Publications by authors named "Sivasish Sindiri"

26 Publications

  • Page 1 of 1

Immuno-transcriptomic profiling of extracranial pediatric solid malignancies.

Cell Rep 2021 Nov;37(8):110047

University of Toronto Musculoskeletal Oncology Unit, Sinai Health System; Department of Surgery, University of Toronto, Toronto, ON, Canada.

We perform an immunogenomics analysis utilizing whole-transcriptome sequencing of 657 pediatric extracranial solid cancer samples representing 14 diagnoses, and additionally utilize transcriptomes of 131 pediatric cancer cell lines and 147 normal tissue samples for comparison. We describe patterns of infiltrating immune cells, T cell receptor (TCR) clonal expansion, and translationally relevant immune checkpoints. We find that tumor-infiltrating lymphocytes and TCR counts vary widely across cancer types and within each diagnosis, and notably are significantly predictive of survival in osteosarcoma patients. We identify potential cancer-specific immunotherapeutic targets for adoptive cell therapies including cell-surface proteins, tumor germline antigens, and lineage-specific transcription factors. Using an orthogonal immunopeptidomics approach, we find several potential immunotherapeutic targets in osteosarcoma and Ewing sarcoma and validated PRAME as a bona fide multi-pediatric cancer target. Importantly, this work provides a critical framework for immune targeting of extracranial solid tumors using parallel immuno-transcriptomic and -peptidomic approaches.
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http://dx.doi.org/10.1016/j.celrep.2021.110047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8642810PMC
November 2021

Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium.

J Clin Oncol 2021 09 24;39(26):2859-2871. Epub 2021 Jun 24.

Department of Pediatrics, Seattle Children's Hospital, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA.

Purpose: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome.

Patients And Methods: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed.

Results: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. (15%), (15%), and (13%) mutations were found at a higher incidence than previously reported and mutations were associated with worse outcomes in both fusion-negative and fusion-positive cases. Interestingly, mutations in isoforms predominated in infants < 1 year (64% of cases). Mutation of was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data.

Conclusion: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.
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http://dx.doi.org/10.1200/JCO.20.03060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425837PMC
September 2021

Notch signaling and efficacy of PD-1/PD-L1 blockade in relapsed small cell lung cancer.

Nat Commun 2021 06 23;12(1):3880. Epub 2021 Jun 23.

Developmental Therapeutics Branch, Center for Cancer Research, NCI, NIH, Bethesda, MD, USA.

Immune checkpoint blockade (ICB) benefits only a small subset of patients with small cell lung cancer (SCLC), yet the mechanisms driving benefit are poorly understood. To identify predictors of clinical benefit to ICB, we performed immunogenomic profiling of tumor samples from patients with relapsed SCLC. Tumors of patients who derive clinical benefit from ICB exhibit cytotoxic T-cell infiltration, high expression of antigen processing and presentation machinery (APM) genes, and low neuroendocrine (NE) differentiation. However, elevated Notch signaling, which positively correlates with low NE differentiation, most significantly predicts clinical benefit to ICB. Activation of Notch signaling in a NE human SCLC cell line induces a low NE phenotype, marked by increased expression of APM genes, demonstrating a mechanistic link between Notch activation, low NE differentiation and increased intrinsic tumor immunity. Our findings suggest Notch signaling as a determinant of response to ICB in SCLC.
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http://dx.doi.org/10.1038/s41467-021-24164-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222224PMC
June 2021

Clonal Evolution and Heterogeneity of Osimertinib Acquired Resistance Mechanisms in EGFR Mutant Lung Cancer.

Cell Rep Med 2020 Apr;1(1)

Genetics Branch, CCR, NCI, NIH, Bethesda, MD 20892, USA.

Clonal evolution of osimertinib-resistance mechanisms in EGFR mutant lung adenocarcinoma is poorly understood. Using multi-region whole-exome and RNA sequencing of prospectively collected pre- and post-osimertinib-resistant tumors, including at rapid autopsies, we identify a likely mechanism driving osimertinib resistance in all patients analyzed. The majority of patients acquire two or more resistance mechanisms either concurrently or in temporal sequence. Focal copy-number amplifications occur subclonally and are spatially and temporally separated from common resistance mutations such as C797S. amplification occurs in 66% (n = 6/9) of first-line osimertinib-treated patients, albeit spatially heterogeneous, often co-occurs with additional acquired focal copy-number amplifications and is associated with early progression. Noteworthy osimertinib-resistance mechanisms discovered include neuroendocrine differentiation without histologic transformation, amplification, and fusions. The subclonal co-occurrence of acquired genomic alterations upon osimertinib resistance will likely require targeting multiple resistance mechanisms by combination therapies.
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http://dx.doi.org/10.1016/j.xcrm.2020.100007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263628PMC
April 2020

Outcome-Related Signatures Identified by Whole Transcriptome Sequencing of Resectable Stage III/IV Melanoma Evaluated after Starting Hu14.18-IL2.

Clin Cancer Res 2020 07 9;26(13):3296-3306. Epub 2020 Mar 9.

Department of Human Oncology, University of Wisconsin-Madison, Madison, Wisconsin.

Purpose: We analyzed whole transcriptome sequencing in tumors from 23 patients with stage III or IV melanoma from a pilot trial of the anti-GD2 immunocytokine, hu14.18-IL2, to identify predictive immune and/or tumor biomarkers in patients with melanoma at high risk for recurrence.

