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Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B- ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.
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Relapsed or refractory B-cell acute lymphoblastic leukemia (B-ALL) is a major cause of pediatric cancer-related deaths. Relapse-specific mutations do not account for all chemotherapy failures in B-ALL patients, suggesting additional mechanisms of resistance. By mining RNA-seq datasets of paired diagnostic/relapse pediatric B-ALL samples, we discovered pervasive alternative splicing (AS) patterns linked to relapse and affecting drivers of resistance to glucocorticoids, anti-folates, and thiopurines. Most splicing variations represented cassette exon skipping, "poison" exon inclusion, and intron retention, phenocopying well-documented loss-of-function mutations. In contrast, relapse-associated AS of NT5C2 mRNA yielded an isoform with the functionally uncharacterized in-frame exon 6a. Incorporation of the 8-amino acid sequence SQVAVQKR into this enzyme created a putative phosphorylation site and resulted in elevated nucleosidase activity, which is a known consequence of gain-of-function mutations in NT5C2 and a common determinant of 6-mercaptopurine (6-MP) resistance. Consistent with this finding, NT5C2ex6a and the R238W hotspot variant conferred comparable levels of resistance to 6-MP in B-ALL cells both in vitro and in vivo. Furthermore, both the NT5C2ex6a and R238W variants induced collateral sensitivity to the inosine monophosphate dehydrogenase (IMPDH) inhibitor mizoribine. These results ascribe an important role for splicing perturbations in chemotherapy resistance in relapsed B-ALL and suggest that IMPDH inhibitors, including the commonly used immunosuppressive agent mycophenolate mofetil, could be a valuable therapeutic option for treating thiopurine-resistant leukemias.
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BACKGROUND: TP53 alterations are common in certain pediatric cancers, making identification of putative germline variants through tumor genomic profiling crucial for disease management. METHODS: We analyzed TP53 alterations in 3123 tumors from 2788 pediatric patients sequenced using tumor-only or tumor-normal paired panels. Germline confirmatory testing was performed when indicated. Somatic and germline variants were classified based on published guidelines. RESULTS: In 248 tumors from 222 patients, 284 tier 1/2 TP53 sequence and small copy number variants were detected. Following germline classification, 86.6% of 142 unique variants were pathogenic or likely pathogenic. Confirmatory testing on 118 patients revealed germline TP53 variants in 28 of them (23 pathogenic or likely pathogenic and 5 of uncertain significance), suggesting a minimum Li-Fraumeni syndrome incidence of 0.8% (23/2788) in this cohort, 10.4% (23/222) in patients with TP53 variant-carrying tumors, and 19.5% (23/118) with available normal samples. About 25% (7/28) of patients with germline TP53 variants did not meet Li-Fraumeni syndrome diagnostic or testing criteria, while 20.9% (28/134) with confirmed or inferred somatic origins did. TP53 biallelic inactivation occurred in 75% of germline carrier tumors and was also prevalent in other groups, causing an elevated tumor-observed variant allelic fraction. Somatic evidence, however, including low variant allele fraction correctly identified only 27.8% (25/90) of patients with confirmed somatic TP53 variants. CONCLUSION: The high incidence and variable phenotype of Li-Fraumeni syndrome in this cohort highlights the importance of assessing germline status of TP53 variants identified in all pediatric tumors. Without clear somatic evidence, distinguishing somatic from germline origins is challenging. Classifying germline and somatic variants should follow appropriate guidelines.
