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1.
Nat Commun ; 11(1): 913, 2020 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-32060267

RESUMO

Aggressive cancers often have activating mutations in growth-controlling oncogenes and inactivating mutations in tumor-suppressor genes. In neuroblastoma, amplification of the MYCN oncogene and inactivation of the ATRX tumor-suppressor gene correlate with high-risk disease and poor prognosis. Here we show that ATRX mutations and MYCN amplification are mutually exclusive across all ages and stages in neuroblastoma. Using human cell lines and mouse models, we found that elevated MYCN expression and ATRX mutations are incompatible. Elevated MYCN levels promote metabolic reprogramming, mitochondrial dysfunction, reactive-oxygen species generation, and DNA-replicative stress. The combination of replicative stress caused by defects in the ATRX-histone chaperone complex, and that induced by MYCN-mediated metabolic reprogramming, leads to synthetic lethality. Therefore, ATRX and MYCN represent an unusual example, where inactivation of a tumor-suppressor gene and activation of an oncogene are incompatible. This synthetic lethality may eventually be exploited to improve outcomes for patients with high-risk neuroblastoma.

3.
Cancer Immunol Res ; 8(3): 409-420, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31907209

RESUMO

Identification of neoantigens is a critical step in predicting response to checkpoint blockade therapy and design of personalized cancer vaccines. This is a cross-disciplinary challenge, involving genomics, proteomics, immunology, and computational approaches. We have built a computational framework called pVACtools that, when paired with a well-established genomics pipeline, produces an end-to-end solution for neoantigen characterization. pVACtools supports identification of altered peptides from different mechanisms, including point mutations, in-frame and frameshift insertions and deletions, and gene fusions. Prediction of peptide:MHC binding is accomplished by supporting an ensemble of MHC Class I and II binding algorithms within a framework designed to facilitate the incorporation of additional algorithms. Prioritization of predicted peptides occurs by integrating diverse data, including mutant allele expression, peptide binding affinities, and determination whether a mutation is clonal or subclonal. Interactive visualization via a Web interface allows clinical users to efficiently generate, review, and interpret results, selecting candidate peptides for individual patient vaccine designs. Additional modules support design choices needed for competing vaccine delivery approaches. One such module optimizes peptide ordering to minimize junctional epitopes in DNA vector vaccines. Downstream analysis commands for synthetic long peptide vaccines are available to assess candidates for factors that influence peptide synthesis. All of the aforementioned steps are executed via a modular workflow consisting of tools for neoantigen prediction from somatic alterations (pVACseq and pVACfuse), prioritization, and selection using a graphical Web-based interface (pVACviz), and design of DNA vector-based vaccines (pVACvector) and synthetic long peptide vaccines. pVACtools is available at http://www.pvactools.org.

4.
Breast Cancer Res Treat ; 179(1): 197-206, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31542876

RESUMO

PURPOSE: Multi-gene signatures provide biological insight and risk stratification in breast cancer. Intrinsic molecular subtypes defined by mRNA expression of 50 genes (PAM50) are prognostic in hormone-receptor positive postmenopausal breast cancer. Yet, for 25-40% in the PAM50 intermediate risk group, long-term risk remains uncertain. Our study aimed to (i) test the long-term prognostic value of the PAM50 signature in pre- and post-menopausal breast cancer; (ii) investigate if the PAM50 model could be improved by addition of other mRNAs implicated in oncogenesis. METHODS: We used archived FFPE samples from 1723 breast cancer survivors; high quality reads were obtained on 1253 samples. Transcript expression was quantified using a custom codeset with probes for > 100 targets. Cox models assessed gene signatures for breast cancer relapse and survival. RESULTS: Over 15 + years of follow-up, PAM50 subtypes were (P < 0.01) associated with breast cancer outcomes after accounting for tumor stage, grade and age at diagnosis. Results did not differ by menopausal status at diagnosis. Women with Luminal B (versus Luminal A) subtype had a > 60% higher hazard. Addition of a 13-gene hypoxia signature improved prognostication with > 40% higher hazard in the highest vs lowest hypoxia tertiles. CONCLUSIONS: PAM50 intrinsic subtypes were independently prognostic for long-term breast cancer survival, irrespective of menopausal status. Addition of hypoxia signatures improved risk prediction. If replicated, incorporating the 13-gene hypoxia signature into the existing PAM50 risk assessment tool, may refine risk stratification and further clarify treatment for breast cancer.