Experimental Design: Patients were randomly assigned to receive the first of three monthly courses of hu14.18-IL2 immunotherapy either before (Group A) or after (Group B) complete surgical resection of all known diseases. Tumors were evaluated by histology and whole transcriptome sequencing.

Results: Tumor-infiltrating lymphocyte (TIL) levels directly associated with relapse-free survival (RFS) and overall survival (OS) in resected tumors from Group A, where early responses to the immunotherapy agent could be assessed. TIL levels directly associated with a previously reported immune signature, which associated with RFS and OS, particularly in Group A tumors. In Group A tumors, there were decreased cell-cycling gene RNA transcripts, but increased RNA transcripts for repair and growth genes. We found that outcome (RFS and OS) was directly associated with several immune signatures and immune-related RNA transcripts and inversely associated with several tumor growth-associated transcripts, particularly in Group A tumors. Most of these associations were not seen in Group B tumors.

Conclusions: We interpret these data to signify that both immunologic and tumoral cell processes, as measured by RNA-sequencing analyses detected shortly after initiation of hu14.18-IL2 therapy, are associated with long-term survival and could potentially be used as prognostic biomarkers in tumor resection specimens obtained after initiating neoadjuvant immunotherapy.
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http://dx.doi.org/10.1158/1078-0432.CCR-19-3294DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7334053PMC
July 2020

Insights into pediatric rhabdomyosarcoma research: Challenges and goals.

Pediatr Blood Cancer 2019 10 21;66(10):e27869. Epub 2019 Jun 21.

Fred Hutchinson Cancer Research Center, Seattle, Washington.

Overall survival rates for pediatric patients with high-risk or relapsed rhabdomyosarcoma (RMS) have not improved significantly since the 1980s. Recent studies have identified a number of targetable vulnerabilities in RMS, but these discoveries have infrequently translated into clinical trials. We propose streamlining the process by which agents are selected for clinical evaluation in RMS. We believe that strong consideration should be given to the development of combination therapies that add biologically targeted agents to conventional cytotoxic drugs. One example of this type of combination is the addition of the WEE1 inhibitor AZD1775 to the conventional cytotoxic chemotherapeutics, vincristine and irinotecan.
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http://dx.doi.org/10.1002/pbc.27869DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6707829PMC
October 2019

APOBEC Mutagenesis and Copy-Number Alterations Are Drivers of Proteogenomic Tumor Evolution and Heterogeneity in Metastatic Thoracic Tumors.

Cell Rep 2019 03;26(10):2651-2666.e6

Thoracic and GI Malignancies Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20814, USA. Electronic address:

Intratumor mutational heterogeneity has been documented in primary non-small-cell lung cancer. Here, we elucidate mechanisms of tumor evolution and heterogeneity in metastatic thoracic tumors (lung adenocarcinoma and thymic carcinoma) using whole-exome and transcriptome sequencing, SNP array for copy-number alterations (CNAs), and mass-spectrometry-based quantitative proteomics of metastases obtained by rapid autopsy. APOBEC mutagenesis, promoted by increased expression of APOBEC3 region transcripts and associated with a high-risk APOBEC3 germline variant, correlated with mutational tumor heterogeneity. TP53 mutation status was associated with APOBEC hypermutator status. Interferon pathways were enriched in tumors with high APOBEC mutagenesis and IFN-γ-induced expression of APOBEC3B in lung adenocarcinoma cells, suggesting that the immune microenvironment may promote mutational heterogeneity. CNAs occurring late in tumor evolution correlated with downstream transcriptomic and proteomic heterogeneity, although global proteomic heterogeneity was significantly greater than transcriptomic and CNA heterogeneity. These results illustrate key mechanisms underlying multi-dimensional heterogeneity in metastatic thoracic tumors.
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http://dx.doi.org/10.1016/j.celrep.2019.02.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461561PMC
March 2019

Low mutation burden and frequent loss of CDKN2A/B and SMARCA2, but not PRC2, define premalignant neurofibromatosis type 1-associated atypical neurofibromas.

Neuro Oncol 2019 08;21(8):981-992

Clinical Genetics Branch, DCEG, NCI, National Institutes of Health (NIH), Rockville, Maryland, USA.

Background: Neurofibromatosis type 1 (NF1) is a tumor-predisposition disorder caused by germline mutations in NF1. NF1 patients have an 8-16% lifetime risk of developing a malignant peripheral nerve sheath tumor (MPNST), a highly aggressive soft-tissue sarcoma, often arising from preexisting benign plexiform neurofibromas (PNs) and atypical neurofibromas (ANFs). ANFs are distinct from both PN and MPNST, representing an intermediate step in malignant transformation.

Methods: In the first comprehensive genomic analysis of ANF originating from multiple patients, we performed tumor/normal whole-exome sequencing (WES) of 16 ANFs. In addition, we conducted WES of 3 MPNSTs, copy-number meta-analysis of 26 ANFs and 28 MPNSTs, and whole transcriptome sequencing analysis of 5 ANFs and 5 MPNSTs.

Results: We identified a low number of mutations (median 1, range 0-5) in the exomes of ANFs (only NF1 somatic mutations were recurrent), and frequent deletions of CDKN2A/B (69%) and SMARCA2 (42%). We determined that polycomb repressor complex 2 (PRC2) genes EED and SUZ12 were frequently mutated, deleted, or downregulated in MPNSTs but not in ANFs. Our pilot gene expression study revealed upregulated NRAS, MDM2, CCND1/2/3, and CDK4/6 in ANFs and MPNSTs, and overexpression of EZH2 in MPNSTs only.

