopoietic stem/progenitor cells. Cytokine profiling revealed that Ptpn11E76K/+ MPN cells produced excessive IL-1β, but not IL-6, T NF-α, IFN-γ, IL-1α, or other inflammatory cytokines. Depletion of the IL-1β receptor effectively restored HSC quiescence, normalized their pool size, and rescued them from exhaustion in Ptpn11E76K/+/IL-1R-/- double mutant mice. These findings suggest IL-1β signaling as a potential therapeutic target for preserving normal hematopoietic development in JMML.
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http://dx.doi.org/10.1182/bloodadvances.2021005089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8753218PMC
January 2022

Successful treatment and integrated genomic analysis of an infant with FIP1L1-RARA fusion-associated myeloid neoplasm.

Blood Adv 2022 02;6(4):1137-1142

Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY.

FIP1L1-RARA-a ssociated neoplasm is a very rare and aggressive disease, with only 3 previously reported cases in the literature. Here, we describe a 9-month-old boy who presented with a FIP1L1-RARA fusion-associated myelodysplastic/myeloproliferative neoplasm-like overlap syndrome, with similarities and distinct features to both acute promyelocytic leukemia and juvenile myelomonocytic leukemia. Using a combined approach of chemotherapy, differentiating agents, and allogeneic hematopoietic stem cell transplant (allo-HCT), this patient remains in remission 20 months after allo-HCT. To our knowledge, this is only the second published pediatric case involving this condition and the only case with a favorable long-term outcome. Given the aggressive disease described in the previously published case report, as well as the successful treatment course described, the combinatorial use of chemotherapy, differentiation therapy, and allo-HCT for treatment of FIP1L1-RARA fusion-associated myeloid neoplasms should be considered.
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http://dx.doi.org/10.1182/bloodadvances.2021004966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8864666PMC
February 2022

Juvenile myelomonocytic leukemia in the molecular era: a clinician's guide to diagnosis, risk stratification, and treatment.

Blood Adv 2021 11;5(22):4783-4793

Department of Pediatrics, Benioff Children's Hospital, University of California, San Francisco, CA; and.

Juvenile myelomonocytic leukemia is an overlapping myeloproliferative and myelodysplastic disorder of early childhood . It is associated with a spectrum of diverse outcomes ranging from spontaneous resolution in rare patients to transformation to acute myeloid leukemia in others that is generally fatal. This unpredictable clinical course, along with initially descriptive diagnostic criteria, led to decades of productive international research. Next-generation sequencing now permits more accurate molecular diagnoses in nearly all patients. However, curative treatment is still reliant on allogeneic hematopoietic cell transplantation for most patients, and additional advances will be required to improve risk stratification algorithms that distinguish those that can be observed expectantly from others who require swift hematopoietic cell transplantation.
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http://dx.doi.org/10.1182/bloodadvances.2021005117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8759142PMC
November 2021

Nf1 and Sh2b3 mutations cooperate in vivo in a mouse model of juvenile myelomonocytic leukemia.

Blood Adv 2021 09;5(18):3587-3591

Department of Pediatrics, Benioff Children's Hospital.

Juvenile myelomonocytic leukemia (JMML) is initiated in early childhood by somatic mutations that activate Ras signaling. Although some patients have only a single identifiable oncogenic mutation, others have 1 or more additional alterations. Such secondary mutations, as a group, are associated with an increased risk of relapse after hematopoietic stem cell transplantation or transformation to acute myeloid leukemia. These clinical observations suggest a cooperative effect between initiating and secondary mutations. However, the roles of specific genes in the prognosis or clinical presentation of JMML have not been described. In this study, we investigate the impact of secondary SH2B3 mutations in JMML. We find that patients with SH2B3 mutations have adverse outcomes, as well as higher white blood cell counts and hemoglobin F levels in the peripheral blood. We further demonstrate this interaction in genetically engineered mice. Deletion of Sh2b3 cooperates with conditional Nf1 deletion in a dose-dependent fashion. These studies illustrate that haploinsufficiency for Sh2b3 contributes to the severity of myeloproliferative disease and provide an experimental system for testing treatments for a high-risk cohort of JMML patients.
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http://dx.doi.org/10.1182/bloodadvances.2020003754DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8945579PMC
September 2021

Exploring the genetic and epigenetic origins of juvenile myelomonocytic leukemia using newborn screening samples.

Leukemia 2022 01 28;36(1):279-282. Epub 2021 Jun 28.

Department of Pediatrics, Benioff Children's Hospital, University of California, San Francisco, San Francisco, CA, USA.

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http://dx.doi.org/10.1038/s41375-021-01331-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8720242PMC
January 2022

Surface Proteomics Reveals CD72 as a Target for -Evolved Nanobody-Based CAR-T Cells in -Rearranged B-ALL.

Cancer Discov 2021 08 16;11(8):2032-2049. Epub 2021 Mar 16.