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Mutação em Linhagem Germinativa , Síndrome de Li-Fraumeni , Neoplasias , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Criança , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/epidemiologia , Neoplasias/genética , Neoplasias/epidemiologia , Masculino , Feminino , Pré-Escolar , Adolescente , Predisposição Genética para Doença , Variações do Número de Cópias de DNA , Testes Genéticos/métodos , Prevalência , LactenteRESUMO
In multicellular organisms, sexed gonads have evolved that facilitate release of sperm versus eggs, and bilaterian animals purposefully combine their gametes via mating behaviors. Distinct neural circuits have evolved that control these physically different mating events for animals producing eggs from ovaries versus sperm from testis. In this review, we will describe the developmental mechanisms that sexually differentiate neural circuits across three major clades of bilaterian animals-Ecdysozoa, Deuterosomia, and Lophotrochozoa. While many of the mechanisms inducing somatic and neuronal sex differentiation across these diverse organisms are clade-specific rather than evolutionarily conserved, we develop a common framework for considering the developmental logic of these events and the types of neuronal differences that produce sex-differentiated behaviors. This article is categorized under: Congenital Diseases > Stem Cells and Development Neurological Diseases > Stem Cells and Development.
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Sêmen , Diferenciação Sexual , Masculino , Animais , Reprodução , Células Germinativas , EspermatozoidesRESUMO
Inherited bone marrow failure syndromes (IBMFS) are a group of heterogeneous disorders that account for â¼30% of pediatric cases of bone marrow failure and are often associated with developmental abnormalities and cancer predisposition. This article reports the laboratory validation and clinical utility of a large-scale, custom-designed next-generation sequencing panel, Children's Hospital of Philadelphia (CHOP) IBMFS panel, for the diagnosis of IBMFS in a cohort of pediatric patients. This panel demonstrated excellent analytic accuracy, with 100% sensitivity, ≥99.99% specificity, and 100% reproducibility on validation samples. In 269 patients with suspected IBMFS, this next-generation sequencing panel was used for identifying single-nucleotide variants, small insertions/deletions, and copy number variations in mosaic or nonmosaic status. Sixty-one pathogenic/likely pathogenic variants (54 single-nucleotide variants/insertions/deletions and 7 copy number variations) and 24 hypomorphic variants were identified, resulting in the molecular diagnosis of IBMFS in 21 cases (7.8%) and exclusion of IBMFS with a diagnosis of a blood disorder in 10 cases (3.7%). Secondary findings, including evidence of early hematologic malignancies and other hereditary cancer-predisposition syndromes, were observed in 9 cases (3.3%). The CHOP IBMFS panel was highly sensitive and specific, with a significant increase in the diagnostic yield of IBMFS. These findings suggest that next-generation sequencing-based panel testing should be a part of routine diagnostics in patients with suspected IBMFS.
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Anemia Aplástica , Doenças da Medula Óssea , Hemoglobinúria Paroxística , Humanos , Criança , Anemia Aplástica/diagnóstico , Anemia Aplástica/genética , Doenças da Medula Óssea/diagnóstico , Doenças da Medula Óssea/genética , Síndrome Congênita de Insuficiência da Medula Óssea , Variações do Número de Cópias de DNA/genética , Reprodutibilidade dos Testes , Hemoglobinúria Paroxística/diagnóstico , Hemoglobinúria Paroxística/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , NucleotídeosRESUMO
Aberrant skipping of coding exons in CD19 and CD22 compromises the response to immunotherapy in B-cell malignancies. Here, we showed that the MS4A1 gene encoding human CD20 also produces several messenger RNA (mRNA) isoforms with distinct 5' untranslated regions. Four variants (V1-4) were detected using RNA sequencing (RNA-seq) at distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma, only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform contained upstream open reading frames and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, whereas V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed chimeric antigen receptor T cells were able to kill both V3- and V1-expressing cells, but the bispecific T-cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on 4 postmosunetuzumab follicular lymphoma relapses and discovered that in 2 of them, the downregulation of CD20 was accompanied by a V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies.