Conclusions: The PN-ANF transition is primarily driven by the deletion of CDKN2A/B. Further progression from ANF to MPNST likely involves broad chromosomal rearrangements and frequent inactivation of the PRC2 genes, loss of the DNA repair genes, and copy-number increase of signal transduction and cell-cycle and pluripotency self-renewal genes.
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http://dx.doi.org/10.1093/neuonc/noz028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682216PMC
August 2019

Pregnancy-Associated Plasma Protein-A (PAPP-A) in Ewing Sarcoma: Role in Tumor Growth and Immune Evasion.

J Natl Cancer Inst 2019 09;111(9):970-982

Background: Ewing sarcoma (EWS) manifests one of the lowest somatic mutation rates of any cancer, leading to a scarcity of druggable mutations and neoantigens. Immunotherapeutics targeting differentially expressed cell surface antigens could provide therapeutic benefit for such tumors. Pregnancy-associated plasma protein A (PAPP-A) is a cell membrane-associated proteinase produced by the placenta that promotes fetal growth by inducing insulinlike growth factor (IGF) signaling.

Methods: By comparing RNA expression of cell surface proteins in EWS (n = 120) versus normal tissues (n = 42), we comprehensively characterized the surfaceome of EWS to identify highly differentially expressed molecules. Using CRISPR/Cas-9 and anti-PAPP-A antibodies, we investigated biological roles for PAPP-A in EWS in vitro and in vivo in NSG xenograft models and performed RNA-sequencing on PAPPA knockout clones (n = 5) and controls (n = 3). All statistical tests were two-sided.

Results: EWS surfaceome analysis identified 11 highly differentially overexpressed genes, with PAPPA ranking second in differential expression. In EWS cell lines, genetic knockout of PAPPA and treatment with anti-PAPP-A antibodies revealed an essential survival role by regulating local IGF-1 bioavailability. MAb-mediated PAPPA inhibition diminished EWS growth in orthotopic xenografts (leg area mm2 at day 49 IgG2a control (CTRL) [n = 14], mean = 397.0, SD = 86.1 vs anti-PAPP-A [n = 14], mean = 311.7, SD = 155.0; P = .03; median OS anti-PAPP-A = 52.5 days, 95% CI = 46.0 to 63.0 days vs IgG2a = 45.0 days, 95% CI = 42.0 to 52.0 days; P = .02) and improved the efficacy of anti-IGF-1R treatment (leg area mm2 at day 49 anti-PAPP-A + anti-IGF-1R [n = 15], mean = 217.9, SD = 148.5 vs IgG2a-CTRL; P < .001; median OS anti-PAPP-A + anti-IGF1R = 63.0 days, 95% CI = 52.0 to 67.0 days vs IgG2a-CTRL; P < .001). Unexpectedly, PAPPA knockout in EWS cell lines induced interferon (IFN)-response genes, including proteins associated with antigen processing/presentation. Consistently, gene expression profiles in PAPPA-low EWS tumors were enriched for immune response pathways.

Conclusion: This work provides a comprehensive characterization of the surfaceome of EWS, credentials PAPP-A as a highly differentially expressed therapeutic target, and discovers a novel link between IGF-1 signaling and immune evasion in cancer, thus implicating shared mechanisms of immune evasion between EWS and the placenta.
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http://dx.doi.org/10.1093/jnci/djy209DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6748813PMC
September 2019

MEK inhibition induces MYOG and remodels super-enhancers in RAS-driven rhabdomyosarcoma.

Sci Transl Med 2018 07;10(448)

Oncogenomics Section, Genetics Branch, National Cancer Institute, National Institutes of Health (NIH), Bethesda, MD 20892, USA.

The RAS isoforms are frequently mutated in many types of human cancers, including PAX3/PAX7 fusion-negative rhabdomyosarcoma. Pediatric RMS arises from skeletal muscle progenitor cells that have failed to differentiate normally. The role of mutant RAS in this differentiation blockade is incompletely understood. We demonstrate that oncogenic RAS, acting through the RAF-MEK [mitogen-activated protein kinase (MAPK) kinase]-ERK (extracellular signal-regulated kinase) MAPK effector pathway, inhibits myogenic differentiation in rhabdomyosarcoma by repressing the expression of the prodifferentiation myogenic transcription factor, MYOG. This repression is mediated by ERK2-dependent promoter-proximal stalling of RNA polymerase II at the locus. Small-molecule screening with a library of mechanistically defined inhibitors showed that RAS-driven RMS is vulnerable to MEK inhibition. MEK inhibition with trametinib leads to the loss of ERK2 at the promoter and releases the transcriptional stalling of expression. MYOG subsequently opens chromatin and establishes super-enhancers at genes required for late myogenic differentiation. Furthermore, trametinib, in combination with an inhibitor of IGF1R, potently decreases rhabdomyosarcoma cell viability and slows tumor growth in xenograft models. Therefore, this combination represents a potential therapeutic for RAS-mutated rhabdomyosarcoma.
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http://dx.doi.org/10.1126/scitranslmed.aan4470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054766PMC
July 2018

Clinically Relevant Cytotoxic Immune Cell Signatures and Clonal Expansion of T-Cell Receptors in High-Risk -Not-Amplified Human Neuroblastoma.

