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Using dasatinib linked to E3 ligase ligands, we identified a potent and selective dual Csk/c-Src PROTAC degrader. We then replaced dasatinib, the c-Src-directed ligand, with a conformation-selective analogue that stabilizes the αC-helix-out conformation of c-Src. Using the αC-helix-out ligand, we identified a PROTAC that is potent and selective for c-Src. We demonstrated a high degree of catalysis with our c-Src PROTACs. Using our c-Src PROTACs, we identified pharmacological advantages of c-Src degradation compared to inhibition with respect to cancer cell proliferation.
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Ubiquitina-Proteína Ligases , Dasatinibe/farmacologia , Proteína Tirosina Quinase CSK/metabolismo , Ligantes , Proliferação de Células , Ubiquitina-Proteína Ligases/metabolismo , ProteóliseRESUMO
The development of novel therapies for brain metastases is an unmet need. Brain metastases may have unique molecular features that could be explored as therapeutic targets. A better understanding of the drug sensitivity of live cells coupled to molecular analyses will lead to a rational prioritization of therapeutic candidates. We evaluated the molecular profiles of 12 breast cancer brain metastases (BCBM) and matched primary breast tumors to identify potential therapeutic targets. We established six novel patient-derived xenograft (PDX) from BCBM from patients undergoing clinically indicated surgical resection of BCBM and used the PDXs as a drug screening platform to interrogate potential molecular targets. Many of the alterations were conserved in brain metastases compared with the matched primary. We observed differential expressions in the immune-related and metabolism pathways. The PDXs from BCBM captured the potentially targetable molecular alterations in the source brain metastases tumor. The alterations in the PI3K pathway were the most predictive for drug efficacy in the PDXs. The PDXs were also treated with a panel of over 350 drugs and demonstrated high sensitivity to histone deacetylase and proteasome inhibitors. Our study revealed significant differences between the paired BCBM and primary breast tumors with the pathways involved in metabolisms and immune functions. While molecular targeted drug therapy based on genomic profiling of tumors is currently evaluated in clinical trials for patients with brain metastases, a functional precision medicine strategy may complement such an approach by expanding potential therapeutic options, even for BCBM without known targetable molecular alterations. Significance: Examining genomic alterations and differentially expressed pathways in brain metastases may inform future therapeutic strategies. This study supports genomically-guided therapy for BCBM and further investigation into incorporating real-time functional evaluation will increase confidence in efficacy estimations during drug development and predictive biomarker assessment for BCBM.
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Neoplasias Encefálicas , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Medicina de Precisão , Fosfatidilinositol 3-Quinases/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológicoRESUMO
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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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 , Mutação , Neoplasias Pancreáticas/genética , Pancreatite Crônica/genética , Adulto , Idade de Início , Criança , Feminino , Humanos , Transplante das Ilhotas Pancreáticas , Masculino , Pancreatectomia , Pancreatite Crônica/complicações , Pancreatite Crônica/cirurgia , Gravidade do Paciente , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas p21(ras)/genética , Tripsina/genéticaRESUMO
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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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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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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Envelhecimento , Músculo Esquelético/lesões , Mioblastos Esqueléticos/fisiologia , Junção Neuromuscular/fisiologia , Superóxido Dismutase-1/deficiência , Animais , Feminino , Masculino , Camundongos KnockoutRESUMO
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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DNA Tumoral Circulante/sangue , Terapia Neoadjuvante , Neoplasias Retais/sangue , Neoplasias Retais/terapia , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Valor Preditivo dos Testes , Neoplasias Retais/patologia , Neoplasias Retais/cirurgia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
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 Mama Triplo Negativas , Negro ou Afro-Americano/genética , Feminino , Gana/epidemiologia , Humanos , Recidiva Local de Neoplasia , Neoplasias de Mama Triplo Negativas/genética , População BrancaRESUMO
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 , Variações do Número de Cópias de DNA , Neoplasias/genética , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Biologia Computacional/métodos , Simulação por Computador , DNA de Neoplasias/genética , Resistencia a Medicamentos Antineoplásicos/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Modelos Genéticos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Filogenia , Medicina de Precisão , Análise de Sequência de DNARESUMO