5.
Proc Natl Acad Sci U S A ; 116(47): 23662-23670, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31685621

RESUMO

The impact of intratumoral heterogeneity (ITH) and the resultant neoantigen landscape on T cell immunity are poorly understood. ITH is a widely recognized feature of solid tumors and poses distinct challenges related to the development of effective therapeutic strategies, including cancer neoantigen vaccines. Here, we performed deep targeted DNA sequencing of multiple metastases from melanoma patients and observed ubiquitous sharing of clonal and subclonal single nucleotide variants (SNVs) encoding putative HLA class I-restricted neoantigen epitopes. However, spontaneous antitumor CD8+ T cell immunity in peripheral blood and tumors was restricted to a few clonal neoantigens featuring an oligo-/monoclonal T cell-receptor (TCR) repertoire. Moreover, in various tumors of the 4 patients examined, no neoantigen-specific TCR clonotypes were identified despite clonal neoantigen expression. Mature dendritic cell (mDC) vaccination with tumor-encoded amino acid-substituted (AAS) peptides revealed diverse neoantigen-specific CD8+ T responses, each composed of multiple TCR clonotypes. Isolation of T cell clones by limiting dilution from tumor-infiltrating lymphocytes (TILs) permitted functional validation regarding neoantigen specificity. Gene transfer of TCRαß heterodimers specific for clonal neoantigens confirmed correct TCR clonotype assignments based on high-throughput TCRBV CDR3 sequencing. Our findings implicate immunological ignorance of clonal neoantigens as the basis for ineffective T cell immunity to melanoma and support the concept that therapeutic vaccination, as an adjunct to checkpoint inhibitor treatment, is required to increase the breadth and diversity of neoantigen-specific CD8+ T cells.

6.
Genome Med ; 11(1): 71, 2019 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-31747945

RESUMO

The resurgence of immune therapies in cancer medicine has elicited a corresponding interest in understanding the basis of patient response or resistance to these treatments. One aspect of patient response clearly lies in the genomic alterations that are associated with cancer onset and progression, including those that contribute to genomic instability and the resulting creation of novel peptide sequences that may present as neoantigens. The immune reaction to these unique 'non-self' peptides is frequently suppressed by the tumor itself, but the use of checkpoint blockade therapies, personalized vaccines, or a combination of these treatments may elicit a tumor-specific immune response that results in cell death. Massively parallel sequencing, coupled with different computational analyses, provides unbiased identification of the germline and somatic alterations that drive cancer development, and of those alterations that lead to neoantigens. These range from simple point mutations that change single amino acids to complex alterations, such as frameshift insertion or deletion mutations, splice-site alterations that lead to exon skipping, structural alterations that lead to the formation of fusion proteins, and other forms of collateral damage caused by genome instability that result in new protein sequences unique to the cancer. The various genome instability phenotypes can be identified as alterations that impact DNA replication or mismatch repair pathways or by their genomic signatures. This review provides an overview of current knowledge regarding the fundamentals of genome replication and of both germline and somatic alterations that disrupt normal replication, leading to various forms of genomic instability in cancers, to the resulting generation of neoantigens and, ultimately, to immune-responsive and resistant phenotypes.

7.
Mol Ther Oncolytics ; 15: 38-48, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31650024

RESUMO

Solid tumors contain a mixture of malignant cells and non-malignant infiltrating cells that often create a chronic inflammatory and immunosuppressive microenvironment that restricts immunotherapeutic approaches. Although childhood and adult cancers share some similarities related to microenvironmental changes, pediatric cancers are unique, and adult cancer practices may not be wholly applicable to our pediatric patients. This review highlights the differences in tumorigenesis, viral infection, and immunologic response between children and adults that need to be considered when trying to apply experiences from experimental therapies in adult cancer patients to pediatric cancers.

9.
iScience ; 18: 1-10, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31271967

RESUMO

Linked-read sequencing enables greatly improves haplotype assembly over standard paired-end analysis. The detection of mosaic single-nucleotide variants benefits from haplotype assembly when the model is informed by the mapping between constituent reads and linked reads. Samovar evaluates haplotype-discordant reads identified through linked-read sequencing, thus enabling phasing and mosaic variant detection across the entire genome. Samovar trains a random forest model to score candidate sites using a dataset that considers read quality, phasing, and linked-read characteristics. Samovar calls mosaic single-nucleotide variants (SNVs) within a single sample with accuracy comparable with what previously required trios or matched tumor/normal pairs and outperforms single-sample mosaic variant callers at minor allele frequency 5%-50% with at least 30X coverage. Samovar finds somatic variants in both tumor and normal whole-genome sequencing from 13 pediatric cancer cases that can be corroborated with high recall with whole exome sequencing. Samovar is available open-source at https://github.com/cdarby/samovar under the MIT license.