Department of Laboratory Medicine, University of California, San Francisco, San Francisco, California.

Alternative strategies are needed for patients with B-cell malignancy relapsing after CD19-targeted immunotherapy. Here, cell surface proteomics revealed CD72 as an optimal target for poor-prognosis /-rearranged (MLLr) B-cell acute lymphoblastic leukemia (B-ALL), which we further found to be expressed in other B-cell malignancies. Using a recently described, fully system, we selected synthetic CD72-specific nanobodies, incorporated them into chimeric antigen receptors (CAR), and demonstrated robust activity against B-cell malignancy models, including CD19 loss. Taking advantage of the role of CD72 in inhibiting B-cell receptor signaling, we found that SHIP1 inhibition increased CD72 surface density. We establish that CD72-nanobody CAR-T cells are a promising therapy for MLLr B-ALL. SIGNIFICANCE: Patients with MLLr B-ALL have poor prognoses despite recent immunotherapy advances. Here, surface proteomics identifies CD72 as being enriched on MLLr B-ALL but also widely expressed across B-cell cancers. We show that a recently described, fully nanobody platform generates binders highly active in CAR-T cells and demonstrate its broad applicability for immunotherapy development..
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http://dx.doi.org/10.1158/2159-8290.CD-20-0242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8338785PMC
August 2021

Distinct genetic pathways define pre-malignant versus compensatory clonal hematopoiesis in Shwachman-Diamond syndrome.

Nat Commun 2021 02 26;12(1):1334. Epub 2021 Feb 26.

Dartmouth-Hitchcock Medical Center, Pediatric Hematology Oncology, Geisel School of Medicine, Lebanon, NH, USA.

To understand the mechanisms that mediate germline genetic leukemia predisposition, we studied the inherited ribosomopathy Shwachman-Diamond syndrome (SDS), a bone marrow failure disorder with high risk of myeloid malignancies at an early age. To define the mechanistic basis of clonal hematopoiesis in SDS, we investigate somatic mutations acquired by patients with SDS followed longitudinally. Here we report that multiple independent somatic hematopoietic clones arise early in life, most commonly harboring heterozygous mutations in EIF6 or TP53. We show that germline SBDS deficiency establishes a fitness constraint that drives selection of somatic clones via two distinct mechanisms with different clinical consequences. EIF6 inactivation mediates a compensatory pathway with limited leukemic potential by ameliorating the underlying SDS ribosome defect and enhancing clone fitness. TP53 mutations define a maladaptive pathway with enhanced leukemic potential by inactivating tumor suppressor checkpoints without correcting the ribosome defect. Subsequent development of leukemia was associated with acquisition of biallelic TP53 alterations. These results mechanistically link leukemia predisposition to germline genetic constraints on cellular fitness, and provide a rational framework for clinical surveillance strategies.
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http://dx.doi.org/10.1038/s41467-021-21588-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7910481PMC
February 2021

Matched Targeted Therapy for Pediatric Patients with Relapsed, Refractory, or High-Risk Leukemias: A Report from the LEAP Consortium.

Cancer Discov 2021 06 9;11(6):1424-1439. Epub 2021 Feb 9.

Division of Oncology and Center for Childhood Cancer Research, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania.

Despite a remarkable increase in the genomic profiling of cancer, integration of genomic discoveries into clinical care has lagged behind. We report the feasibility of rapid identification of targetable mutations in 153 pediatric patients with relapsed/refractory or high-risk leukemias enrolled on a prospective clinical trial conducted by the LEAP Consortium. Eighteen percent of patients had a high confidence Tier 1 or 2 recommendation. We describe clinical responses in the 14% of patients with relapsed/refractory leukemia who received the matched targeted therapy. Further, in order to inform future targeted therapy for patients, we validated variants of uncertain significance, performed drug-sensitivity testing in patient leukemia samples, and identified new combinations of targeted therapies in cell lines and patient-derived xenograft models. These data and our collaborative approach should inform the design of future precision medicine trials. SIGNIFICANCE: Patients with relapsed/refractory leukemias face limited treatment options. Systematic integration of precision medicine efforts can inform therapy. We report the feasibility of identifying targetable mutations in children with leukemia and describe correlative biology studies validating therapeutic hypotheses and novel mutations...
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http://dx.doi.org/10.1158/2159-8290.CD-20-0564DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8178162PMC
June 2021

Targeting the Ras pathway in pediatric hematologic malignancies.

Curr Opin Pediatr 2021 02 29;33(1):49-58. Epub 2020 Dec 29.

Department of Pediatrics, Benioff Children's Hospital, Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, California, USA.