Clin Cancer Res 2018 11 21;24(22):5673-5684. Epub 2018 May 21.

Oncogenomics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland.

High-risk neuroblastoma is an aggressive disease. DNA sequencing studies have revealed a paucity of actionable genomic alterations and a low mutation burden, posing challenges to develop effective novel therapies. We used RNA sequencing (RNA-seq) to investigate the biology of this disease, including a focus on tumor-infiltrating lymphocytes (TIL). We performed deep RNA-seq on pretreatment diagnostic tumors from 129 high-risk and 21 low- or intermediate-risk patients with neuroblastomas. We used single-sample gene set enrichment analysis to detect gene expression signatures of TILs in tumors and examined their association with clinical and molecular parameters, including patient outcome. The expression profiles of 190 additional pretreatment diagnostic neuroblastomas, a neuroblastoma tissue microarray, and T-cell receptor (TCR) sequencing were used to validate our findings. We found that -not-amplified (-NA) tumors had significantly higher cytotoxic TIL signatures compared with -amplified (-A) tumors. A reported MYCN activation signature was significantly associated with poor outcome for high-risk patients with -NA tumors; however, a subgroup of these patients who had elevated activated natural killer (NK) cells, CD8 T cells, and cytolytic signatures showed improved outcome and expansion of infiltrating TCR clones. Furthermore, we observed upregulation of immune exhaustion marker genes, indicating an immune-suppressive microenvironment in these neuroblastomas. This study provides evidence that RNA signatures of cytotoxic TIL are associated with the presence of activated NK/T cells and improved outcomes in high-risk neuroblastoma patients harboring -NA tumors. Our findings suggest that these high-risk patients with -NA neuroblastoma may benefit from additional immunotherapies incorporated into the current therapeutic strategies. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6504934PMC
November 2018

Vangl2/RhoA Signaling Pathway Regulates Stem Cell Self-Renewal Programs and Growth in Rhabdomyosarcoma.

Cell Stem Cell 2018 03;22(3):414-427.e6

Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts General Hospital Research Institute, Boston, MA 02129, USA; Harvard Stem Cell Institute, Cambridge, MA 02139, USA. Electronic address:

Tumor growth and relapse are driven by tumor propagating cells (TPCs). However, mechanisms regulating TPC fate choices, maintenance, and self-renewal are not fully understood. Here, we show that Van Gogh-like 2 (Vangl2), a core regulator of the non-canonical Wnt/planar cell polarity (Wnt/PCP) pathway, affects TPC self-renewal in rhabdomyosarcoma (RMS)-a pediatric cancer of muscle. VANGL2 is expressed in a majority of human RMS and within early mononuclear progenitor cells. VANGL2 depletion inhibited cell proliferation, reduced TPC numbers, and induced differentiation of human RMS in vitro and in mouse xenografts. Using a zebrafish model of embryonal rhabdomyosarcoma (ERMS), we determined that Vangl2 expression enriches for TPCs and promotes their self-renewal. Expression of constitutively active and dominant-negative isoforms of RHOA revealed that it acts downstream of VANGL2 to regulate proliferation and maintenance of TPCs in human RMS. Our studies offer insights into pathways that control TPCs and identify new potential therapeutic targets.
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http://dx.doi.org/10.1016/j.stem.2018.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6354590PMC
March 2018

Functional screening of FGFR4-driven tumorigenesis identifies PI3K/mTOR inhibition as a therapeutic strategy in rhabdomyosarcoma.

Oncogene 2018 05 28;37(20):2630-2644. Epub 2018 Feb 28.

Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and outcomes have stagnated, highlighting a need for novel therapies. Genomic analysis of RMS has revealed that alterations in the receptor tyrosine kinase (RTK)/RAS/PI3K axis are common and that FGFR4 is frequently mutated or overexpressed. Although FGFR4 is a potentially druggable receptor tyrosine kinase, its functions in RMS are undefined. This study tested FGFR4-activating mutations and overexpression for the ability to generate RMS in mice. Murine tumor models were subsequently used to discover potential therapeutic targets and to test a dual PI3K/mTOR inhibitor in a preclinical setting. Specifically, we provide the first mechanistic evidence of differential potency in the most common human RMS mutations, V550E or N535K, compared to FGFR4 overexpression as murine myoblasts expressing FGFR4 undergo higher rates of cellular transformation, engraftment into mice, and rapidly form sarcomas that highly resemble human RMS. Murine tumor cells overexpressing FGFR4 were tested in an in vitro dose-response drug screen along with human RMS cell lines. Compounds were grouped by target class, and potency was determined using average percentage of area under the dose-response curve (AUC). RMS cells were highly sensitive to PI3K/mTOR inhibitors, in particular, GSK2126458 (omipalisib) was a potent inhibitor of FGFR4 tumor-derived cell and human RMS cell viability. FGFR4-overexpressing myoblasts and tumor cells had low nanomolar GSK2126458 EC values. Mass cytometry using mouse and human RMS cell lines validated GSK2126458 specificity at single-cell resolution, decreasing the abundance of phosphorylated Akt as well as decreasing phosphorylation of the downstream mTOR effectors 4ebp1, Eif4e, and S6. Moreover, PI3K/mTOR inhibition also robustly decreased the growth of RMS tumors in vivo. Thus, by developing a preclinical platform for testing novel therapies, we identified PI3K/mTOR inhibition as a promising new therapy for this devastating pediatric cancer.
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http://dx.doi.org/10.1038/s41388-017-0122-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054765PMC
May 2018

A mutational comparison of adult and adolescent and young adult (AYA) colon cancer.