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Infecções por Vírus Epstein-Barr , Neoplasias , Humanos , Processamento Alternativo , RNA Mensageiro/genética , Regiões 5' não Traduzidas , Infecções por Vírus Epstein-Barr/genética , Herpesvirus Humano 4/genética , Antígenos CD20/genética , Isoformas de Proteínas/genética , Imunoterapia , Biossíntese de Proteínas , Neoplasias/genéticaRESUMO
Aberrant skipping of coding exons in CD19 and CD22 compromises responses to immunotherapy for B-cell malignancies. Here, we show that the MS4A1 gene encoding human CD20 also produces several mRNA isoforms with distinct 5' untranslated regions (5'-UTR). Four variants (V1-4) were detectable by RNA-seq in distinct stages of normal B-cell differentiation and B-lymphoid malignancies, with V1 and V3 being the most abundant by far. During B-cell activation and Epstein-Barr virus infection, redirection of splicing from V1 to V3 coincided with increased CD20 positivity. Similarly, in diffuse large B-cell lymphoma only V3, but not V1, correlated with CD20 protein levels, suggesting that V1 might be translation-deficient. Indeed, the longer V1 isoform was found to contain upstream open reading frames (uORFs) and a stem-loop structure, which cooperatively inhibited polysome recruitment. By modulating CD20 isoforms with splice-switching Morpholino oligomers, we enhanced CD20 expression and anti-CD20 antibody rituximab-mediated cytotoxicity in a panel of B-cell lines. Furthermore, reconstitution of CD20-knockout cells with V3 mRNA led to the recovery of CD20 positivity, while V1-reconstituted cells had undetectable levels of CD20 protein. Surprisingly, in vitro CD20-directed CAR T cells were able to kill both V3- and V1-expressing cells, but the bispecific T cell engager mosunetuzumab was only effective against V3-expressing cells. To determine whether CD20 splicing is involved in immunotherapy resistance, we performed RNA-seq on four post-mosunetuzumab follicular lymphoma relapses and discovered that in two of them downregulation of CD20 was accompanied by the V3-to-V1 shift. Thus, splicing-mediated mechanisms of epitope loss extend to CD20-directed immunotherapies. Key Points: In normal & malignant human B cells, CD20 mRNA is alternatively spliced into four 5'-UTR isoforms, some of which are translation-deficient.The balance between translation-deficient and -competent isoforms modulates CD20 protein levels & responses to CD20-directed immunotherapies. Explanation of Novelty: We discovered that in normal and malignant B-cells, CD20 mRNA is alternatively spliced to generate four distinct 5'-UTRs, including the longer translation-deficient V1 variant. Cells predominantly expressing V1 were still sensitive to CD20-targeting chimeric antigen receptor T-cells. However, they were resistant to the bispecific anti-CD3/CD20 antibody mosunetuzumab, and the shift to V1 were observed in CD20-negative post-mosunetuzumab relapses of follicular lymphoma.
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BACKGROUND: It remains an important challenge to predict the functional consequences or clinical impacts of genetic variants in human diseases, such as cancer. An increasing number of genetic variants in cancer have been discovered and documented in public databases such as COSMIC, but the vast majority of them have no functional or clinical annotations. Some databases, such as CiVIC are available with manual annotation of functional mutations, but the size of the database is small due to the use of human annotation. Since the unlabeled data (millions of variants) typically outnumber labeled data (thousands of variants), computational tools that take advantage of unlabeled data may improve prediction accuracy. RESULT: To leverage unlabeled data to predict functional importance of genetic variants, we introduced a method using semi-supervised generative adversarial networks (SGAN), incorporating features from both labeled and unlabeled data. Our SGAN model incorporated features from clinical guidelines and predictive scores from other computational tools. We also performed comparative analysis to study factors that influence prediction accuracy, such as using different algorithms, types of features, and training sample size, to provide more insights into variant prioritization. We found that SGAN can achieve competitive performances with small labeled training samples by incorporating unlabeled samples, which is a unique advantage compared to traditional machine learning methods. We also found that manually curated samples can achieve a more stable predictive performance than publicly available datasets. CONCLUSIONS: By incorporating much larger samples of unlabeled data, the SGAN method can improve the ability to detect novel oncogenic variants, compared to other machine-learning algorithms that use only labeled datasets. SGAN can be potentially used to predict the pathogenicity of more complex variants such as structural variants or non-coding variants, with the availability of more training samples and informative features.