Purpose Of Review: Ras pathway mutations are one of the most common type of alterations in pediatric hematologic malignancies and are frequently associated with adverse outcomes. Despite ongoing efforts to use targeted treatments, there remain no Food and Drug Administration (FDA)-approved medications specifically for children with Ras pathway-mutated leukemia. This review will summarize the role of Ras pathway mutations in pediatric leukemia, discuss the current state of Ras pathway inhibitors and highlight the most promising agents currently being evaluated in clinical trials.

Recent Findings: Efficacy using RAF and MEK inhibitors has been demonstrated across multiple solid and brain tumors, and these are now considered standard-of-care for certain tumor types in adults and children. Clinical trials are now testing these medications for the first time in pediatric hematologic disorders, such as acute lymphoblastic leukemia, juvenile myelomonocytic leukemia, and histiocytic disorders. Novel inhibitors of the Ras pathway, including direct RAS inhibitors, are also being tested in clinical trials across a spectrum of pediatric and adult malignancies.

Summary: Activation of the Ras pathway is a common finding in pediatric hematologic neoplasms. Implementation of precision medicine with a goal of improving outcomes for these patients will require testing of Ras pathway inhibitors in combination with other drugs in the context of current and future clinical trials.
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http://dx.doi.org/10.1097/MOP.0000000000000981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880552PMC
February 2021

Molecular and phenotypic diversity of CBL-mutated juvenile myelomonocytic leukemia.

Haematologica 2022 01 1;107(1):178-186. Epub 2022 Jan 1.

Department of Pediatrics, Benioff Children's Hospital, University of California, San Francisco, San Francisco, CA; Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco.

Mutations in the gene CBL were first identified in adults with various myeloid malignancies. Some patients with juvenile myelomonocytic leukemia (JMML) were also noted to harbor mutations in CBL, but were found to have generally less aggressive disease courses compared to other forms of Ras pathway-mutant JMML. Importantly, and in contrast to most reports in adults, the majority of CBL mutations in JMML patients are germline with acquired uniparental disomy occurring in affected marrow cells. Here, we systematically studied a large cohort of 33 JMML patients with CBL mutations and found this disease to be highly diverse in presentation and overall outcome. Moreover, we discovered somatically-acquired CBL mutations in 15% of pediatric patients who presented with more aggressive disease. Neither clinical features nor methylation profiling were able to distinguish somatic CBL patients from germline CBL patients, highlighting the need for germline testing. Overall, we demonstrate that disease courses are quite heterogeneous even among germline CBL patients. Prospective clinical trials are warranted to find ideal treatment strategies for this diverse cohort of patients.
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http://dx.doi.org/10.3324/haematol.2020.270595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8719097PMC
January 2022

International Consensus Definition of DNA Methylation Subgroups in Juvenile Myelomonocytic Leukemia.

Clin Cancer Res 2021 01 2;27(1):158-168. Epub 2020 Nov 2.

Section Translational Cancer Epigenomics, Division Translational Medical Oncology, German Cancer Research Center (DKFZ) & National Center for Tumor Diseases (NCT), Heidelberg, Germany.

Purpose: Known clinical and genetic markers have limitations in predicting disease course and outcome in juvenile myelomonocytic leukemia (JMML). DNA methylation patterns in JMML have correlated with outcome across multiple studies, suggesting it as a biomarker to improve patient stratification. However, standardized approaches to classify JMML on the basis of DNA methylation patterns are lacking. We, therefore, sought to define an international consensus for DNA methylation subgroups in JMML and develop classification methods for clinical implementation.

Experimental Design: Published DNA methylation data from 255 patients with JMML were used to develop and internally validate a classifier model. Accuracy across platforms (EPIC-arrays and MethylSeq) was tested using a technical validation cohort (32 patients). The suitability of both methods for single-patient classification was demonstrated using an independent cohort (47 patients).

Results: Analysis of pooled, published data established three DNA methylation subgroups as a standard. Unfavorable prognostic parameters ( mutation, elevated fetal hemoglobin, and older age) were significantly enriched in the high methylation (HM) subgroup. A classifier was then developed that predicted subgroups with 98% accuracy across different technological platforms. Applying the classifier to an independent validation cohort confirmed an association of HM with secondary mutations, high relapse incidence, and inferior overall survival (OS), while the low methylation subgroup was associated with a favorable disease course. Multivariable analysis established DNA methylation subgroups as the only significant factor predicting OS.

Conclusions: This study provides an international consensus definition for DNA methylation subgroups in JMML. We developed and validated methods which will facilitate the design of risk-stratified clinical trials in JMML.
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http://dx.doi.org/10.1158/1078-0432.CCR-20-3184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7785676PMC
January 2021

Clinical utilization of blinatumomab and inotuzumab immunotherapy in children with relapsed or refractory B-acute lymphoblastic leukemia.

Pediatr Blood Cancer 2021 01 24;68(1):e28718. Epub 2020 Oct 24.

Children's Hospital of Philadelphia, Division of Oncology and Center for Childhood Cancer Research, Philadelphia, Pennsylvania.