Cancer 2018 03 30;124(5):1070-1082. Epub 2017 Nov 30.

Center for Cancer Research, National Cancer Institute, Bethesda, Maryland.

Background: It is possible that the relative lack of progress in treatment outcomes among adolescent and young adult (AYA) patients with cancer is caused by a difference in disease biology compared with the corresponding diseases in younger and older individuals. There is evidence that colon cancer is more aggressive and has a poorer prognosis in AYA patients than in older adult patients.

Methods: To further understand the molecular basis for this difference, whole-exome sequencing was conducted on a cohort of 30 adult, 30 AYA, and 2 pediatric colon cancers.

Results: A statistically significant difference in mutational frequency was observed between AYA and adult samples in 43 genes, including ROBO1, MYC binding protein 2 (MYCBP2), breast cancer 2 (early onset) (BRCA2), MAP3K3, MCPH1, RASGRP3, PTCH1, RAD9B, CTNND1, ATM, NF1; KIT, PTEN, and FBXW7. Many of these mutations were nonsynonymous, missense, stop-gain, or frameshift mutations that were damaging. Next, RNA sequencing was performed on a subset of the samples to confirm the mutations identified by exome sequencing. This confirmation study verified the presence of a significantly greater frequency of damaging mutations in AYA compared with adult colon cancers for 5 of the 43 genes (MYCBP2, BRCA2, PHLPP1, TOPORS, and ATR).

Conclusions: The current results provide the rationale for a more comprehensive study with a larger sample set and experimental validation of the functional impact of the identified variants along with their contribution to the biologic and clinical characteristics of AYA colon cancer. Cancer 2018;124:1070-82. © 2017 American Cancer Society.
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http://dx.doi.org/10.1002/cncr.31136DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5821537PMC
March 2018

Paired Expression Analysis of Tumor Cell Surface Antigens.

Front Oncol 2017 21;7:173. Epub 2017 Aug 21.

Genetics Branch, National Cancer Institute, Center for Cancer Research, National Institutes of Health, Bethesda, MD, United States.

Adoptive immunotherapy with antibody-based therapy or with T cells transduced to express chimeric antigen receptors (CARs) is useful to the extent that the cell surface membrane protein being targeted is not expressed on normal tissues. The most successful CAR-based (anti-CD19) or antibody-based therapy (anti-CD20) in hematologic malignancies has the side effect of eliminating the normal B cell compartment. Targeting solid tumors may not provide a similar expendable marker. Beyond antibody to Her2/NEU and EGFR, very few antibody-based and no CAR-based therapies have seen broad clinical application for solid tumors. To expand the way in which the surfaceome of solid tumors can be analyzed, we created an algorithm that defines the pairwise relative overexpression of surface antigens. This enables the development of specific immunotherapies that require the expression of two discrete antigens on the surface of the tumor target. This dyad analysis was facilitated by employing the Hotelling's -squared test (Hotelling-Lawley multivariate analysis of variance) for two independent variables in comparison to a third constant entity (i.e., gene expression levels in normal tissues). We also present a unique consensus scoring mechanism for identifying transcripts that encode cell surface proteins. The unique application of our bioinformatics processing pipeline and statistical tools allowed us to compare the expression of two membrane protein targets as a pair, and to propose a new strategy based on implementing immunotherapies that require both antigens to be expressed on the tumor cell surface to trigger therapeutic effector mechanisms. Specifically, we found that, for MYCN amplified neuroblastoma, pairwise expression of ACVR2B or anaplastic lymphoma kinase (ALK) with GFRA3, GFRA2, Cadherin 24, or with one another provided the strongest hits. For MYCN, non-amplified stage 4 neuroblastoma, neurotrophic tyrosine kinase 1, or ALK paired with GFRA2, GFRA3, SSK1, GPR173, or with one another provided the most promising paired-hits. We propose that targeting these markers together would increase the specificity and thereby the safety of CAR-based therapy for neuroblastoma.
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http://dx.doi.org/10.3389/fonc.2017.00173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5566986PMC
August 2017

The NOTCH1/SNAIL1/MEF2C Pathway Regulates Growth and Self-Renewal in Embryonal Rhabdomyosarcoma.

Cell Rep 2017 06;19(11):2304-2318

Department of Pathology, Massachusetts General Hospital, Boston, MA 02114, USA; Center of Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA; Harvard Stem Cell Institute, Boston, MA 02114, USA. Electronic address:

Tumor-propagating cells (TPCs) share self-renewal properties with normal stem cells and drive continued tumor growth. However, mechanisms regulating TPC self-renewal are largely unknown, especially in embryonal rhabdomyosarcoma (ERMS)-a common pediatric cancer of muscle. Here, we used a zebrafish transgenic model of ERMS to identify a role for intracellular NOTCH1 (ICN1) in increasing TPCs by 23-fold. ICN1 expanded TPCs by enabling the de-differentiation of zebrafish ERMS cells into self-renewing myf5+ TPCs, breaking the rigid differentiation hierarchies reported in normal muscle. ICN1 also had conserved roles in regulating human ERMS self-renewal and growth. Mechanistically, ICN1 upregulated expression of SNAIL1, a transcriptional repressor, to increase TPC number in human ERMS and to block muscle differentiation through suppressing MEF2C, a myogenic differentiation transcription factor. Our data implicate the NOTCH1/SNAI1/MEF2C signaling axis as a major determinant of TPC self-renewal and differentiation in ERMS, raising hope of therapeutically targeting this pathway in the future.
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http://dx.doi.org/10.1016/j.celrep.2017.05.061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563075PMC
June 2017

PAX3-FOXO1 Establishes Myogenic Super Enhancers and Confers BET Bromodomain Vulnerability.

