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1.
Acta Neuropathol Commun ; 9(1): 61, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33827698

RESUMO

Retinoblastoma is a childhood cancer of the retina involving germline or somatic alterations of the RB Transcriptional Corepressor 1 gene, RB1. Rare cases of sellar-suprasellar region retinoblastoma without evidence of ocular or pineal tumors have been described. A nine-month-old male presented with a sellar-suprasellar region mass. Histopathology showed an embryonal tumor with focal Flexner-Wintersteiner-like rosettes and loss of retinoblastoma protein (RB1) expression by immunohistochemistry. DNA array-based methylation profiling confidently classified the tumor as pineoblastoma group A/intracranial retinoblastoma. The patient was subsequently enrolled on an institutional translational cancer research protocol and underwent comprehensive molecular profiling, including paired tumor/normal exome and genome sequencing and RNA-sequencing of the tumor. Additionally, Pacific Biosciences (PacBio) Single Molecule Real Time (SMRT) sequencing was performed from comparator normal and disease-involved tissue to resolve complex structural variations. RNA-sequencing revealed multiple fusions clustered within 13q14.1-q21.3, including a novel in-frame fusion of RB1-SIAH3 predicted to prematurely truncate the RB1 protein. SMRT sequencing revealed a complex structural rearrangement spanning 13q14.11-q31.3, including two somatic structural variants within intron 17 of RB1. These events corresponded to the RB1-SIAH3 fusion and a novel RB1 rearrangement expected to correlate with the complete absence of RB1 protein expression. Comprehensive molecular analysis, including DNA array-based methylation profiling and sequencing-based methodologies, were critical for classification and understanding the complex mechanism of RB1 inactivation in this diagnostically challenging tumor.

2.
Artigo em Inglês | MEDLINE | ID: mdl-33637608

RESUMO

In this follow-up report, we present updated information regarding a previously reported pediatric patient with a World Health Organization grade I ganglioglioma harboring a BRAF p.T599dup mutation (Cold Spring Harb Mol Case Stud 4: a002618). This patient, based on our initial finding, is receiving combination targeted therapy with a selective BRAF inhibitor (dabrafenib) plus MEK inhibitor (trametinib). The combination therapy was started after the patient experienced progressive tumor growth and worsening neurological symptoms, including visual changes, headaches, and peripheral neuropathy, despite 9 months of treatment with adjuvant chemotherapy (vinblastine). The patient has been receiving dabrafenib plus trametinib for 15 months and continues to have stable disease as well as improved neurological symptoms. Although combinatorial therapy targeting BRAF and MEK using dabrafenib and trametinib, respectively, is indicated for tumors harboring a BRAF p.V600E/K mutation, our report demonstrates efficacy of this combination in a non-V600E BRAF-mutated tumor. The identification of BRAF alterations may assist clinicians in determining alternative targeted treatment strategies, especially considering the paucity of effective treatments for primary brain tumors and the poor prognosis associated with many central nervous system (CNS) diagnoses. Additional case studies or larger cohort reports will continue to clarify the efficacy of BRAF and/or MEK inhibitors in patients whose tumors harbor a BRAF alteration.

3.
Ann N Y Acad Sci ; 2020 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-33184911

RESUMO

Cancer immunotherapy has dramatically changed the approach to cancer treatment. The aim of targeting the immune system to recognize and destroy cancer cells has afforded many patients the prospect of achieving deep, long-term remission and potential cures. However, many challenges remain for achieving the goal of effective immunotherapy for all cancer patients. Checkpoint inhibitors have been able to achieve long-term responses in a minority of patients, yet improving response rates with combination therapies increases the possibility of toxicity. Chimeric antigen receptor T cells have demonstrated high response rates in hematological cancers, although most patients experience relapse. In addition, some cancers are notoriously immunologically "cold" and typically are not effective targets for immunotherapy. Overcoming these obstacles will require new strategies to improve upon the efficacy of current agents, identify biomarkers to select appropriate therapies, and discover new modalities to expand the accessibility of immunotherapy to additional tumor types and patient populations.