Background: The treatment paradigm for patients with relapsed/refractory B-cell acute lymphoblastic leukemia (rrALL) has been revolutionized given recent clinical trials demonstrating remarkable success of immunotherapies and leading to drug approvals by United States and European agencies. We report experience with commercial blinatumomab and inotuzumab use at two North American pediatric oncology centers in children and adolescents/young adults with B-ALL.

Procedure: Patients 0-25 years old treated with the CD19 × CD3 bispecific T cell-engaging antibody blinatumomab and/or the CD22 antibody-drug conjugate inotuzumab from January 1, 2010, to June 1, 2018, were eligible. Disease status included relapsed B-ALL in second or greater relapse, primary chemotherapy-refractory B-ALL, or B-ALL complicated by severe infection precluding delivery of conventional chemotherapy.

Results: We identified 27 patients who received blinatumomab and/or inotuzumab outside of clinical trials during the study period. Four of the 13 patients (31%) with relapsed disease achieved minimal residual disease (MRD)-negative remission, and five patients (39%) underwent hematopoietic stem cell transplant (HSCT). In the 12 patients with primary chemorefractory B-ALL treated with immunotherapy, 11 (92%) achieved MRD-negative remission as assessed by flow cytometry; 10 patients (83%) underwent subsequent HSCT. Two patients with B-ALL in MRD-negative remission received blinatumomab due to severe infection and remained in remission after chemotherapy continuation.

Conclusions: Blinatumomab and inotuzumab can induce deep remissions in patients with rrALL and facilitate subsequent HSCT or other cellular therapies. Blinatumomab can also serve as an effective bridging therapy during severe infection. The optimal timing, choice of immunotherapeutic agent(s), and duration of responses require further investigation via larger-scale clinical trials.
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http://dx.doi.org/10.1002/pbc.28718DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7688575PMC
January 2021

NUP98-NSD1 Driven MDS/MPN in Childhood Masquerading as JMML.

J Pediatr Hematol Oncol 2021 08;43(6):e808-e811

Department of Pediatrics, Benioff Children's Hospital.

Overlapping myelodysplastic/myeloproliferative neoplasms (MDS/MPN) are clonal hematopoietic disorders with features of myelodysplasia and myeloproliferation. The only well-characterized MDS/MPN in children is juvenile myelomonocytic leukemia, an aggressive disorder of infants and toddlers. The biochemical hallmark of this disease is hyperactivation of the Ras/MAPK signaling pathway caused by mutations in Ras pathway genes in more than 90% of patients. Translocations involving receptor tyrosine kinases have been identified in rare cases. Here, we report a 2-year-old patient who presented with MDS/MPN driven by a cytogenetically cryptic NUP98-NSD1 fusion, a translocation thought to exclusively occur in patients with acute myeloid leukemia.
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http://dx.doi.org/10.1097/MPH.0000000000001913DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7889745PMC
August 2021

Low toxicity and favorable overall survival in relapsed/refractory B-ALL following CAR T cells and CD34-selected T-cell depleted allogeneic hematopoietic cell transplant.

Bone Marrow Transplant 2020 11 10;55(11):2160-2169. Epub 2020 May 10.

Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.

To define the tolerability and outcome of allogeneic hematopoietic stem cell transplant (allo-HSCT) following CAR T-cell therapy, we retrospectively reviewed pediatric/young adult patients with relapsed/refractory B-ALL who underwent this treatment. Fifteen patients (median age 13 years; range 1-20 years) with a median potential follow-up of 39 months demonstrated 24-month cumulative incidence of relapse, cumulative incidence of TRM, and OS of 16% (95% CI: 0-37%), 20% (95% CI: 0-40%), and 80% (95% CI: 60-100%), respectively. Severe toxicity following CAR T cells did not impact OS (p = 0.27), while greater time from CAR T cells to allo-HSCT (>80 days) was associated with a decrease in OS. In comparing CD34-selected T-cell depleted (TCD; n = 9) vs unmodified (n = 6) allo-HSCT, the cumulative incidence of relapse, TRM, and OS at 24 months was 22% (95% CI: 0-49%) vs 0% (p = 0.14), 0% vs 50% [95% CI: 10-90%] (p = 0.02) and 100% vs 50% [95% CI: 10-90%] (p = 0.02). In this small cohort of patients, CAR T cells followed by a CD34-selected TCD allo-HSCT appears to result in less TRM and favorable OS when compared with unmodified allo-HSCT. There was no evidence that disease control was impacted by the type of consolidative allo-HSCT, which demonstrates the feasibility of this approach.
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http://dx.doi.org/10.1038/s41409-020-0926-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7606268PMC
November 2020

Precision cancer monitoring using a novel, fully integrated, microfluidic array partitioning digital PCR platform.

Sci Rep 2019 12 20;9(1):19606. Epub 2019 Dec 20.