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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INTRODUCTION: We aimed to estimate the effects of a family history of colorectal cancer (CRC) or esophageal cancer on the risk of Barrett's esophagus (BE) and identify variants in cancer genes that may explain the association. METHODS: Men scheduled for screening colonoscopy were recruited to undergo upper endoscopy. Cases and noncases were screenees with and without BE, respectively. The effects of family histories on BE were estimated with logistic regression, adjusting for the potential confounders. We additionally recruited men recently diagnosed with BE by clinically indicated endoscopies. Banked germline DNA from cases of BE with ≥2 first-degree relatives (FDRs) with CRC and/or an FDR with esophageal cancer underwent next-generation sequencing using a panel of 275 cancer genes. RESULTS: Of the 822 men screened for CRC who underwent upper endoscopy, 70 were newly diagnosed with BE (8.5%). BE was associated with family histories of esophageal cancer (odds ratio = 2.63; 95% confidence interval = 1.07-6.47) and CRC in ≥2 vs 0 FDRs (odds ratio = 3.73; 95% confidence interval = 0.898-15.4). DNA analysis of subjects with both BE and a family history of cancer identified one or more germline variants of interest in genes associated with cancer predisposition in 10 of 14 subjects, including the same novel variant in EPHA5 in 2 unrelated individuals. DISCUSSION: We found an increased risk for BE associated with a family history of esophageal cancer or CRC. Although analysis of germline DNA yielded no clinically actionable findings, discovery of the same EPHA5 variant of uncertain significance in 2 of 14 cases merits additional investigation.
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Esôfago de Barrett/genética , Biomarcadores Tumorais/genética , Neoplasias Colorretais/epidemiologia , Neoplasias Esofágicas/epidemiologia , Anamnese/estatística & dados numéricos , Idoso , Esôfago de Barrett/diagnóstico , Esôfago de Barrett/epidemiologia , Esôfago de Barrett/patologia , Estudos de Casos e Controles , Colonoscopia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Neoplasias Esofágicas/genética , Esofagoscopia , Esôfago/diagnóstico por imagem , Esôfago/patologia , Predisposição Genética para Doença , Testes Genéticos , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
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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Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica/genética , Neoplasias de Mama Triplo Negativas/genética , Biomarcadores Tumorais/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Ontologia Genética , Heterogeneidade Genética , Humanos , Invasividade Neoplásica/patologia , Transcriptoma , Neoplasias de Mama Triplo Negativas/patologia , Células Tumorais CultivadasRESUMO
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/farmacologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/etiologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Biologia Computacional/métodos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/genética , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Perfilação da Expressão Gênica , Humanos , Mutação , Proteômica , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Treatment of locally advanced rectal cancer includes chemotherapy, radiation, and surgery but patient responses to neoadjuvant treatment are variable. We have shown that rectal tumors are comprised of multiple genetically distinct sub-clones. Unique sub-clones within tumors may harbor mutations which contribute to inter-patient variation in response to neoadjuvant chemoradiotherapy (nCRT). Analysis of the influence of nCRT on the extent and nature of intra-tumoral genetic heterogeneity in rectal cancer may provide insights into mechanisms of resistance. Locally advanced rectal cancer patients underwent pre-treatment biopsies. At the time of surgery, tissue from the treated tumor was obtained and analyzed. Pre- and post-treatment specimens were subjected to whole exome and confirmatory deep sequencing for somatic mutations. Copy number variation was assessed using OncoScan SNP arrays. Genomic data were analyzed using PyClone to identify sub-clonal tumor population following nCRT. Alterations that persisted or were enriched in the post-treatment tumor specimen following nCRT were defined for each patient. Thirty-two samples were obtained from ten patients. PyClone identified 2 to 10 genetic sub-clones per tumor. Substantial changes in the proportions of individual sub-clones in pre- versus post-treatment tumor material were found in all patients. Resistant sub-clones recurrently contained mutations in TP53, APC, ABCA13, MUC16, and THSD4. Recurrent copy number variation was observed across multiple chromosome regions after nCRT. Pathway analysis including variant alleles and copy number changes associated with resistant sub-clones revealed significantly altered pathways, especially those linked to the APC and TP53 genes, which were the two most frequently mutated genes. Intra-tumoral heterogeneity is evident in pre-treatment rectal cancer. Following treatment, sub-clonal populations are selectively modified and enrichment of a subset of pre-treatment sub-clones is seen. Further studies are needed to define recurrent alterations at diagnosis that may contribute to resistance to nCRT.
