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PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive subtype most prevalent among women of Western Sub-Saharan African ancestry. It accounts for 15-25% of African American (AA) breast cancers (BC) and up to 80% of Ghanaian breast cancers, thus contributing to outcome disparities in BC for black women. The aggressive biology of TNBC has been shown to be regulated partially by breast cancer stem cells (BCSC) which mediate tumor recurrence and metastasis and are more abundant in African breast tumors. METHODS: We studied the biological differences between TNBC in women with African ancestry and those of Caucasian women by comparing the gene expression of the BCSC. From low-passage patient derived xenografts (PDX) from Ghanaian (GH), AA, and Caucasian American (CA) TNBCs, we sorted for and sequenced the stem cell populations and analyzed for differential gene enrichment. RESULTS: In our cohort of TNBC tumors, we observed that the ALDH expressing stem cells display distinct ethnic specific gene expression patterns, with the largest difference existing between the GH and AA ALDH+ cells. Furthermore, the tumors from the women of African ancestry [GH/AA] had ALDH stem cell (SC) enrichment for expression of immune related genes and processes. Among the significantly upregulated genes were CD274 (PD-L1), CXCR9, CXCR10 and IFI27, which could serve as potential drug targets. CONCLUSIONS: Further exploration of the role of immune regulated genes and biological processes in BCSC may offer insight into developing novel approaches to treating TNBC to help ameliorate survival disparities in women with African ancestry.
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Neoplasias de la Mama Triple Negativas , Negro o Afroamericano/genética , Femenino , Ghana/epidemiología , Humanos , Recurrencia Local de Neoplasia , Neoplasias de la Mama Triple Negativas/genética , Población BlancaRESUMEN
PURPOSE: There is a need for biomarkers of drug efficacy for targeted therapies in triple-negative breast cancer (TNBC). As a step toward this, we identify multi-omic molecular determinants of anti-TNBC efficacy in cell lines for a panel of oncology drugs. METHODS: Using 23 TNBC cell lines, drug sensitivity scores (DSS3) were determined using a panel of investigational drugs and drugs approved for other indications. Molecular readouts were generated for each cell line using RNA sequencing, RNA targeted panels, DNA sequencing, and functional proteomics. DSS3 values were correlated with molecular readouts using a FDR-corrected significance cutoff of p* < 0.05 and yielded molecular determinant panels that predict anti-TNBC efficacy. RESULTS: Six molecular determinant panels were obtained from 12 drugs we prioritized based on their efficacy. Determinant panels were largely devoid of DNA mutations of the targeted pathway. Molecular determinants were obtained by correlating DSS3 with molecular readouts. We found that co-inhibiting molecular correlate pathways leads to robust synergy across many cell lines. CONCLUSIONS: These findings demonstrate an integrated method to identify biomarkers of drug efficacy in TNBC where DNA predictions correlate poorly with drug response. Our work outlines a framework for the identification of novel molecular determinants and optimal companion drugs for combination therapy based on these correlates.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/etiología , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Biología Computacional/métodos , Relación Dosis-Respuesta a Droga , Resistencia a Antineoplásicos/genética , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Perfilación de la Expresión Génica , Humanos , Mutación , Proteómica , Resultado del Tratamiento , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is becoming the major chronic liver disease in many countries. Its pathogenesis is multifactorial, but twin and familial studies indicate significant heritability, which is not fully explained by currently known genetic susceptibility loci. Notably, mutations in genes encoding nuclear lamina proteins, including lamins, cause lipodystrophy syndromes that include NAFLD. We hypothesized that variants in lamina-associated proteins predispose to NAFLD and used a candidate gene-sequencing approach to test for variants in 10 nuclear lamina-related genes in a cohort of 37 twin and sibling pairs: 21 individuals with and 53 without NAFLD. Twelve heterozygous sequence variants were identified in four lamina-related genes (ZMPSTE24, TMPO, SREBF1, SREBF2). The majority of NAFLD patients (>90%) had at least one variant compared to <40% of controls (P < 0.0001). When only insertions/deletions and changes in conserved residues were considered, the difference between the groups was similarly striking (>80% versus <25%; P < 0.0001). Presence of a lamina variant segregated with NAFLD independently of the PNPLA3 I148M polymorphism. Several variants were found in TMPO, which encodes the lamina-associated polypeptide-2 (LAP2) that has not been associated with liver disease. One of these, a frameshift insertion that generates truncated LAP2, abrogated lamin-LAP2 binding, caused LAP2 mislocalization, altered endogenous lamin distribution, increased lipid droplet accumulation after oleic acid treatment in transfected cells, and led to cytoplasmic association with the ubiquitin-binding protein p62/SQSTM1. CONCLUSION: Several variants in nuclear lamina-related genes were identified in a cohort of twins and siblings with NAFLD; one such variant, which results in a truncated LAP2 protein and a dramatic phenotype in cell culture, represents an association of TMPO/LAP2 variants with NAFLD and underscores the potential importance of the nuclear lamina in NAFLD. (Hepatology 2018;67:1710-1725).