Cancer Discov 2017 08 26;7(8):884-899. Epub 2017 Apr 26.

Genetics Branch, NCI, NIH, Bethesda, Maryland.

Alveolar rhabdomyosarcoma is a life-threatening myogenic cancer of children and adolescent young adults, driven primarily by the chimeric transcription factor PAX3-FOXO1. The mechanisms by which PAX3-FOXO1 dysregulates chromatin are unknown. We find PAX3-FOXO1 reprograms the -regulatory landscape by inducing super enhancers. PAX3-FOXO1 uses super enhancers to set up autoregulatory loops in collaboration with the master transcription factors MYOG, MYOD, and MYCN. This myogenic super enhancer circuitry is consistent across cell lines and primary tumors. Cells harboring the fusion gene are selectively sensitive to small-molecule inhibition of protein targets induced by, or bound to, PAX3-FOXO1-occupied super enhancers. Furthermore, PAX3-FOXO1 recruits and requires the BET bromodomain protein BRD4 to function at super enhancers, resulting in a complete dependence on BRD4 and a significant susceptibility to BRD inhibition. These results yield insights into the epigenetic functions of PAX3-FOXO1 and reveal a specific vulnerability that can be exploited for precision therapy. PAX3-FOXO1 drives pediatric fusion-positive rhabdomyosarcoma, and its chromatin-level functions are critical to understanding its oncogenic activity. We find that PAX3-FOXO1 establishes a myoblastic super enhancer landscape and creates a profound subtype-unique dependence on BET bromodomains, the inhibition of which ablates PAX3-FOXO1 function, providing a mechanistic rationale for exploring BET inhibitors for patients bearing PAX-fusion rhabdomyosarcoma. .
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http://dx.doi.org/10.1158/2159-8290.CD-16-1297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802885PMC
August 2017

Algorithm-driven high-throughput screening of colloidal nanoparticles under simulated physiological and therapeutic conditions.

Nanoscale 2017 Feb;9(6):2291-2300

Laboratory of Biological Chemistry, Division of Biotechnology Review and Research IV, Center for Drug Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.

Colloidal nanoparticles have shown tremendous potential as cancer drug carriers and as phototherapeutics. However, the stability of nanoparticles under physiological and phototherapeutic conditions is a daunting issue, which needs to be addressed in order to ensure a successful clinical translation. The design, development and implementation of unique algorithms are described herein for high-throughput hydrodynamic size measurements of colloidal nanoparticles. The data obtained from such measurements provide clinically-relevant particle size distribution assessments that are directly related to the stability and aggregation profiles of the nanoparticles under putative physiological and phototherapeutic conditions; those profiles are not only dependent on the size and surface coating of the nanoparticles, but also on their composition. Uncoated nanoparticles showed varying degrees of association with bovine serum albumin, whereas PEGylated nanoparticles did not exhibit significant association with the protein. The algorithm-driven, high-throughput size screening method described in this report provides highly meaningful size measurement patterns stemming from the association of colloidal particles with bovine serum albumin used as a protein model. Noteworthy is that this algorithm-based high-throughput method can accomplish sophisticated hydrodynamic size measurement protocols within days instead of years it would take conventional hydrodynamic size measurement techniques to achieve a similar task.
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http://dx.doi.org/10.1039/c6nr08579bDOI Listing
February 2017

Myogenic regulatory transcription factors regulate growth in rhabdomyosarcoma.

Elife 2017 01 12;6. Epub 2017 Jan 12.

Molecular Pathology, Cancer Center, and Regenerative Medicine, Massachusetts General Hospital, Boston, United States.

Rhabdomyosarcoma (RMS) is a pediatric malignacy of muscle with myogenic regulatory transcription factors MYOD and MYF5 being expressed in this disease. Consensus in the field has been that expression of these factors likely reflects the target cell of transformation rather than being required for continued tumor growth. Here, we used a transgenic zebrafish model to show that Myf5 is sufficient to confer tumor-propagating potential to RMS cells and caused tumors to initiate earlier and have higher penetrance. Analysis of human RMS revealed that MYF5 and MYOD are mutually-exclusively expressed and each is required for sustained tumor growth. ChIP-seq and mechanistic studies in human RMS uncovered that MYF5 and MYOD bind common DNA regulatory elements to alter transcription of genes that regulate muscle development and cell cycle progression. Our data support unappreciated and dominant oncogenic roles for MYF5 and MYOD convergence on common transcriptional targets to regulate human RMS growth.
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http://dx.doi.org/10.7554/eLife.19214DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5231408PMC
January 2017

MultiDimensional ClinOmics for Precision Therapy of Children and Adolescent Young Adults with Relapsed and Refractory Cancer: A Report from the Center for Cancer Research.