Department of Pediatrics, UCSF Benioff Children's Hospital, San Francisco, CA, USA.

A novel digital PCR (dPCR) platform combining off-the-shelf reagents, a micro-molded plastic microfluidic consumable with a fully integrated single dPCR instrument was developed to address the needs for routine clinical diagnostics. This new platform offers a simplified workflow that enables: rapid time-to-answer; low potential for cross contamination; minimal sample waste; all within a single integrated instrument. Here we showcase the capability of this fully integrated platform to detect and quantify non-small cell lung carcinoma (NSCLC) rare genetic mutants (EGFR T790M) with precision cell-free DNA (cfDNA) standards. Next, we validated the platform with an established chronic myeloid leukemia (CML) fusion gene (BCR-ABL1) assay down to 0.01% mutant allele frequency to highlight the platform's utility for precision cancer monitoring. Thirdly, using a juvenile myelomonocytic leukemia (JMML) patient-specific assay we demonstrate the ability to precisely track an individual cancer patient's response to therapy and show the patient's achievement of complete molecular remission. These three applications highlight the flexibility and utility of this novel fully integrated dPCR platform that has the potential to transform personalized medicine for cancer recurrence monitoring.
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http://dx.doi.org/10.1038/s41598-019-55872-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6925289PMC
December 2019

The impact of total body irradiation-based regimens on outcomes in children and young adults with acute lymphoblastic leukemia undergoing allogeneic hematopoietic stem cell transplantation.

Pediatr Blood Cancer 2020 02 14;67(2):e28079. Epub 2019 Nov 14.

Division of Pediatric Allergy, Immunology, and Blood & Marrow Transplantation, UCSF Benioff Children's Hospital, San Francisco, California.

Introduction: Total body irradiation (TBI)-based conditioning is the standard of care in the treatment of acute lymphoblastic leukemia (ALL) that requires allogeneic hematopoietic stem cell transplantation (HSCT). However, TBI is known to be associated with an increased risk of late effects, and therefore, non-TBI regimens have also been utilized successfully. A recent study showed that patients that were next-generation sequencing-minimal residual disease (NGS-MRD) negative prior to allogeneic HSCT had a very low risk of relapse, and perhaps could avoid exposure to TBI without compromising disease control. We examined outcomes at our institution in patients that received a TBI or non-TBI regimen, as well as explored the impact of NGS-MRD status in predicting risk of relapse post transplant.

Procedures: This retrospective analysis included 57 children and young adults with ALL that received their first myeloablative allogeneic HSCT from 2012 to 2017 at the University of California San Francisco. Our primary endpoint was the cumulative incidence of relapse at 3 years post transplant.

Results: We demonstrated similar cumulative incidence of relapse for patients treated with either a TBI or non-TBI conditioning regimen, while NGS-MRD positivity prior to transplant was highly predictive of relapse. The presence of acute graft-versus-host disease was associated with decreased relapse rates, particularly among patients that received a TBI conditioning regimen and patients that were NGS-MRD positive prior to HSCT.

Conclusions: Our data suggest that the decision to use either a TBI or non-TBI regimens in ALL should depend on NGS-MRD status, with conditioning regimens based on TBI reserved for patients that cannot achieve NGS-MRD negativity prior to allogeneic HSCT.
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http://dx.doi.org/10.1002/pbc.28079DOI Listing
February 2020

Molecular assessment of pretransplant chemotherapy in the treatment of juvenile myelomonocytic leukemia.

Pediatr Blood Cancer 2019 11 26;66(11):e27948. Epub 2019 Jul 26.

Department of Pediatrics, Benioff Children's Hospital, University of California, San Francisco, California.

Background: Despite the intensity of hematopoietic stem cell transplantation (HCT), relapse remains the most common cause of death in juvenile myelomonocytic leukemia (JMML). In contrast to other leukemias where therapy is used to reduce leukemic burden prior to transplant, many patients with JMML proceed directly to HCT with active disease. The objective of this study was to elucidate whether pre-HCT therapy has an effect on the molecular burden of disease and how this affects outcome post-HCT.

Procedure: Twenty-one patients with JMML who received pre-HCT therapy and were transplanted at UCSF were analyzed in this study. The mutant allele frequency of the driver mutation was assessed before and after pre-HCT therapy, using custom amplicon next-generation sequencing.

Results: Of the 21 patients, seven patients (33%) responded to therapy with a significant reduction in their mutant allele frequency and were classified as molecular responders. Six of these patients received moderate-intensity chemotherapy, one patient received only azacitidine. The 5-year progression-free survival after HCT of molecular responders was 100% versus 61% for nonresponders (P = .12). Survival of molecular nonresponders was not improved by use of high-intensity conditioning, but patients were salvaged if they experienced severe graft versus host disease. There were no baseline clinical characteristics that were associated with response to pre-HCT therapy.