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Algoritmos , Neoplasias , Humanos , Aprendizado de Máquina , Neoplasias/genética , Bases de Dados Factuais , Aprendizado de Máquina SupervisionadoRESUMO
Congenital pulmonary airway malformations (CPAMs) have a range of morphologies with varying cyst sizes and histologic features (types 1 to 3). Evidence suggested they arise secondary to bronchial atresia, however, we recently showed that cases with type 1 and 3 morphology are driven by mosaic KRAS mutations. We hypothesized that 2 distinct mechanisms account for most CPAMs: one subset is secondary to KRAS mosaicism and another is due to bronchial atresia. Cases with type 2 histology, similar to sequestrations, would be related to obstruction and therefore negative for KRAS mutations regardless of cyst size. We sequenced KRAS exon 2 in type 2 CPAMs, cystic intralobar and extralobar sequestrations, and intrapulmonary bronchogenic cysts. All were negative. Most sequestrations had a large airway in the subpleural parenchyma adjacent to the systemic vessel, anatomically confirming bronchial obstruction. We compared morphology to type 1 and 3 CPAMs. On average, type 1 CPAMs had significantly larger cysts, but there remained substantial size overlap between KRAS mutant and wild-type lesions. Features of mucostasis were frequent in sequestrations and type 2 CPAMs, while their cysts were generally simple and round with flat epithelium. Features of cyst architectural and epithelial complexity were more common in type 1 and 3 CPAMs, which rarely showed mucostasis. Similarity in histologic features among cases that are negative for KRAS mutation support the hypothesis that, like sequestrations, the malformation of type 2 CPAMs is related to obstruction during development. A mechanistic approach to classification may improve existing subjective morphologic methods.
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Sequestro Broncopulmonar , Malformação Adenomatoide Cística Congênita do Pulmão , Cistos , Humanos , Sequestro Broncopulmonar/patologia , Proteínas Proto-Oncogênicas p21(ras) , Malformação Adenomatoide Cística Congênita do Pulmão/genética , Malformação Adenomatoide Cística Congênita do Pulmão/patologia , Cistos/patologia , Aberrações Cromossômicas , Pulmão/patologiaRESUMO
To assess the clinical implementation of the 2017 Standards and Guidelines for the Interpretation and Reporting of Sequence Variants in Cancer: A Joint Consensus Recommendation of the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists, identify content that may result in classification inconsistencies, and evaluate implementation barriers, an Association for Molecular Pathology Working Group conducted variant interpretation challenges and a guideline implementation survey. A total of 134 participants participated in the variant interpretation challenges, consisting of 11 variants in four cancer cases. Results demonstrate 86% (range, 54% to 94%) of the respondents correctly classified clinically significant variants, variants of uncertain significance, and benign/likely benign variants; however, only 59% (range, 39% to 84%) of responses agreed with the working group's consensus intended responses regarding both tiers and categories of clinical significance. In the implementation survey, 71% (157/220) of respondents have implemented the 2017 guidelines for variant classification and reporting either with or without modifications. Collectively, this study demonstrates that, although they may not yet be optimized, the 2017 guideline recommendations are being adopted for standardized somatic variant classification. The working group identified significant areas for future guideline improvement, including the need for a more granular and comprehensive classification system and education resources to meet the growing needs of both laboratory professionals and medical oncologists.