4.
Artigo em Inglês | MEDLINE | ID: mdl-33028644

RESUMO

Infantile fibrosarcoma (IFS) is nearly universally driven by gene fusions involving the NTRK family. ETV6-NTRK3 fusions account for ∼85% of alterations; the remainder are attributed to NTRK-variant fusions. Rarely, other genomic aberrations have been described in association with tumors identified as IFS or IFS-like. We describe the utility of genomic characterization of an IFS-like tumor. We also describe the successful treatment combination of VAC (vincristine, actinomycin, cyclophosphamide) with tyrosine kinase inhibitor (TKI) maintenance in this entity. This patient presented at birth with a right facial mass, enlarging at 1 mo to 4.9 × 4.5 × 6.3 cm. Biopsy demonstrated hypercellular fascicles of spindle cells with patchy positivity for smooth muscle actin (SMA) and negativity for S100, desmin, myogenin, and MyoD1. Targeted RNA sequencing identified a novel RBPMS-MET fusion with confirmed absence of ETV6-NTRK3, and the patient was diagnosed with an IFS-like tumor. A positron emission tomography (PET) scan was negative for metastatic disease. VAC was given for a duration of 10 mo. Resection at 13 mo of age demonstrated positive margins. Cabozantinib, a MET-targeting TKI, was initiated. The patient tolerated cabozantinib well and has no evidence of disease at 24 mo of age. We describe a novel RBPMS-MET driver fusion in association with a locally aggressive IFS-like tumor. MET functions as an oncogene and, when associated with the RNA binding protein RBPMS, forms an in-frame fusion product that retains the MET kinase domain. This fusion is associated with aberrant cell signaling pathway expression and subsequent malignancy. We describe treatment with cabozantinib in a patient with an IFS-like neoplasm.

5.
Am J Surg Pathol ; 2020 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-33074854

RESUMO

Meningiomas are a central nervous system tumor primarily afflicting adults, with <1% of cases diagnosed during childhood or adolescence. Somatic variation in NF2 may be found in ∼50% of meningiomas, with other genetic drivers (eg, SMO, AKT1, TRAF7) contributing to NF2 wild-type tumors. NF2 is an upstream negative regulator of YAP signaling and loss of the NF2 protein product, Merlin, results in YAP overexpression and target gene transcription. This mechanism of dysregulation is described in NF2-driven meningiomas, but further work is necessary to understand the NF2-independent mechanism of tumorigenesis. Amid our institutional patient-centric comprehensive molecular profiling study, we identified an individual with meningioma harboring a YAP1-FAM118B fusion, previously reported only in supratentorial ependymoma. The tumor histopathology was remarkable, characterized by prominent islands of calcifying fibrous nodules within an overall collagen-rich matrix. To gain insight into this finding, we subsequently evaluated the genetic landscape of 11 additional pediatric and adolescent/young adulthood meningioma patients within the Children's Brain Tumor Tissue Consortium. A second individual harboring a YAP1-FAM118B gene fusion was identified within this database. Transcriptomic profiling suggested that YAP1-fusion meningiomas are biologically distinct from NF2-driven meningiomas. Similar to other meningiomas, however, YAP1-fusion meningiomas demonstrated overexpression of EGFR and MET. DNA methylation profiling further distinguished YAP1-fusion meningiomas from those observed in ependymomas. In summary, we expand the genetic spectrum of somatic alteration associated with NF2 wild-type meningioma to include the YAP1-FAM118B fusion and provide support for aberrant signaling pathways potentially targetable by therapeutic intervention.

6.
J Mol Diagn ; 22(10): 1238-1245, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32745614

RESUMO

Somatic variants, primarily fusion genes and single-nucleotide variants (SNVs) or insertions/deletions (indels), are prevalent among sarcomas. In many cases, accurate diagnosis of these tumors incorporates genetic findings that may also carry prognostic or therapeutic significance. Using the anchored multiplex PCR-based FusionPlex system, a custom RNA sequencing panel was developed that simultaneously detects fusion genes, SNVs, and indels in 112 genes found to be recurrently mutated in solid tumors. Using this assay, a retrospective analysis was conducted to identify somatic variants that may have assisted with classifying a cohort of 90 previously uncharacterized primarily pediatric sarcoma specimens. In total, somatic variants were identified in 45.5% (41/90) of the samples tested, including 22 cases with fusion genes and 19 cases with SNVs or indels. In addition, two of these findings represent novel alterations: a WHSC1L1/NCOA2 fusion and a novel in-frame deletion in the NRAS gene (NM_002524: c.174_176delAGC p.Ala59del). These sequencing results, taken in context with the available clinical data, indicate a potential change in the initial diagnosis, prognosis, or management in 27 of the 90 cases. This study presents a custom RNA sequencing assay that detects fusion genes and SNVs in tandem and has the ability to identify novel fusion partners. These features highlight the advantages associated with utilizing anchored multiplex PCR technology for the rapid and highly sensitive detection of somatic variants.