Conclusions: Despite the myelodysplastic nature of JMML, patients treated with pre-HCT therapy can achieve molecular remissions. These patients experienced a trend toward improved outcomes post-HCT. Importantly, molecular testing can be helpful to distinguish between responders and nonresponders and should become an integral part of clinical care.
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http://dx.doi.org/10.1002/pbc.27948DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6754267PMC
November 2019

Activating Mutations of RRAS2 Are a Rare Cause of Noonan Syndrome.

Am J Hum Genet 2019 06 23;104(6):1223-1232. Epub 2019 May 23.

Département de Génétique, Assistance Publique des Hôpitaux de Paris (AP-HP) Hôpital Robert Debré, 75019 Paris, France; INSERM UMR 1141 - Université de Paris, 75019 Paris, France.

Aberrant signaling through pathways controlling cell response to extracellular stimuli constitutes a central theme in disorders affecting development. Signaling through RAS and the MAPK cascade controls a variety of cell decisions in response to cytokines, hormones, and growth factors, and its upregulation causes Noonan syndrome (NS), a developmental disorder whose major features include a distinctive facies, a wide spectrum of cardiac defects, short stature, variable cognitive impairment, and predisposition to malignancies. NS is genetically heterogeneous, and mutations in more than ten genes have been reported to underlie this disorder. Despite the large number of genes implicated, about 10%-20% of affected individuals with a clinical diagnosis of NS do not have mutations in known RASopathy-associated genes, indicating that additional unidentified genes contribute to the disease, when mutated. By using a mixed strategy of functional candidacy and exome sequencing, we identify RRAS2 as a gene implicated in NS in six unrelated subjects/families. We show that the NS-causing RRAS2 variants affect highly conserved residues localized around the nucleotide binding pocket of the GTPase and are predicted to variably affect diverse aspects of RRAS2 biochemical behavior, including nucleotide binding, GTP hydrolysis, and interaction with effectors. Additionally, all pathogenic variants increase activation of the MAPK cascade and variably impact cell morphology and cytoskeletal rearrangement. Finally, we provide a characterization of the clinical phenotype associated with RRAS2 mutations.
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http://dx.doi.org/10.1016/j.ajhg.2019.04.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562003PMC
June 2019

Predisposing germline mutations in high hyperdiploid acute lymphoblastic leukemia in children.

Genes Chromosomes Cancer 2019 10 27;58(10):723-730. Epub 2019 May 27.

Center for Genetic Epidemiology, Department of Preventive Medicine, USC Keck School of Medicine, Los Angeles, California.

High hyperdiploidy (HD) is the most common cytogenetic subtype of childhood acute lymphoblastic leukemia (ALL), and a higher incidence of HD has been reported in ALL patients with congenital cancer syndromes. We assessed the frequency of predisposing germline mutations in 57 HD-ALL patients from the California Childhood Leukemia Study via targeted sequencing of cancer-relevant genes. Three out of 57 patients (5.3%) harbored confirmed germline mutations that were likely causal, in NBN, ETV6, and FLT3, with an additional six patients (10.5%) harboring putative predisposing mutations that were rare in unselected individuals (<0.01% allele frequency in the Exome Aggregation Consortium, ExAC) and predicted functional (scaled CADD score ≥ 20) in known or potential ALL predisposition genes (SH2B3, CREBBP, PMS2, MLL, ABL1, and MYH9). Three additional patients carried rare and predicted damaging germline mutations in GAB2, a known activator of the ERK/MAPK and PI3K/AKT pathways and binding partner of PTPN11-encoded SHP2. The frequency of rare and predicted functional germline GAB2 mutations was significantly higher in our patients (2.6%) than in ExAC (0.28%, P = 4.4 × 10 ), an observation that was replicated in ALL patients from the TARGET project (P = .034). We cloned patient GAB2 mutations and expressed mutant proteins in HEK293 cells and found that frameshift mutation P621fs led to reduced SHP2 binding and ERK1/2 phosphorylation but significantly increased AKT phosphorylation, suggesting possible RAS-independent leukemogenic effects. Our results support a significant contribution of rare, high penetrance germline mutations to HD-ALL etiology, and pinpoint GAB2 as a putative novel ALL predisposition gene.
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http://dx.doi.org/10.1002/gcc.22765DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6684857PMC
October 2019

Genomic subtyping and therapeutic targeting of acute erythroleukemia.

Nat Genet 2019 04 29;51(4):694-704. Epub 2019 Mar 29.