Clin Cancer Res 2016 08 18;22(15):3810-20. Epub 2016 Mar 18.

Oncogenomics Section, Genetics Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland.

Purpose: We undertook a multidimensional clinical genomics study of children and adolescent young adults with relapsed and refractory cancers to determine the feasibility of genome-guided precision therapy.

Experimental Design: Patients with non-central nervous system solid tumors underwent a combination of whole exome sequencing (WES), whole transcriptome sequencing (WTS), and high-density single-nucleotide polymorphism array analysis of the tumor, with WES of matched germline DNA. Clinically actionable alterations were identified as a reportable germline mutation, a diagnosis change, or a somatic event (including a single nucleotide variant, an indel, an amplification, a deletion, or a fusion gene), which could be targeted with drugs in existing clinical trials or with FDA-approved drugs.

Results: Fifty-nine patients in 20 diagnostic categories were enrolled from 2010 to 2014. Ages ranged from 7 months to 25 years old. Seventy-three percent of the patients had prior chemotherapy, and the tumors from these patients with relapsed or refractory cancers had a higher mutational burden than that reported in the literature. Thirty patients (51% of total) had clinically actionable mutations, of which 24 (41%) had a mutation that was currently targetable in a clinical trial setting, 4 patients (7%) had a change in diagnosis, and 7 patients (12%) had a reportable germline mutation.

Conclusions: We found a remarkably high number of clinically actionable mutations in 51% of the patients, and 12% with significant germline mutations. We demonstrated the clinical feasibility of next-generation sequencing in a diverse population of relapsed and refractory pediatric solid tumors. Clin Cancer Res; 22(15); 3810-20. ©2016 AACR.
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http://dx.doi.org/10.1158/1078-0432.CCR-15-2717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4970946PMC
August 2016

Recurrent inactivating RASA2 mutations in melanoma.

Nat Genet 2015 Dec 26;47(12):1408-10. Epub 2015 Oct 26.

Molecular Cell Biology Department, Weizmann Institute of Science, Rehovot, Israel.

Analysis of 501 melanoma exomes identified RASA2, encoding a RasGAP, as a tumor-suppressor gene mutated in 5% of melanomas. Recurrent loss-of-function mutations in RASA2 were found to increase RAS activation, melanoma cell growth and migration. RASA2 expression was lost in ≥30% of human melanomas and was associated with reduced patient survival. These findings identify RASA2 inactivation as a melanoma driver and highlight the importance of RasGAPs in cancer.
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http://dx.doi.org/10.1038/ng.3427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4954601PMC
December 2015

Germline CARD11 Mutation in a Patient with Severe Congenital B Cell Lymphocytosis.

J Clin Immunol 2015 Jan 29;35(1):32-46. Epub 2014 Oct 29.

Department of Pharmacology, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Rd., Bethesda, MD, 20814, USA.

Purpose: Activating germline mutations in CARD11 have recently been linked to a rare genetic disorder associated with congenital B cell lymphocytosis. We describe a patient with a similar clinical phenotype who had a de novo germline G123D CARD11 mutation.

Methods: Whole exome sequencing was performed on DNA from the patient and his biological parents. Laboratory studies examined characteristics of the patient's B and T lymphocytes. A CARD11 cDNA containing the mutation was transfected into a lymphocyte cell line to gain an understanding of its function. RNA sequencing was performed on samples from the patient and from patients with alternate germline CARD11 mutations and differential gene expression analysis was performed.

Results: The patient had a decade-long history of severe polyclonal B lymphocytosis in the 20,000-90,000 lymphocytes/mm(3) range, which was markedly exacerbated by EBV infection and splenectomy at different times. He had a heterozygous germline CARD11 mutation causing a G123D amino acid substitution, which was demonstrated to induce NF-κB activation in unstimulated lymphocytes. In contrast to previous patients with CARD11 mutations, this patient's B cells exhibited higher expression of several cell cycle progression genes, as well as enhanced proliferation and improved survival following B cell receptor stimulation.

Conclusions: This is the third reported germline and first de novo CARD11 mutation shown to cause congenital B cell lymphocytosis. The mutation was associated with a dramatically greater lymphocytosis than in previously described cases, disproportionate to the level of constitutive NF-κB activation. However, comparative review of the patient's clinical history, combined with additional genomic and functional analyses, underscore other important variables that may affect pathophysiology or regulate mutant CARD11 function in B cell proliferation and disease. We now refer to these patients as having BENTA disease (B cell Expansion with NF-κB and T cell Anergy).
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http://dx.doi.org/10.1007/s10875-014-0106-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466218PMC
January 2015

The genomic landscape of the Ewing Sarcoma family of tumors reveals recurrent STAG2 mutation.

PLoS Genet 2014 Jul 10;10(7):e1004475. Epub 2014 Jul 10.

Oncogenomics Section, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, United States of America.