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Neoplasias , Patologia Molecular , Humanos , Estados Unidos , Patologistas , Neoplasias/diagnóstico , Neoplasias/genética , Sequenciamento de Nucleotídeos em Larga Escala , OncologiaRESUMO
BACKGROUND: The incidence and biology of IDH1/2 mutations in pediatric gliomas are unclear. Notably, current treatment approaches by pediatric and adult providers vary significantly. We describe the frequency and clinical outcomes of IDH1/2-mutant gliomas in pediatrics. METHODS: We performed a multi-institutional analysis of the frequency of pediatric IDH1/2-mutant gliomas, identified by next-generation sequencing (NGS). In parallel, we retrospectively reviewed pediatric IDH1/2-mutant gliomas, analyzing clinico-genomic features, treatment approaches, and outcomes. RESULTS: Incidence: Among 851 patients with pediatric glioma who underwent NGS, we identified 78 with IDH1/2 mutations. Among patients 0-9 and 10-21 years old, 2/378 (0.5%) and 76/473 (16.1%) had IDH1/2-mutant tumors, respectively. Frequency of IDH mutations was similar between low-grade glioma (52/570, 9.1%) and high-grade glioma (25/277, 9.0%). Four tumors were graded as intermediate histologically, with one IDH1 mutation. Outcome: Seventy-six patients with IDH1/2-mutant glioma had outcome data available. Eighty-four percent of patients with low-grade glioma (LGG) were managed observantly without additional therapy. For low-grade astrocytoma, 5-year progression-free survival (PFS) was 42.9% (95%CI:20.3-63.8) and, despite excellent short-term overall survival (OS), numerous disease-related deaths after year 10 were reported. Patients with high-grade astrocytoma had a 5-year PFS/OS of 36.8% (95%CI:8.8-66.4) and 84% (95%CI:50.1-95.6), respectively. Patients with oligodendroglioma had excellent OS. CONCLUSIONS: A subset of pediatric gliomas is driven by IDH1/2 mutations, with a higher rate among adolescents. The majority of patients underwent upfront observant management without adjuvant therapy. Findings suggest that the natural history of pediatric IDH1/2-mutant glioma may be similar to that of adults, though additional studies are needed.
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Astrocitoma , Neoplasias Encefálicas , Glioma , Adulto , Adolescente , Humanos , Criança , Estudos Retrospectivos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/terapia , Glioma/genética , Glioma/terapia , Astrocitoma/genética , Mutação , Genômica , Isocitrato Desidrogenase/genéticaRESUMO
BACKGROUND: To achieve replicative immortality, most cancers develop a telomere maintenance mechanism, such as reactivation of telomerase or alternative lengthening of telomeres (ALT). There are limited data on the prevalence and clinical significance of ALT in pediatric brain tumors, and ALT-directed therapy is not available. METHODS: We performed C-circle analysis (CCA) on 579 pediatric brain tumors that had corresponding tumor/normal whole genome sequencing through the Open Pediatric Brain Tumor Atlas (OpenPBTA). We detected ALT in 6.9% (n = 40/579) of these tumors and completed additional validation by ultrabright telomeric foci in situ on a subset of these tumors. We used CCA to validate TelomereHunter for computational prediction of ALT status and focus subsequent analyses on pediatric high-grade gliomas (pHGGs) Finally, we examined whether ALT is associated with recurrent somatic or germline alterations. RESULTS: ALT is common in pHGGs (n = 24/63, 38.1%), but occurs infrequently in other pediatric brain tumors (<3%). Somatic ATRX mutations occur in 50% of ALT+ pHGGs and in 30% of ALT- pHGGs. Rare pathogenic germline variants in mismatch repair (MMR) genes are significantly associated with an increased occurrence of ALT. CONCLUSIONS: We demonstrate that ATRX is mutated in only a subset of ALT+ pHGGs, suggesting other mechanisms of ATRX loss of function or alterations in other genes may be associated with the development of ALT in these patients. We show that germline variants in MMR are associated with the development of ALT in patients with pHGG.