7.
Neurol Genet ; 6(4): e460, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32637635

RESUMO

Objective: Many genetic studies of intractable epilepsy in pediatric patients primarily focus on inherited, constitutional genetic deficiencies identified in patient blood. Recently, studies have revealed somatic mosaicism associated with epilepsy in which genetic variants are present only in a subset of brain cells. We hypothesize that tissue-specific, somatic mosaicism represents an important genetic etiology in epilepsy and aim to discover somatic alterations in epilepsy-affected brain tissue. Methods: We have pursued a research study to identify brain somatic mosaicism, using next-generation sequencing (NGS) technologies, in patients with treatment refractory epilepsy who have undergone surgical resection of affected brain tissue. Results: We used an integrated combination of NGS techniques and conventional approaches (radiology, histopathology, and electrophysiology) to comprehensively characterize multiple brain regions from a single patient with intractable epilepsy. We present a 3-year-old male patient with West syndrome and intractable tonic seizures in whom we identified a pathogenic frameshift somatic variant in SLC35A2, present at a range of variant allele fractions (4.2%-19.5%) in 12 different brain tissues detected by targeted sequencing. The proportion of the SLC35A2 variant correlated with severity and location of neurophysiology and neuroimaging abnormalities for each tissue. Conclusions: Our findings support the importance of tissue-based sequencing and highlight a correlation in our patient between SLC35A2 variant allele fractions and the severity of epileptogenic phenotypes in different brain tissues obtained from a grid-based resection of clinically defined epileptogenic regions.

8.
Nat Commun ; 11(1): 3644, 2020 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-32686686

RESUMO

Colorectal cancer (CRC) is a biologically heterogeneous disease. To characterize its mutational profile, we conduct targeted sequencing of 205 genes for 2,105 CRC cases with survival data. Our data shows several findings in addition to enhancing the existing knowledge of CRC. We identify PRKCI, SPZ1, MUTYH, MAP2K4, FETUB, and TGFBR2 as additional genes significantly mutated in CRC. We find that among hypermutated tumors, an increased mutation burden is associated with improved CRC-specific survival (HR = 0.42, 95% CI: 0.21-0.82). Mutations in TP53 are associated with poorer CRC-specific survival, which is most pronounced in cases carrying TP53 mutations with predicted 0% transcriptional activity (HR = 1.53, 95% CI: 1.21-1.94). Furthermore, we observe differences in mutational frequency of several genes and pathways by tumor location, stage, and sex. Overall, this large study provides deep insights into somatic mutations in CRC, and their potential relationships with survival and tumor features.


Assuntos
Neoplasias Colorretais/genética , Proteínas de Neoplasias/genética , Neoplasias do Colo/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação INDEL , Mutação , Prognóstico , Proteína Supressora de Tumor p53/genética
9.
Sci Adv ; 6(24): eaay9691, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32577507

RESUMO

Tumor heterogeneity and evolution drive treatment resistance in metastatic colorectal cancer (mCRC). Patient-derived xenografts (PDXs) can model mCRC biology; however, their ability to accurately mimic human tumor heterogeneity is unclear. Current genomic studies in mCRC have limited scope and lack matched PDXs. Therefore, the landscape of tumor heterogeneity and its impact on the evolution of metastasis and PDXs remain undefined. We performed whole-genome, deep exome, and targeted validation sequencing of multiple primary regions, matched distant metastases, and PDXs from 11 patients with mCRC. We observed intricate clonal heterogeneity and evolution affecting metastasis dissemination and PDX clonal selection. Metastasis formation followed both monoclonal and polyclonal seeding models. In four cases, metastasis-seeding clones were not identified in any primary region, consistent with a metastasis-seeding-metastasis model. PDXs underrepresented the subclonal heterogeneity of parental tumors. These suggest that single sample tumor sequencing and current PDX models may be insufficient to guide precision medicine.

10.
Nat Commun ; 11(1): 2156, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358485

RESUMO

Colorectal cancer (CRC) is the most common gastrointestinal malignancy in the U.S.A. and approximately 50% of patients develop metastatic disease (mCRC). Despite our understanding of long non-coding RNAs (lncRNAs) in primary colon cancer, their role in mCRC and treatment resistance remains poorly characterized. Therefore, through transcriptome sequencing of normal, primary, and distant mCRC tissues we find 148 differentially expressed RNAs Associated with Metastasis (RAMS). We prioritize RAMS11 due to its association with poor disease-free survival and promotion of aggressive phenotypes in vitro and in vivo. A FDA-approved drug high-throughput viability assay shows that elevated RAMS11 expression increases resistance to topoisomerase inhibitors. Subsequent experiments demonstrate RAMS11-dependent recruitment of Chromobox protein 4 (CBX4) transcriptionally activates Topoisomerase II alpha (TOP2α). Overall, recent clinical trials using topoisomerase inhibitors coupled with our findings of RAMS11-dependent regulation of TOP2α supports the potential use of RAMS11 as a biomarker and therapeutic target for mCRC.