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Antineoplásicos/farmacologia , Evolução Clonal/efeitos dos fármacos , Evolução Clonal/genética , Heterogeneidade Genética , Neoplasias Retais/genética , Adulto , Idoso , Antineoplásicos/uso terapêutico , Biomarcadores Tumorais , Quimiorradioterapia , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Terapia Neoadjuvante , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Retais/metabolismo , Neoplasias Retais/patologia , Neoplasias Retais/terapia , Transdução de Sinais , Resultado do Tratamento , Sequenciamento do ExomaRESUMO
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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Sequenciamento do Exoma/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Biologia Computacional/métodos , Variação Genética , Genoma Humano , Humanos , Anotação de Sequência Molecular , Neoplasias/genética , SoftwareRESUMO
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 Ligação a DNA/genética , Proteínas de Membrana/genética , Hepatopatia Gordurosa não Alcoólica/genética , Lâmina Nuclear/genética , Adulto , Feminino , Imunofluorescência/métodos , Predisposição Genética para Doença , Variação Genética , Técnicas de Genotipagem/métodos , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Immunoblotting/métodos , Imunoprecipitação/métodos , Fígado/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Mutação , Estudos Prospectivos , Irmãos , Espectrometria de Massas em Tandem/métodos , GêmeosRESUMO
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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Evolução Clonal , Neoplasias Colorretais/etiologia , Neoplasias Colorretais/patologia , Linfonodos/patologia , Adulto , Idoso , Mapeamento Cromossômico , Evolução Clonal/genética , Biologia Computacional/métodos , Variações do Número de Cópias de DNA , Feminino , Perfilação da Expressão Gênica , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Estadiamento de Neoplasias , Filogenia , Polimorfismo de Nucleotídeo Único , TranscriptomaRESUMO
BACKGROUND & AIMS: The incidence of colorectal cancer (CRC) in individuals younger than 50 years is increasing. We sought to ascertain the proportion of young CRC cases associated with genetic predisposition. METHODS: We performed a retrospective study of individuals diagnosed with CRC at an age younger than 50 years, evaluated by the clinical genetics service at a single tertiary care cancer center from 1998 through 2015. We collected data on patient histories, tumor phenotypes, and results of germline DNA sequencing. For subjects with uninformative clinical evaluations, germline DNA samples were (re)sequenced using a research-based next-generation sequencing multigene panel. The primary outcome was identification of a pathogenic germline mutation associated with cancer predisposition. RESULTS: Of 430 young CRC cases, 111 (26%) had a first-degree relative with CRC. Forty-one of the subjects with CRC (10%) had tumors with histologic evidence for mismatch repair deficiency. Of 315 subjects who underwent clinical germline sequencing, 79 had mutations associated with a hereditary cancer syndrome and 21 had variants of uncertain significance. Fifty-six subjects had pathogenic variants associated with Lynch syndrome (25 with mutations in MSH2, 24 with mutations in MLH1, 5 with mutations in MSH6, and 2 with mutations in PMS2) and 10 subjects had pathogenic variants associated with familial adenomatous polyposis. Thirteen subjects had mutations in other cancer-associated genes (8 in MUTYH, 2 in SMAD4, 1 in BRCA1, 1 in TP53, and 1 in CHEK2), all identified through multigene panel tests. Among 117 patients with uninformative clinical evaluations, next-generation sequence analysis using a multigene panel detected actionable germline variants in 6 patients (5%). Only 43 of the 85 subjects with germline mutations associated with a hereditary cancer syndrome (51%) reported a CRC diagnosis in a first-degree relative. CONCLUSIONS: Approximately 1 in 5 individuals diagnosed with CRC at age younger than 50 years carries a germline mutation associated with cancer; nearly half of these do not have clinical histories typically associated with the identified syndrome. Germline testing with multigene cancer panels should be considered for all young patients with CRC.
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Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Mutação em Linhagem Germinativa , Adolescente , Adulto , Idade de Início , Neoplasias Colorretais/diagnóstico , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Hereditariedade , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Valor Preditivo dos Testes , Estudos Retrospectivos , Fatores de Risco , Centros de Atenção Terciária , Adulto JovemRESUMO
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.