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Proteínas de Unión al ADN/genética , Proteínas de la Membrana/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Lámina Nuclear/genética , Adulto , Femenino , Técnica del Anticuerpo Fluorescente/métodos , Predisposición Genética a la Enfermedad , Variación Genética , Técnicas de Genotipaje/métodos , Heterocigoto , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Immunoblotting/métodos , Inmunoprecipitación/métodos , Hígado/metabolismo , Hígado/patología , Masculino , Persona de Mediana Edad , Mutación , Estudios Prospectivos , Hermanos , Espectrometría de Masas en Tándem/métodos , GemelosRESUMEN
Colorectal cancer arises in part from the cumulative effects of multiple gene lesions. Recent studies in selected cancer types have revealed significant intra-tumor genetic heterogeneity and highlighted its potential role in disease progression and resistance to therapy. We hypothesized the existence of significant intra-tumor genetic heterogeneity in rectal cancers involving variations in localized somatic mutations and copy number abnormalities. Two or three spatially disparate regions from each of six rectal tumors were dissected and subjected to the next-generation whole-exome DNA sequencing, Oncoscan SNP arrays, and targeted confirmatory sequencing and analysis. The resulting data were integrated to define subclones using SciClone. Mutant-allele tumor heterogeneity (MATH) scores, mutant allele frequency correlation, and mutation percent concordance were calculated, and copy number analysis including measurement of correlation between samples was performed. Somatic mutations profiles in individual cancers were similar to prior studies, with some variants found in previously reported significantly mutated genes and many patient-specific mutations in each tumor. Significant intra-tumor heterogeneity was identified in the spatially disparate regions of individual cancers. All tumors had some heterogeneity but the degree of heterogeneity was quite variable in the samples studied. We found that 67-97% of exonic somatic mutations were shared among all regions of an individual's tumor. The SciClone computational method identified 2-8 shared and unshared subclones in the spatially disparate areas in each tumor. MATH scores ranged from 7 to 41. Allele frequency correlation scores ranged from R(2)=0.69-0.96. Measurements of correlation between samples for copy number changes varied from R(2)=0.74-0.93. All tumors had some heterogeneity, but the degree was highly variable in the samples studied. The occurrence of significant intra-tumor heterogeneity may allow selected tumors to have a genetic reservoir to draw from in their evolutionary response to therapy and other challenges.