Assuntos
Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Animais , Western Blotting , Células CACO-2 , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Biologia Computacional , DNA Topoisomerases Tipo II/metabolismo , Progressão da Doença , Éxons/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Células HCT116 , Células HT29 , Humanos , Ligases/metabolismo , Camundongos , Proteínas do Grupo Polycomb/metabolismo , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Inibidores da Topoisomerase/farmacologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-32371413

RESUMO

Exome sequencing (ES) has become an important tool in pediatric genomic medicine, improving identification of disease-associated variation due to assay breadth. Depth is also afforded by ES, enabling detection of lower-frequency mosaic variation compared to Sanger sequencing in the studied tissue, thus enhancing diagnostic yield. Within a pediatric tertiary-care hospital, we report two years of clinical ES data from probands evaluated for genetic disease to assess diagnostic yield, characteristics of causal variants, and prevalence of mosaicism among disease-causing variants. Exome-derived, phenotype-driven variant data from 357 probands was analyzed concurrent with parental ES data, when available. Blood was the source of nucleic acid. Sequence read alignments were manually reviewed for all assessed variants. Sanger sequencing was used for suspected de novo or mosaic variation. Clinical provider notes were reviewed to determine concordance between laboratory-reported data and the ordering provider's interpretation of variant-associated disease causality. Laboratory-derived diagnostic yield and provider-substantiated diagnoses had 91.4% concordance. The cohort returned 117 provider-substantiated diagnoses among 115 probands for a diagnostic yield of 32.2%. De novo variants represented 64.9% of disease-associated variation within trio analyses. Among the 115 probands, five harbored disease-associated somatic mosaic variation. Two additional probands were observed to inherit a disease-associated variant from an unaffected mosaic parent. Among inheritance patterns, de novo variation was the most frequent disease etiology. Somatic mosaicism is increasingly recognized as a significant contributor to genetic disease, particularly with increased sequence depth attainable from ES. This report highlights the potential and importance of detecting mosaicism in ES.

12.
Nat Commun ; 11(1): 913, 2020 02 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.


Assuntos
Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/metabolismo , Proteína Nuclear Ligada ao X/genética , Animais , Pré-Escolar , Estudos de Coortes , Feminino , Amplificação de Genes , Humanos , Lactente , Masculino , Camundongos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mutação , Proteína Proto-Oncogênica N-Myc/metabolismo , Neuroblastoma/genética , Espécies Reativas de Oxigênio/metabolismo , Proteína Nuclear Ligada ao X/metabolismo
13.
Cancer Immunol Res ; 8(3): 409-420, 2020 03.
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.


Assuntos
Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/imunologia , Biologia Computacional/métodos , Mineração de Dados , Neoplasias/imunologia , Redes Neurais de Computação , Algoritmos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Inteligência Artificial/normas , Vacinas Anticâncer/administração & dosagem , Humanos , Imunoterapia/métodos , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Software
15.
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.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , Sobreviventes de Câncer/estatística & dados numéricos , Perfilação da Expressão Gênica/métodos , Adulto , Idoso , Neoplasias da Mama/mortalidade , Neoplasias da Mama/patologia , Hipóxia Celular , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Análise de Sobrevida
16.
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.


Assuntos
Antígenos de Neoplasias/imunologia , Genoma Humano , Instabilidade Genômica , Neoplasias/etiologia , Fenótipo , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Células Germinativas , Humanos , Imunoterapia , Neoplasias/diagnóstico , Neoplasias/metabolismo , Neoplasias/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo
17.
Proc Natl Acad Sci U S A ; 116(47): 23662-23670, 2019 11 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.


Assuntos
Antígenos de Neoplasias/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma/imunologia , Subpopulações de Linfócitos T/imunologia , Substituição de Aminoácidos , Antígenos de Neoplasias/genética , Vacinas Anticâncer/imunologia , Células Clonais , DNA de Neoplasias/genética , Células Dendríticas/imunologia , Antígenos HLA/imunologia , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Melanoma/genética , Melanoma/secundário , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Neoplasias Retroperitoneais/imunologia , Neoplasias Retroperitoneais/secundário , Análise de Sequência de DNA , Especificidade do Receptor de Antígeno de Linfócitos T , Evasão Tumoral , Vacinação
19.
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.

20.
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.

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