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

Acute erythroid leukemia (AEL) is a high-risk leukemia of poorly understood genetic basis, with controversy regarding diagnosis in the spectrum of myelodysplasia and myeloid leukemia. We compared genomic features of 159 childhood and adult AEL cases with non-AEL myeloid disorders and defined five age-related subgroups with distinct transcriptional profiles: adult, TP53 mutated; NPM1 mutated; KMT2A mutated/rearranged; adult, DDX41 mutated; and pediatric, NUP98 rearranged. Genomic features influenced outcome, with NPM1 mutations and HOXB9 overexpression being associated with a favorable prognosis and TP53, FLT3 or RB1 alterations associated with poor survival. Targetable signaling mutations were present in 45% of cases and included recurrent mutations of ALK and NTRK1, the latter of which drives erythroid leukemogenesis sensitive to TRK inhibition. This genomic landscape of AEL provides the framework for accurate diagnosis and risk stratification of this disease, and the rationale for testing targeted therapies in this high-risk leukemia.
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http://dx.doi.org/10.1038/s41588-019-0375-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828160PMC
April 2019

Downregulating Notch counteracts Kras-induced ERK activation and oxidative phosphorylation in myeloproliferative neoplasm.

Leukemia 2019 03 11;33(3):671-685. Epub 2018 Sep 11.

McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, Madison, WI, USA.

The Notch signaling pathway contributes to the pathogenesis of a wide spectrum of human cancers, including hematopoietic malignancies. Its functions are highly dependent on the specific cellular context. Gain-of-function NOTCH1 mutations are prevalent in human T-cell leukemia, while loss of Notch signaling is reported in myeloid leukemias. Here, we report a novel oncogenic function of Notch signaling in oncogenic Kras-induced myeloproliferative neoplasm (MPN). We find that downregulation of Notch signaling in hematopoietic cells via DNMAML expression or Pofut1 deletion significantly blocks MPN development in Kras mice in a cell-autonomous manner. Further mechanistic studies indicate that inhibition of Notch signaling upregulates Dusp1, a dual phosphatase that inactivates p-ERK, and downregulates cytokine-evoked ERK activation in Kras cells. Moreover, mitochondrial metabolism is greatly enhanced in Kras cells but significantly reprogrammed by DNMAML close to that in control cells. Consequently, cell proliferation and expanded myeloid compartment in Kras mice are significantly reduced. Consistent with these findings, combined inhibition of the MEK/ERK pathway and mitochondrial oxidative phosphorylation effectively inhibited the growth of human and mouse leukemia cells in vitro. Our study provides a strong rational to target both ERK signaling and aberrant metabolism in oncogenic Ras-driven myeloid leukemia.
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http://dx.doi.org/10.1038/s41375-018-0248-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405304PMC
March 2019

Disease burden and conditioning regimens in ASCT1221, a randomized phase II trial in children with juvenile myelomonocytic leukemia: A Children's Oncology Group study.

Pediatr Blood Cancer 2018 07 12;65(7):e27034. Epub 2018 Mar 12.

Division of Pediatric Hematology/Oncology, Benioff Children's Hospital, University of California San Francisco, San Francisco, California.

Background: Most patients with juvenile myelomonocytic leukemia (JMML) are curable only with allogeneic hematopoietic cell transplantation (HCT). However, the current standard conditioning regimen, busulfan-cyclophosphamide-melphalan (Bu-Cy-Mel), may be associated with higher risks of morbidity and mortality. ASCT1221 was designed to test whether the potentially less-toxic myeloablative conditioning regimen containing busulfan-fludarabine (Bu-Flu) would be associated with equivalent outcomes.

Procedure: Twenty-seven patients were enrolled on ASCT1221 from 2013 to 2015. Pre- and post-HCT (starting Day +30) mutant allele burden was measured in all and pre-HCT therapy was administered according to physician discretion.

Results: Fifteen patients were randomized (six to Bu-Cy-Mel and nine to Bu-Flu) after meeting diagnostic criteria for JMML. Pre-HCT low-dose chemotherapy did not appear to reduce pre-HCT disease burden. Two patients, however, received aggressive chemotherapy pre-HCT and achieved low disease-burden state; both are long-term survivors. All four patients with detectable mutant allele burden at Day +30 post-HCT eventually progressed compared to two of nine patients with unmeasurable allele burden (P = 0.04). The 18-month event-free survival of the entire cohort was 47% (95% CI, 21-69%), and was 83% (95% CI, 27-97%) and 22% (95% CI, 03-51%) for Bu-Cy-Mel and Bu-Flu, respectively (P = 0.04). ASCT1221 was terminated early due to concerns that the Bu-Flu arm had inferior outcomes.

Conclusions: The regimen of Bu-Flu is inadequate to provide disease control in patients with JMML who present to HCT with large burdens of disease. Advances in molecular testing may allow better characterization of biologic risk, pre-HCT responses to chemotherapy, and post-HCT management.
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http://dx.doi.org/10.1002/pbc.27034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980696PMC
July 2018

ABVD Without Radiation for Newly Diagnosed Pediatric and Young Adult Patients With Hodgkin Lymphoma: A Single Center Retrospective Analysis of 28 Consecutive Patients.