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Frecuencia de los Genes/genética , Heterogeneidad Genética , Neoplasias del Recto/genética , Anciano , Biología Computacional , Femenino , Dosificación de Gen/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Polimorfismo de Nucleótido Simple/genética , Neoplasias del Recto/química , Recto/químicaRESUMEN
BACKGROUND AND OBJECTIVE: Predicting response to therapy for each patient's tumor is critical to improving long-term outcomes for muscle-invasive bladder cancer. This study aims to establish ex vivo bladder cancer patient-derived organoid (PDO) models that are representative of patients' tumors and determine the potential efficacy of standard of care and curated experimental therapies. METHODS: Tumor material was collected prospectively from consented bladder cancer patients to generate short-term PDO models, which were screened against a panel of clinically relevant drugs in ex vivo three-dimensional culture. Multiomic profiling was utilized to validate the PDO models, establish the molecular characteristics of each tumor, and identify potential biomarkers of drug response. Gene expression (GEX) patterns between paired primary tissue and PDO samples were assessed using Spearman's rank correlation coefficients. Molecular correlates of therapy response were identified using Pearson correlation coefficients and Kruskal-Wallis tests with Dunn's post hoc pairwise comparison testing. KEY FINDINGS AND LIMITATIONS: A total of 106 tumors were collected from 97 patients, with 65 samples yielding sufficient material for complete multiomic molecular characterization and PDO screening with six to 32 drugs/combinations. Short-term PDOs faithfully represent the tumor molecular characteristics, maintain diverse cell types, and avoid shifts in GEX-based subtyping that accompany long-term PDO cultures. Utilizing an integrative approach, novel correlations between ex vivo drug responses and genomic alterations, GEX, and protein expression were identified, including a multiomic signature of gemcitabine response. The positive predictive value of ex vivo drug responses and the novel multiomic gemcitabine response signature need to be validated in future studies. CONCLUSIONS AND CLINICAL IMPLICATIONS: Short-term PDO cultures retain the molecular characteristics of tumor tissue and avoid shifts in expression-based subtyping that have plagued long-term cultures. Integration of multiomic profiling and ex vivo drug screening data identifies potential predictive biomarkers, including a novel signature of gemcitabine response. PATIENT SUMMARY: Better models are needed to predict patient response to therapy in bladder cancer. We developed a platform that uses short-term culture to best mimic each patient's tumor and assess potential sensitivity to therapeutics.
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Brain metastases are the most lethal progression event, in part because the biological processes underpinning brain metastases are poorly understood. There is a paucity of realistic models of metastasis, as current in vivo murine models are slow to manifest metastasis. We set out to delineate metabolic and secretory modulators of brain metastases by utilizing two models consisting of in vitro microfluidic devices: 1) a blood brain niche (BBN) chip that recapitulates the blood-brain-barrier and niche; and 2) a migration chip that assesses cell migration. We report secretory cues provided by the brain niche that attract metastatic cancer cells to colonize the brain niche region. Astrocytic Dkk-1 is increased in response to brain-seeking breast cancer cells and stimulates cancer cell migration. Brain-metastatic cancer cells under Dkk-1 stimulation increase gene expression of FGF-13 and PLCB1. Further, extracellular Dkk-1 modulates cancer cell migration upon entering the brain niche.
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Metastases are the leading cause of death in cancer patients. RhoC, a member of the Rho GTPase family, has been shown to facilitate metastasis of aggressive breast cancer cells by influencing motility, invasion, and chemokine secretion, but as yet there is no integrated model of the precise mechanism of how RhoC promotes metastasis. A common phenotypic characteristic of metastatic cells influenced by these mechanisms is dysregulation of cell-cell junctions. Thus, we set out to study how RhoA- and RhoC-GTPase influence the cell-cell junctions in aggressive breast cancers. We demonstrate that CRISPR-Cas9 knockout of RhoC in SUM 149 and MDA 231 breast cancer cells results in increased normalization of junctional integrity denoted by junction protein expression/colocalization. In functional assessments of junction stability, RhoC knockout cells have increased barrier integrity and increased cell-cell adhesion compared to wild-type cells. Whole exome RNA sequencing and targeted gene expression profiling demonstrate decreased expression of Type I interferon-stimulated genes in RhoC knockout cells compared to wild-type, and subsequent treatment with interferon-alpha resulted in significant increases in adhesion and decreases in invasiveness of wild-type cells and a dampened response to interferon-alpha stimulation with respect to adhesion and invasiveness in RhoC knockout cells. We delineate a key role of RhoC-GTPase in modulation of junctions and response to interferon, which supports inhibition of RhoC as a potential anti-invasion therapeutic strategy.