10.
Cancer Epidemiol Biomarkers Prev ; 28(9): 1525-1533, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31186261

RESUMO

BACKGROUND: There is substantial variation in breast cancer survival rates, even among patients with similar clinical and genomic profiles. New biomarkers are needed to improve risk stratification and inform treatment options. Our aim was to identify novel miRNAs associated with breast cancer survival and quantify their prognostic value after adjusting for established clinical factors and genomic markers. METHODS: Using the Women's Healthy Eating and Living (WHEL) breast cancer cohort with >15 years of follow-up and archived tumor specimens, we assayed PAM50 mRNAs and 25 miRNAs using the Nanostring nCounter platform. RESULTS: We obtained high-quality reads on 1,253 samples (75% of available specimens) and used an existing research-use algorithm to ascertain PAM50 subtypes and risk scores (ROR-PT). We identified miRNAs significantly associated with breast cancer outcomes and then tested these in independent TCGA samples. miRNAs that were also prognostic in TCGA samples were further evaluated in multiple regression Cox models. We also used penalized regression for unbiased discovery. CONCLUSIONS: Two miRNAs, 210 and 29c, were associated with breast cancer outcomes in the WHEL and TCGA studies and further improved risk stratification within PAM50 risk groups: 10-year survival was 62% in the node-negative high miR-210-high ROR-PT group versus 75% in the low miR-210- high ROR-PT group. Similar results were obtained for miR-29c. We identified three additional miRNAs, 187-3p, 143-3p, and 205-5p, via penalized regression. IMPACT: Our findings suggest that miRNAs might be prognostic for long-term breast cancer survival and might improve risk stratification. Further research to incorporate miRNAs into existing clinicogenomic signatures is needed.

11.
Eur J Med Genet ; 62(8): 103701, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31195167

RESUMO

Klippel-Feil syndrome (KFS) is an exceedingly rare constitutional disorder in which a paucity of knowledge exists about the disease and its associated morbidity and mortality. We present a 4-year-old male with KFS, who notably was also diagnosed with large-cell anaplastic medulloblastoma. We evaluated the genetic basis of co-occurring KFS and medulloblastoma and the role of MYO18B as related to medulloblastoma. Constitutional and somatic variant and copy number analyses were performed from DNA-based exome studies, along with RNA-sequencing of tumor tissue, to elucidate the genetic etiology of the co-existing disease states. We identified novel constitutional compound heterozygous frameshift variants (NM_032608.5: p.Leu2257SerfsTer16 and p.Arg2220SerfsTer74) each encoding a premature stop of translation in MYO18B, consistent with a diagnosis of KFS. We did not identify any somatic variants of known relevance or disease-relevant therapeutic targets in the tumor. The somatic copy number profile was suggestive of Group 3γ medulloblastoma. Relative to pediatric brain tumors, medulloblastoma, particularly, Group 3, had increased gene expression of MYO18B. In summary, coexisting constitutional and somatic diagnoses in this patient enabled the elucidation of the genetic etiology of KFS and provided support for the role of MYO18B in tumor suppression.


Assuntos
Síndrome de Klippel-Feil/genética , Meduloblastoma/genética , Miosinas/genética , Proteínas Supressoras de Tumor/genética , Sequenciamento Completo do Exoma , Pré-Escolar , Exoma/genética , Mutação da Fase de Leitura/genética , Heterozigoto , Humanos , Síndrome de Klippel-Feil/complicações , Síndrome de Klippel-Feil/diagnóstico , Síndrome de Klippel-Feil/patologia , Masculino , Meduloblastoma/complicações , Meduloblastoma/diagnóstico , Meduloblastoma/patologia
12.
13.
Oncoimmunology ; 8(4): e1561106, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30906654

RESUMO

Neoantigens represent promising targets for personalized cancer vaccine strategies. However, the feasibility of this approach in lower mutational burden tumors like glioblastoma (GBM) remains unknown. We have previously reported the use of an immunogenomics pipeline to identify candidate neoantigens in preclinical models of GBM. Here, we report the application of the same immunogenomics pipeline to identify candidate neoantigens and guide screening for neoantigen-specific T cell responses in a patient with GBM treated with a personalized synthetic long peptide vaccine following autologous tumor lysate DC vaccination. Following vaccination, reactivity to three HLA class I- and five HLA class II-restricted candidate neoantigens were detected by IFN-γ ELISPOT in peripheral blood. A similar pattern of reactivity was observed among isolated post-treatment tumor-infiltrating lymphocytes. Genomic analysis of pre- and post-treatment GBM reflected clonal remodeling. These data demonstrate the feasibility and translational potential of a therapeutic neoantigen-based vaccine approach in patients with primary CNS tumors.