The Ewing sarcoma family of tumors (EFT) is a group of highly malignant small round blue cell tumors occurring in children and young adults. We report here the largest genomic survey to date of 101 EFT (65 tumors and 36 cell lines). Using a combination of whole genome sequencing and targeted sequencing approaches, we discover that EFT has a very low mutational burden (0.15 mutations/Mb) but frequent deleterious mutations in the cohesin complex subunit STAG2 (21.5% tumors, 44.4% cell lines), homozygous deletion of CDKN2A (13.8% and 50%) and mutations of TP53 (6.2% and 71.9%). We additionally note an increased prevalence of the BRCA2 K3326X polymorphism in EFT patient samples (7.3%) compared to population data (OR 7.1, p = 0.006). Using whole transcriptome sequencing, we find that 11% of tumors pathologically diagnosed as EFT lack a typical EWSR1 fusion oncogene and that these tumors do not have a characteristic Ewing sarcoma gene expression signature. We identify samples harboring novel fusion genes including FUS-NCATc2 and CIC-FOXO4 that may represent distinct small round blue cell tumor variants. In an independent EFT tissue microarray cohort, we show that STAG2 loss as detected by immunohistochemistry may be associated with more advanced disease (p = 0.15) and a modest decrease in overall survival (p = 0.10). These results significantly advance our understanding of the genomic and molecular underpinnings of Ewing sarcoma and provide a foundation towards further efforts to improve diagnosis, prognosis, and precision therapeutics testing.
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http://dx.doi.org/10.1371/journal.pgen.1004475DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4091782PMC
July 2014

Bioinformatic description of immunotherapy targets for pediatric T-cell leukemia and the impact of normal gene sets used for comparison.

Front Oncol 2014 10;4:134. Epub 2014 Jun 10.

Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, MD , USA.

Pediatric lymphoid leukemia has the highest cure rate of all pediatric malignancies, yet due to its prevalence, still accounts for the majority of childhood cancer deaths and requires long-term highly toxic therapy. The ability to target B-cell ALL with immunoglobulin-like binders, whether anti-CD22 antibody or anti-CD19 CAR-Ts, has impacted treatment options for some patients. The development of new ways to target B-cell antigens continues at rapid pace. T-cell ALL accounts for up to 20% of childhood leukemia but has yet to see a set of high-value immunotherapeutic targets identified. To find new targets for T-ALL immunotherapy, we employed a bioinformatic comparison to broad normal tissue arrays, hematopoietic stem cells (HSC), and mature lymphocytes, then filtered the results for transcripts encoding plasma membrane proteins. T-ALL bears a core T-cell signature and transcripts encoding TCR/CD3 components and canonical markers of T-cell development predominate, especially when comparison was made to normal tissue or HSC. However, when comparison to mature lymphocytes was also undertaken, we identified two antigens that may drive, or be associated with leukemogenesis; TALLA-1 and hedgehog interacting protein. In addition, TCR subfamilies, CD1, activation and adhesion markers, membrane-organizing molecules, and receptors linked to metabolism and inflammation were also identified. Of these, only CD52, CD37, and CD98 are currently being targeted clinically. This work provides a set of targets to be considered for future development of immunotherapies for T-ALL.
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http://dx.doi.org/10.3389/fonc.2014.00134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050364PMC
June 2014

Comprehensive genomic analysis of rhabdomyosarcoma reveals a landscape of alterations affecting a common genetic axis in fusion-positive and fusion-negative tumors.

Cancer Discov 2014 Feb 23;4(2):216-31. Epub 2014 Jan 23.

1Pediatric Oncology Branch, Oncogenomics Section, Center for Cancer Research, NIH; 2Laboratory of Pathology, National Cancer Institute, Bethesda, Maryland; 3Broad Institute of MIT and Harvard, Cambridge; 4Medical Oncology and Center for Cancer Genome Discovery, Dana-Farber Cancer Institute; 5Department of Pathology, Harvard Medical School, Boston, Massachusetts; 6University of Nebraska Medical Center, Omaha, Nebraska; 7Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, Texas; and 8Department of Pediatrics, Seattle Children's Hospital, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, Washington; 9The Tumour Bank, The Children's Cancer Research Unit, The Children's Hospital at Westmead, Westmead, New South Wales, Australia; 10Department of Oncology, Hospital Sant Joan de Deu de Barcelona, Barcelona, Spain.

Unlabelled: Despite gains in survival, outcomes for patients with metastatic or recurrent rhabdomyosarcoma remain dismal. In a collaboration between the National Cancer Institute, Children's Oncology Group, and Broad Institute, we performed whole-genome, whole-exome, and transcriptome sequencing to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Two genotypes are evident in rhabdomyosarcoma tumors: those characterized by the PAX3 or PAX7 fusion and those that lack these fusions but harbor mutations in key signaling pathways. The overall burden of somatic mutations in rhabdomyosarcoma is relatively low, especially in tumors that harbor a PAX3/7 gene fusion. In addition to previously reported mutations in NRAS, KRAS, HRAS, FGFR4, PIK3CA, and CTNNB1, we found novel recurrent mutations in FBXW7 and BCOR, providing potential new avenues for therapeutic intervention. Furthermore, alteration of the receptor tyrosine kinase/RAS/PIK3CA axis affects 93% of cases, providing a framework for genomics-directed therapies that might improve outcomes for patients with rhabdomyosarcoma.

Significance: This is the most comprehensive genomic analysis of rhabdomyosarcoma to date. Despite a relatively low mutation rate, multiple genes were recurrently altered, including NRAS, KRAS, HRAS, FGFR4, PIK3CA, CTNNB1, FBXW7, and BCOR. In addition, a majority of rhabdomyosarcoma tumors alter the receptor tyrosine kinase/RAS/PIK3CA axis, providing an opportunity for genomics-guided intervention.
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http://dx.doi.org/10.1158/2159-8290.CD-13-0639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462130PMC
February 2014
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