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INTRODUCTION: Chronic pancreatitis is associated with an increased risk of developing pancreatic cancer, and patients with inherited forms of pancreatitis are at greatest risk. We investigated whether clinical severity of pancreatitis could also be an indicator of cancer risk independent of etiology by performing targeted DNA sequencing to assess the mutational burden in 55 cancer-associated genes. METHODS: Using picodroplet digital polymerase chain reaction and next-generation sequencing, we reported the genomic profiles of pancreases from severe clinical cases of chronic pancreatitis that necessitated palliative total pancreatectomy with islet autotransplantation. RESULTS: We assessed 57 tissue samples from 39 patients with genetic and idiopathic etiologies and found that despite the clinical severity of disease, there was no corresponding increase in mutational burden. The average allele frequency of somatic variants was 1.19% (range 1.00%-5.97%), and distinct regions from the same patient displayed genomic heterogeneity, suggesting that these variants are subclonal. Few oncogenic KRAS mutations were discovered (7% of all samples), although we detected evidence of frequent cancer-related variants in other genes such as TP53, CDKN2A, and SMAD4. Of note, tissue samples with oncogenic KRAS mutations and samples from patients with PRSS1 mutations harbored an increased total number of somatic variants, suggesting that these patients may have increased genomic instability and could be at an increased risk of developing pancreatic cancer. DISCUSSION: Overall, we showed that even in those patients with chronic pancreatitis severe enough to warrant total pancreatectomy with islet autotransplantation, pancreatic cancer-related mutational burden is not appreciably increased.
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Carcinoma Ductal Pancreático/genética , Mutación , Neoplasias Pancreáticas/genética , Pancreatitis Crónica/genética , Adulto , Edad de Inicio , Niño , Femenino , Humanos , Trasplante de Islotes Pancreáticos , Masculino , Pancreatectomía , Pancreatitis Crónica/complicaciones , Pancreatitis Crónica/cirugía , Gravedad del Paciente , Polimorfismo de Nucleótido Simple , Proteínas Proto-Oncogénicas p21(ras)/genética , Tripsina/genéticaRESUMEN
During aging and neuromuscular diseases, there is a progressive loss of skeletal muscle volume and function impacting mobility and quality of life. Muscle loss is often associated with denervation and a loss of resident muscle stem cells (satellite cells or MuSCs); however, the relationship between MuSCs and innervation has not been established. Herein, we administered severe neuromuscular trauma to a transgenic murine model that permits MuSC lineage tracing. We show that a subset of MuSCs specifically engraft in a position proximal to the neuromuscular junction (NMJ), the synapse between myofibers and motor neurons, in healthy young adult muscles. In aging and in a mouse model of neuromuscular degeneration (Cu/Zn superoxide dismutase knockout - Sod1-/-), this localized engraftment behavior was reduced. Genetic rescue of motor neurons in Sod1-/- mice reestablished integrity of the NMJ in a manner akin to young muscle and partially restored MuSC ability to engraft into positions proximal to the NMJ. Using single cell RNA-sequencing of MuSCs isolated from aged muscle, we demonstrate that a subset of MuSCs are molecularly distinguishable from MuSCs responding to myofiber injury and share similarity to synaptic myonuclei. Collectively, these data reveal unique features of MuSCs that respond to synaptic perturbations caused by aging and other stressors.