14.
Pediatr Blood Cancer ; 66(6): e27681, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30848065

RESUMO

BACKGROUND/OBJECTIVES: Anthracyclines are used in induction therapy of pediatric acute lymphoblastic leukemia (ALL) and are known to generate oxidative stress; whether this translates into enhanced antileukemic activity or hemolytic effects in patients with glucose-6-phosphate dehydrogenase (G6PD) deficiency is unknown. DESIGN/METHODS: Among 726 pediatric patients with newly diagnosed ALL treated at St. Jude Children's Research Hospital, 22 had deficient G6PD activity. We compared the prevalence of positive minimal residual disease (MRD) ≥1% at Day 15/Day 19 of induction or ≥0.01% at Day 42/Day 46 (end of induction) and the number of red blood cell (RBC) transfusions after daunorubicin in induction between patients with or without G6PD deficiency, adjusting for ALL risk group, treatment protocol, age, and gender. RESULTS: There was no difference in Day 15/19 (P = 1) or end of induction MRD (P = 0.76) nor in the number of RBC transfusions (P = 0.73); the lack of association with MRD was confirmed in a dataset of 1192 newly diagnosed male patients enrolled in a Children's Oncology Group trial (P = 0.78). CONCLUSION: We found no evidence that G6PD deficiency affects daunorubicin activity during induction treatment for ALL.


Assuntos
Antibióticos Antineoplásicos/uso terapêutico , Daunorrubicina/uso terapêutico , Glucosefosfato Desidrogenase/metabolismo , Neoplasia Residual/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Criança , Estudos de Coortes , Feminino , Seguimentos , Humanos , Quimioterapia de Indução , Masculino , Terapia Neoadjuvante , Neoplasia Residual/tratamento farmacológico , Neoplasia Residual/enzimologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/enzimologia , Prognóstico , Fatores de Risco , Segurança
15.
Cancer Immunol Res ; 7(3): 458-465, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30635271

RESUMO

To understand prognostic factors for outcome between differentially sequenced nivolumab and ipilimumab in a randomized phase II trial, we measured T-cell infiltration and PD-L1 by IHC, T-cell repertoire metrics, and mutational load within the tumor. We used next-generation sequencing (NGS) and assessed the association of those parameters with response and overall survival. Immunosequencing of the T-cell receptor ß-chain locus (TCRß) from DNA of 91 pretreatment tumor samples and an additional 22 pairs of matched pre- and posttreatment samples from patients who received nivolumab followed by ipilimumab (nivo/ipi), or the reverse (ipi/nivo), was performed to measure T-cell clonality and fraction. Mutational and neoantigen load were also assessed by NGS in 82 of the 91 patients. Tumors were stained using IHC for PD-L1+ and CD8+ T cells. Pretreatment tumor TCR clonality and neoantigen load were marginally associated with best response with nivo/ipi (P = 0.04 and 0.05, respectively), but not with ipi/nivo. Amalgamated pretreatment mutational load and tumor T-cell fraction were significantly associated with best response with nivo/ipi (P = 0.002). Pretreatment PD-L1 staining intensity and CD8+ T-cell counts were correlated with T-cell fraction and clonality, but not mutational or neoantigen load. Patients with increased T-cell fraction posttreatment at week 13 had a 30-fold increased likelihood of survival (P = 0.002). Mutational and neoantigen load, and T-cell infiltrate within the tumor, were associated with outcome of sequential checkpoint inhibition using nivolumab then ipilimumab, but not when ipilimumab was administered before nivolumab.