J Pediatr Hematol Oncol 2018 05;40(4):290-294

Department of Pediatrics, Benioff Children's Hospital.

Hodgkin lymphoma (HL) is the most common malignancy affecting adolescents and young adults. Treatment with a combination of chemotherapy and radiation results in cure rates of >90%. However, radiation therapy causes significant late effects and avoiding radiation entirely for patients who respond to chemotherapy is an accepted strategy. Since 2011, 28 consecutive patients diagnosed with classic HL have been treated with doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) for 4 to 6 cycles. Patients who achieved a complete metabolic response (CMR) as assessed by [F] fluorodeoxyglucose positron emission tomography by the end of chemotherapy did not receive radiation. Among the 27 evaluable patients, 26/27 (96.2%) achieved a CMR with ABVD alone with 24/27 (88.9%) having achieved a CMR after 2 cycles. Event-free survival at 5 years is 90.5% and overall survival is 100% with a median follow-up time of 22.4 and 22.1 months, respectively. Treating pediatric and young adult HL patients with ABVD alone results in CMRs in >95% of patients. Patients who were refractory to ABVD or relapsed after treatment eventually achieved remission with a combination of standard and novel salvage therapies. This regimen demonstrates the feasibility of avoiding upfront radiation in newly diagnosed pediatric HL patients.
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http://dx.doi.org/10.1097/MPH.0000000000001094DOI Listing
May 2018

Genome-wide DNA methylation is predictive of outcome in juvenile myelomonocytic leukemia.

Nat Commun 2017 12 19;8(1):2127. Epub 2017 Dec 19.

Department of Pediatrics, Benioff Children's Hospital, University of California, San Francisco, 1450 3rd Street, San Francisco, CA, 94158, USA.

Juvenile myelomonocytic leukemia (JMML) is a myeloproliferative disorder of childhood caused by mutations in the Ras pathway. Outcomes in JMML vary markedly from spontaneous resolution to rapid relapse after hematopoietic stem cell transplantation. Here, we hypothesized that DNA methylation patterns would help predict disease outcome and therefore performed genome-wide DNA methylation profiling in a cohort of 39 patients. Unsupervised hierarchical clustering identifies three clusters of patients. Importantly, these clusters differ significantly in terms of 4-year event-free survival, with the lowest methylation cluster having the highest rates of survival. These findings were validated in an independent cohort of 40 patients. Notably, all but one of 14 patients experiencing spontaneous resolution cluster together and closer to 22 healthy controls than to other JMML cases. Thus, we show that DNA methylation patterns in JMML are predictive of outcome and can identify the patients most likely to experience spontaneous resolution.
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http://dx.doi.org/10.1038/s41467-017-02178-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736624PMC
December 2017

Robust patient-derived xenografts of MDS/MPN overlap syndromes capture the unique characteristics of CMML and JMML.

Blood 2017 07 2;130(4):397-407. Epub 2017 Jun 2.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY.

Chronic myelomonocytic leukemia (CMML) and juvenile myelomonocytic leukemia (JMML) are myelodysplastic syndrome (MDS)/myeloproliferative neoplasm (MPN) overlap disorders characterized by monocytosis, myelodysplasia, and a characteristic hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF). Currently, there are no available disease-modifying therapies for CMML, nor are there preclinical models that fully recapitulate the unique features of CMML. Through use of immunocompromised mice with transgenic expression of human GM-CSF, interleukin-3, and stem cell factor in a NOD/SCID-IL2Rγ background (NSGS mice), we demonstrate remarkable engraftment of CMML and JMML providing the first examples of serially transplantable and genetically accurate models of CMML. Xenotransplantation of CD34 cells (n = 8 patients) or unfractionated bone marrow (BM) or peripheral blood mononuclear cells (n = 10) resulted in robust engraftment of CMML in BM, spleen, liver, and lung of recipients (n = 82 total mice). Engrafted cells were myeloid-restricted and matched the immunophenotype, morphology, and genetic mutations of the corresponding patient. Similar levels of engraftment were seen upon serial transplantation of human CD34 cells in secondary NSGS recipients (2/5 patients, 6/11 mice), demonstrating the durability of CMML grafts and functionally validating CD34 cells as harboring the disease-initiating compartment in vivo. Successful engraftments of JMML primary samples were also achieved in all NSGS recipients (n = 4 patients, n = 12 mice). Engraftment of CMML and JMML resulted in overt phenotypic abnormalities and lethality in recipients, which facilitated evaluation of the JAK2/FLT3 inhibitor pacritinib in vivo. These data reveal that NSGS mice support the development of CMML and JMML disease-initiating and mature leukemic cells in vivo, allowing creation of genetically accurate preclinical models of these disorders.
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http://dx.doi.org/10.1182/blood-2017-01-763219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5533204PMC
July 2017
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