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Envejecimiento , Músculo Esquelético/lesiones , Mioblastos Esqueléticos/fisiología , Unión Neuromuscular/fisiología , Superóxido Dismutasa-1/deficiencia , Animales , Femenino , Masculino , Ratones NoqueadosRESUMEN
PURPOSE: This study was designed to assess the ability of perioperative circulating tumor DNA (ctDNA) to predict surgical outcome and recurrence following neoadjuvant chemoradiation for locally advanced rectal cancer (LARC). MATERIALS AND METHODS: Twenty-nine patients with newly diagnosed LARC treated between January 2014 and February 2018 were enrolled. Patients received long-course neoadjuvant chemoradiation prior to surgery. Plasma ctDNA was collected at baseline, preoperatively, and postoperatively. Next-generation sequencing was used to identify mutations in the primary tumor, and mutation-specific droplet digital polymerase chain reaction was used to assess mutation fraction in ctDNA. RESULTS: The median age was 54 years. The overall margin-negative, node-negative resection rate was 73% and was significantly higher among patients with undetectable preoperative ctDNA (n = 17, 88%) versus patients with detectable preoperative ctDNA (n = 9, 44%; P = .028). Undetectable ctDNA was also associated with more favorable neoadjuvant rectal scores (univariate linear regression, P = .029). Recurrence-free survival (RFS) was calculated for the subset (n = 19) who both underwent surgery and had postoperative ctDNA available. At a median follow-up of 20 months, patients with detectable postoperative ctDNA experienced poorer RFS (hazard ratio, 11.56; P = .007). All patients (4 of 4) with detectable postoperative ctDNA recurred (positive predictive value = 100%), whereas only 2 of 15 patients with undetectable ctDNA recurred (negative predictive value = 87%). CONCLUSION: Among patients treated with neoadjuvant chemoradiation for LARC, patients with undetectable preoperative ctDNA were more likely to have a favorable surgical outcome as measured by the rate of margin-negative, node-negative resections and neoadjuvant rectal score. Furthermore, we have confirmed prior reports indicating that detectable postoperative ctDNA is associated with worse RFS. Future prospective study is needed to assess the potential for ctDNA to assist with personalizing treatment for LARC.
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ADN Tumoral Circulante/sangre , Terapia Neoadyuvante , Neoplasias del Recto/sangre , Neoplasias del Recto/terapia , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Valor Predictivo de las Pruebas , Neoplasias del Recto/patología , Neoplasias del Recto/cirugía , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Mutant isocitrate-dehydrogenase 1 (mIDH1) synthesizes the oncometabolite 2-hydroxyglutarate (2HG), which elicits epigenetic reprogramming of the glioma cells' transcriptome by inhibiting DNA and histone demethylases. We show that the efficacy of immune-stimulatory gene therapy (TK/Flt3L) is enhanced in mIDH1 gliomas, due to the reprogramming of the myeloid cells' compartment infiltrating the tumor microenvironment (TME). We uncovered that the immature myeloid cells infiltrating the mIDH1 TME are mainly nonsuppressive neutrophils and preneutrophils. Myeloid cell reprogramming was triggered by granulocyte colony-stimulating factor (G-CSF) secreted by mIDH1 glioma stem/progenitor-like cells. Blocking G-CSF in mIDH1 gliomabearing mice restores the inhibitory potential of the tumor-infiltrating myeloid cells, accelerating tumor progression. We demonstrate that G-CSF reprograms bone marrow granulopoiesis, resulting in noninhibitory myeloid cells within mIDH1 glioma TME and enhancing the efficacy of immune-stimulatory gene therapy.