16.
Mol Cancer Res ; 17(4): 895-906, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30651371

RESUMO

To investigate the genomic evolution of metastatic pediatric osteosarcoma, we performed whole-genome and targeted deep sequencing on 14 osteosarcoma metastases and two primary tumors from four patients (two to eight samples per patient). All four patients harbored ancestral (truncal) somatic variants resulting in TP53 inactivation and cell-cycle aberrations, followed by divergence into relapse-specific lineages exhibiting a cisplatin-induced mutation signature. In three of the four patients, the cisplatin signature accounted for >40% of mutations detected in the metastatic samples. Mutations potentially acquired during cisplatin treatment included NF1 missense mutations of uncertain significance in two patients and a KIT G565R activating mutation in one patient. Three of four patients demonstrated widespread ploidy differences between samples from the sample patient. Single-cell seeding of metastasis was detected in most metastatic samples. Cross-seeding between metastatic sites was observed in one patient, whereas in another patient a minor clone from the primary tumor seeded both metastases analyzed. These results reveal extensive clonal heterogeneity in metastatic osteosarcoma, much of which is likely cisplatin-induced. IMPLICATIONS: The extent and consequences of chemotherapy-induced damage in pediatric cancers is unknown. We found that cisplatin treatment can potentially double the mutational burden in osteosarcoma, which has implications for optimizing therapy for recurrent, chemotherapy-resistant disease.


Assuntos
Neoplasias Ósseas/tratamento farmacológico , Neoplasias Ósseas/genética , Cisplatino/uso terapêutico , Osteossarcoma/tratamento farmacológico , Osteossarcoma/genética , Antineoplásicos/farmacologia , Neoplasias Ósseas/patologia , Cisplatino/farmacologia , Evolução Clonal/efeitos dos fármacos , Análise Mutacional de DNA , Feminino , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Masculino , Modelos Genéticos , Mutagênese/efeitos dos fármacos , Metástase Neoplásica , Osteossarcoma/patologia , Sequenciamento Completo do Genoma
17.
Artigo em Inglês | MEDLINE | ID: mdl-30478097

RESUMO

Although DNA and RNA sequencing has a history spanning five decades, large-scale massively parallel sequencing, or next-generation sequencing (NGS), has only been commercially available for about 10 years. Nonetheless, the meteoric increase in sequencing throughput with NGS has dramatically changed our understanding of our genome and ourselves. Sequencing the first human genome as a haploid reference took nearly 10 years but now a full diploid human genome sequence can be accomplished in just a few days. NGS has also reduced the cost of generating sequence data and a plethora of sequence-based methods for probing a genome have emerged using NGS as the readout and have been applied to many species. NGS methods have also entered the medical realm and will see an increasing use in diagnosis and treatment. NGS has largely been driven by short-read generation (150 bp) but new platforms have emerged and are now capable of generating long multikilobase reads. These latter platforms enable reference-independent genome assemblies and long-range haplotype generation. Rapid DNA and RNA sequencing is now mainstream and will continue to have an increasing impact on biology and medicine.

18.
Artigo em Inglês | MEDLINE | ID: mdl-30397020

RESUMO

The application of next-generation sequencing (NGS) technology to the study of cancer genomes has been transformational. Not only has this technology revealed the genetic and epigenetic underpinnings of disease onset and progression, but also has redefined our clinical diagnosis and treatment paradigms. This rapid translation from discovery to clinical platform has occurred in the context of new pharmaceutical paradigms, enabling the use of NGS for the diagnosis and definition of therapeutic vulnerabilities of cancer. This review explores this transformation and identifies cutting-edge applications of NGS that will result in its additional utility in cancer care.

19.
Nat Genet ; 51(1): 175-179, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30510237

RESUMO

Recent efforts to design personalized cancer immunotherapies use predicted neoantigens, but most neoantigen prediction strategies do not consider proximal (nearby) variants that alter the peptide sequence and may influence neoantigen binding. We evaluated somatic variants from 430 tumors to understand how proximal somatic and germline alterations change the neoantigenic peptide sequence and also affect neoantigen binding predictions. On average, 241 missense somatic variants were analyzed per sample. Of these somatic variants, 5% had one or more in-phase missense proximal variants. Without incorporating proximal variant correction for major histocompatibility complex class I neoantigen peptides, the overall false discovery rate (incorrect neoantigens predicted) and the false negative rate (strong-binding neoantigens missed) across peptides of lengths 8-11 were estimated as 0.069 (6.9%) and 0.026 (2.6%), respectively.


Assuntos
Antígenos de Neoplasias/genética , Variação Genética/genética , Neoplasias/genética , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Imunoterapia/métodos
20.
Nat Commun ; 9(1): 4850, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30429476

RESUMO

The original version of this Article contained errors in the depiction of confidence intervals in the NF1 BCSS data illustrated in Figure 3b. These have now been corrected in both the PDF and HTML versions of the Article. The incorrect version of Figure 3b is presented in the associated Author Correction.

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