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Neoplasias Encefálicas , Glioma , Humanos , Criança , Reparo de Erro de Pareamento de DNA , Homeostase do Telômero/genética , Proteína Nuclear Ligada ao X/genética , Glioma/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Mutação , Telômero/genética , Telômero/patologiaRESUMO
Angiosarcomas are rare, malignant soft tissue tumors in children that arise in a wide range of anatomical locations and have limited targeted therapies available. Here, we report a rare case of a pediatric angiosarcoma (pAS) with Li-Fraumeni syndrome (LFS) expressing a novel NOTCH1-ROS1 gene fusion. Although both NOTCH1 and ROS1 are established proto-oncogenes, our study is the first to describe the mechanistic role of NOTCH1-ROS1 fusion arising via intrachromosomal rearrangement. NOTCH1-ROS1 displayed potent neoplastic transformation propensity in vitro, and harbors tumorigenic potential in vivo, where it induced oncogenic activation of the MAPK, PI3K/mTOR, and JAK-STAT signaling pathways in a murine allograft model. We found an unexpected contribution of the NOTCH1 extracellular region in mediating NOTCH1-ROS1 activation and oncogenic function, highlighting the contribution of both NOTCH1 and ROS1 fusion partners in driving tumorigenicity. Interestingly, neither membrane localization nor fusion protein dimerization were found to be essential for NOTCH1-ROS1 fusion oncogenicity. To target NOTCH1-ROS1-driven tumors, we tested both NOTCH1-directed inhibitors and ROS1-targeted tyrosine kinase inhibitors (TKI) in heterologous models (NIH3T3, Ba/F3). Although NOTCH1 inhibitors did not suppress NOTCH1-ROS1-driven oncogenic growth, we found that oral entrectinib treatment effectively suppressed the growth of NOTCH-ROS1-driven tumors. Taken together, we report the first known pAS case with a novel NOTCH1-ROS1 alteration along with a detailed report on the function and therapeutic targeting of NOTCH1-ROS1. Our study highlights the importance of genomic profiling of rare cancers such as pAS to reveal actionable drivers and improve patient outcomes.
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Hemangiossarcoma , Proteínas Tirosina Quinases , Criança , Humanos , Camundongos , Animais , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Hemangiossarcoma/tratamento farmacológico , Hemangiossarcoma/genética , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas/genética , Células NIH 3T3 , Fusão Gênica , Receptor Notch1/genéticaRESUMO
Because the clinical impact of cancer genomics is being increasingly recognized, tumor sequencing will likely continue to expand in breadth and scope. Therefore, it is vital for laboratory professionals to adopt the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists guidelines and create a standardized system of classification and nomenclature for somatic variants. Combining robust bioinformatics pipelines with thorough data analysis is necessary to efficiently and reproducibly identify and assess the impact of clinically relevant variants.
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Testes Genéticos , Neoplasias , Biologia Computacional , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias/diagnóstico , Neoplasias/genética , Patologia MolecularRESUMO
Neuroblastoma evolution, heterogeneity, and resistance remain inadequately defined, suggesting a role for circulating tumor DNA (ctDNA) sequencing. To define the utility of ctDNA profiling in neuroblastoma, 167 blood samples from 48 high-risk patients were evaluated for ctDNA using comprehensive genomic profiling. At least one pathogenic genomic alteration was identified in 56% of samples and 73% of evaluable patients, including clinically actionable ALK and RAS-MAPK pathway variants. Fifteen patients received ALK inhibition (ALKi), and ctDNA data revealed dynamic genomic evolution under ALKi therapeutic pressure. Serial ctDNA profiling detected disease evolution in 15 of 16 patients with a recurrently identified variant-in some cases confirming disease progression prior to standard surveillance methods. Finally, ctDNA-defined ERRFI1 loss-of-function variants were validated in neuroblastoma cellular models, with the mutant proteins exhibiting loss of wild-type ERRFI1's tumor-suppressive functions. Taken together, ctDNA is prevalent in children with high-risk neuroblastoma and should be followed throughout neuroblastoma treatment. SIGNIFICANCE: ctDNA is prevalent in children with neuroblastoma. Serial ctDNA profiling in patients with neuroblastoma improves the detection of potentially clinically actionable and functionally relevant variants in cancer driver genes and delineates dynamic tumor evolution and disease progression beyond that of standard tumor sequencing and clinical surveillance practices. See related commentary by Deubzer et al., p. 2727. This article is highlighted in the In This Issue feature, p. 2711.