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The zymogen granule is the specialized organelle in pancreatic acinar cells for digestive enzyme storage and regulated secretion and is a classic model for studying secretory granule function. Our long term goal is to develop a comprehensive architectural model for zymogen granule membrane (ZGM) proteins that would direct new hypotheses for subsequent functional studies. Our initial proteomics analysis focused on identification of proteins from purified ZGM (Chen, X., Walker, A. K., Strahler, J. R., Simon, E. S., Tomanicek-Volk, S. L., Nelson, B. B., Hurley, M. C., Ernst, S. A., Williams, J. A., and Andrews, P. C. (2006) Organellar proteomics: analysis of pancreatic zymogen granule membranes. Mol. Cell. Proteomics 5, 306-312). In the current study, a new global topology analysis of ZGM proteins is described that applies isotope enrichment methods to a protease protection protocol. Our results showed that tryptic peptides of ZGM proteins were separated into two distinct clusters according to their isobaric tag for relative and absolute quantification (iTRAQ) ratios for proteinase K-treated versus control zymogen granules. The low iTRAQ ratio cluster included cytoplasm-orientated membrane and membrane-associated proteins including myosin V, vesicle-associated membrane proteins, syntaxins, and all the Rab proteins. The second cluster having unchanged ratios included predominantly luminal proteins. Because quantification is at the peptide level, this technique is also capable of mapping both cytoplasm- and lumen-orientated domains from the same transmembrane protein. To more accurately assign the topology, we developed a statistical mixture model to provide probabilities for identified peptides to be cytoplasmic or luminal based on their iTRAQ ratios. By implementing this approach to global topology analysis of ZGM proteins, we report here an experimentally constrained, comprehensive topology model of identified zymogen granule membrane proteins. This model contributes to a firm foundation for developing a higher order architecture model of the ZGM and for future functional studies of individual ZGM proteins.
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Proteínas de la Membrana/análisis , Proteínas de la Membrana/química , Páncreas/química , Vesículas Secretoras/química , Secuencia de Aminoácidos , Western Blotting , Citoplasma/metabolismo , Endopeptidasa K/metabolismo , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/metabolismo , Proteómica , Reproducibilidad de los Resultados , Tripsina/metabolismoRESUMEN
Dissecting tumor heterogeneity is a key to understanding the complex mechanisms underlying drug resistance in cancers. The rich literature of pioneering studies on tumor heterogeneity analysis spurred a recent community-wide benchmark study that compares diverse modeling algorithms. Here we present FastClone, a top-performing algorithm in accuracy in this benchmark. FastClone improves over existing methods by allowing the deconvolution of subclones that have independent copy number variation events within the same chromosome regions. We characterize the behavior of FastClone in identifying subclones using stage III colon cancer primary tumor samples as well as simulated data. It achieves approximately 100-fold acceleration in computation for both simulated and patient data. The efficacy of FastClone will allow its application to large-scale data and clinical data, and facilitate personalized medicine in cancers.
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Algoritmos , Variaciones en el Número de Copia de ADN , Neoplasias/genética , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Biología Computacional/métodos , Simulación por Computador , ADN de Neoplasias/genética , Resistencia a Antineoplásicos/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Genéticos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Filogenia , Medicina de Precisión , Análisis de Secuencia de ADNRESUMEN
Identifying better predictive and prognostic biomarkers for the diagnosis and treatment of triple negative breast cancer (TNBC) is complicated by tumor heterogeneity ranging from responses to therapy, mutational burden, and clonal evolution. To overcome the gap in our understanding of tumor heterogeneity, we hypothesized that isolating and studying the gene expression profile of invasive tumor cell subpopulations would be a crucial step towards achieving this goal. In this report, we utilized a fluidic device previously reported to be capable of supporting long-term three-dimensional growth and invasion dynamics of cancer cells. Live invading and matched non-invading SUM149 inflammatory breast cancer cells were enriched using this device and these two functionally distinct subpopulations were tested for differences in gene expression using a gene expression microarray. 305 target genes were identified to have altered expression in the invading cells compared to the non-invading tumoroid cells. Gene ontology analysis of the gene panel identified multiple biological roles ranging from extracellular matrix reorganization to modulation of the immune response and Rho signaling. Interestingly, the genes associated with the invasion front differ between different samples, consistent with inter- and intra-tumor heterogeneity. This work suggests the impact of heterogeneity in biomarker discovery should be considered as cancer therapy increasingly heads towards a personalized approach.
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Regulación Neoplásica de la Expresión Génica , Invasividad Neoplásica/genética , Neoplasias de la Mama Triple Negativas/genética , Biomarcadores de Tumor/genética , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Heterogeneidad Genética , Humanos , Invasividad Neoplásica/patología , Transcriptoma , Neoplasias de la Mama Triple Negativas/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Gliomas are the most common primary brain tumors. High-Grade Gliomas have a median survival (MS) of 18 months, while Low-Grade Gliomas (LGGs) have an MS of approximately 7.3 years. Seventy-six percent of patients with LGG express mutated isocitrate dehydrogenase (mIDH) enzyme. Survival of these patients ranges from 1 to 15 years, and tumor mutational burden ranges from 0.28 to 3.85 somatic mutations/megabase per tumor. We tested the hypothesis that the tumor mutational burden would predict the survival of patients with tumors bearing mIDH. METHODS: We analyzed the effect of tumor mutational burden on patients' survival using clinical and genomic data of 1199 glioma patients from The Cancer Genome Atlas and validated our results using the Glioma Longitudinal AnalySiS consortium. RESULTS: High tumor mutational burden negatively correlates with the survival of patients with LGG harboring mIDH (P = .005). This effect was significant for both Oligodendroglioma (LGG-mIDH-O; MS = 2379 vs 4459 days in high vs low, respectively; P = .005) and Astrocytoma (LGG-mIDH-A; MS = 2286 vs 4412 days in high vs low respectively; P = .005). There was no differential representation of frequently mutated genes (eg, TP53, ATRX, CIC, and FUBP) in either group. Gene set enrichment analysis revealed an enrichment in Gene Ontologies related to cell cycle, DNA-damage response in high versus low tumor mutational burden. Finally, we identified 6 gene sets that predict survival for LGG-mIDH-A and LGG-mIDH-O. CONCLUSIONS: we demonstrate that tumor mutational burden is a powerful, robust, and clinically relevant prognostic factor of MS in mIDH patients.
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This chapter contains a step-by-step protocol for identifying somatic SNPs and small Indels from next-generation sequencing data of tumor samples and matching normal samples. The workflow presented here is largely based on the Broad Institute's "Best Practices" guidelines and makes use of their Genome Analysis Toolkit (GATK) platform. Variants are annotated with population allele frequencies and curated resources such as GnomAD and ClinVar and curated effect predictions from dbNSFP using VCFtools, SnpEff, and SnpSift.
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Secuenciación del Exoma/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Biología Computacional/métodos , Variación Genética , Genoma Humano , Humanos , Anotación de Secuencia Molecular , Neoplasias/genética , Programas InformáticosRESUMEN
Purpose: Recent studies have highlighted the existence of subclones in tumors. Lymph nodes are generally the first location of metastasis for most solid epithelial tumors, including colorectal cancer. We sought to understand the genetic origin of lymph node metastasis in colorectal cancer by evaluating the relationship between colorectal cancer subclones present in primary tumors and lymph nodes.Experimental Design: A total of 33 samples from seven colorectal cancers, including two or three spatially disparate regions from each primary tumor and one to four matched lymph nodes for each tumor, underwent next-generation whole-exome DNA sequencing, Affymetrix OncoScan SNP arrays, and targeted deep confirmatory sequencing. We performed mapping between SNPs and copy number events from the primary tumor and matched lymph node samples, allowing us to profile heterogeneity and the mutational origin of lymph node metastases. The computational method PyClone was used to define subclones within each tumor. The method Clonality Inference in Tumors Using Phylogeny (CITUP) was subsequently used to infer phylogenetic relationships among subclones.Results: We found that there was substantial heterogeneity in mutations and copy number changes among all samples from any given patient. For each patient, the primary tumor regions and matched lymph node metastases were each polyclonal, and the clonal populations differed from one lymph node to another. In some patients, the cancer cell populations in a given lymph node originated from multiple distinct regions of a tumor.Conclusions: Our data support a model of lymph node metastatic spread in colorectal cancer whereby metastases originate from multiple waves of seeding from the primary tumor over time. Clin Cancer Res; 24(9); 2214-24. ©2017 AACRSee related commentary by Gerlinger, p. 2032.
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Evolución Clonal , Neoplasias Colorrectales/etiología , Neoplasias Colorrectales/patología , Ganglios Linfáticos/patología , Adulto , Anciano , Mapeo Cromosómico , Evolución Clonal/genética , Biología Computacional/métodos , Variaciones en el Número de Copia de ADN , Femenino , Perfilación de la Expresión Génica , Heterogeneidad Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Modelos Biológicos , Estadificación de Neoplasias , Filogenia , Polimorfismo de Nucleótido Simple , TranscriptomaRESUMEN
Current mass spectrometers provide a number of alternative methodologies for producing tandem mass spectra specifically for phosphopeptide analysis. In particular, generation of MS(3) spectra in a data-dependent manner upon detection of the neutral loss of a phosphoric acid in MS(2) spectra is a popular technique for circumventing the problem of poor phosphopeptide backbone fragmentation. The newer Multistage Activation method provides another option. Both these strategies require additional cycle time on the instrument and therefore reduce the number of spectra that can be measured in the same amount of time. Additional informatics is often required to make most efficient use of the additional information provided by these spectra as well. This work presents a comparison of several commonly used mass spectrometry methods for the study of phosphopeptide-enriched samples: an MS(2)-only method, a Multistage Activation method, and an MS(2)/MS(3) data-dependent neutral loss method. Several strategies for dealing effectively with the resulting MS(3) data in the latter approach are also presented and compared. The overall goal is to infer whether any one methodology performs significantly better than another for identifying phosphopeptides. On data presented here, the Multistage Activation methodology is demonstrated to perform optimally and does not result in significant loss of unique peptide identifications.
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Espectrometría de Masas , Fosfopéptidos/análisis , Algoritmos , Secuencia de Aminoácidos , Espectrometría de Masas/instrumentación , Espectrometría de Masas/métodos , Fosfopéptidos/genéticaRESUMEN
Improvements in ion trap instrumentation have made n-dimensional mass spectrometry more practical. The overall goal of the study was to describe a model for making use of MS(2) and MS(3) information in mass spectrometry experiments. We present a statistical model for adjusting peptide identification probabilities based on the combined information obtained by coupling peptide assignments of consecutive MS(2) and MS(3) spectra. Using two data sets, a mixture of known proteins and a complex phosphopeptide-enriched sample, we demonstrate an increase in discriminating power of the adjusted probabilities compared with models using MS(2) or MS(3) data only. This work also addresses the overall value of generating MS(3) data as compared with an MS(2)-only approach with a focus on the analysis of phosphopeptide data.
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Espectrometría de Masas/métodos , Modelos Estadísticos , Péptidos/análisis , Secuencia de Aminoácidos , Animales , Bases de Datos de Proteínas , Datos de Secuencia Molecular , Péptidos/química , Fosfopéptidos/análisis , Fosfopéptidos/química , ProbabilidadRESUMEN
Manual analysis of mass spectrometry data is a current bottleneck in high throughput proteomics. In particular, the need to manually validate the results of mass spectrometry database searching algorithms can be prohibitively time-consuming. Development of software tools that attempt to quantify the confidence in the assignment of a protein or peptide identity to a mass spectrum is an area of active interest. We sought to extend work in this area by investigating the potential of recent machine learning algorithms to improve the accuracy of these approaches and as a flexible framework for accommodating new data features. Specifically we demonstrated the ability of boosting and random forest approaches to improve the discrimination of true hits from false positive identifications in the results of mass spectrometry database search engines compared with thresholding and other machine learning approaches. We accommodated additional attributes obtainable from database search results, including a factor addressing proton mobility. Performance was evaluated using publically available electrospray data and a new collection of MALDI data generated from purified human reference proteins.