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2.
J Neurol Sci ; 411: 116687, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32018185

RESUMO

Oxaliplatin therapy can be complicated by chemotherapy-induced peripheral neuropathy (CIPN). Other neurotoxic chemotherapies have been linked to single nucleotide variants (SNV) in Charcot-Marie-Tooth disease (CMT) genes. Whether oxaliplatin carries increased risks of CIPN due to SNV in CMT-associated genes is unknown. 353 patients receiving oxaliplatin in NCCTG N08CB were serially evaluated for CIPN using a validated patient-reported outcome (PRO) instrument, the CIPN20 questionnaire (sensory scale). 49 canonical CMT-associated genes were analyzed for rare and common SNV by nextgen sequencing. The 157 patients with the highest and lowest susceptibility to CIPN (cases and controls) harbored 270 non-synonymous SNV in CMT-associated genes (coding regions). 143 of these were rare, occurring only once ("singletons"). CIPN cases had 0.84 singletons per patient compared with 0.98 in controls. An imbalance in favor of cases was noted only in few genes including PRX, which was previously highlighted as a candidate CIPN gene in patients receiving paclitaxel. However, the imbalance was only modest (5 singleton SNV in cases and 2 in controls). Therefore, while singleton SNV were common, they did overall not portend an increased risk of CIPN. Furthermore, testing CMT-associated genes using recurrent non-synonymous SNV did not reveal any significant association with CIPN. Genetic analysis of patients from N08CB provides clinical guidance that oxaliplatin chemotherapy decisions should not be altered by the majority of SNV that may be encountered in CMT-associated genes when common genetic tests are performed, such as exome or genome sequencing. Oxaliplatin's CIPN risk appears unrelated to CMT-associated genes.


Assuntos
Doença de Charcot-Marie-Tooth , Doença de Charcot-Marie-Tooth/genética , Testes Genéticos , Humanos , Oxaliplatina/efeitos adversos , Paclitaxel , Sequenciamento do Exoma
3.
Pain Med ; 20(5): 897-906, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30590777

RESUMO

OBJECTIVE: This study modeled image-guided epidural drug delivery to test whether intraprocedural distribution of pre-injected contrast reliably predicts the neuroanatomical reach of resiniferatoxin-mediated nociceptive neurolysis. METHODS: Swine (N = 12) received unilateral L4-S2 computed tomography fluoroscopy injections by a blinded neuroradiologist; 0.25 mL of contrast was pre-injected to confirm dorsal periganglionic targeting, followed by a 0.5-mL injection of 5 µg of resiniferatoxin/Tween80 or vehicle control. Epidural contrast distribution was graded according to maximum medial excursion. Spinal cord substance P immunostaining quantified the magnitude and anatomical range of resiniferatoxin activity. RESULTS: Periganglionic injection was well tolerated by all animals without development of neurological deficits or other complications. Swine were a suitable model of human clinical spinal intervention. The transforaminal approach was used at all L4 and 50% of L5 segments; the remaining segments were approached by the interlaminar route. All injections were successful with unilateral contrast distribution for all resiniferatoxin injections (N = 28). Immunohistochemistry showed bilateral ablation of substance P+ fibers entering the spinal cord of all resiniferatoxin-treated segments. The intensity of substance P immunostaining in treated segments fell below the lower 99% confidence interval of controls, defining the knockout phenotype. Substance P knockout occurred over a narrow range and was uncorrelated to the anatomical distribution of pre-injected contrast. CONCLUSIONS: Periganglionic resiniferatoxin/Tween80 induced bilateral ablation of spinal cord substance P despite exclusively unilateral targeting. These data suggest that the location of pre-injected contrast is an imperfect surrogate for the neuroanatomical range of drugs delivered to the dorsal epidural compartment that may fail to predict contralateral drug effects.


Assuntos
Diterpenos/administração & dosagem , Bloqueio Nervoso/métodos , Neurotoxinas/administração & dosagem , Animais , Feminino , Fluoroscopia/métodos , Injeções Epidurais , Raízes Nervosas Espinhais/efeitos dos fármacos , Suínos , Terapia Assistida por Computador/métodos
4.
Support Care Cancer ; 25(11): 3537-3544, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28634656

RESUMO

PURPOSE: Clinical practice guidelines on chemotherapy-induced peripheral neuropathy (CIPN) use the NCI Common Terminology Criteria for Adverse Events (CTCAE), while recent clinical trials employ a potentially superior measure, the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire-CIPN twenty-item scale (QLQ-CIPN20), a patient-reported outcome (PRO). Practitioners and researchers lack guidance, regarding how QLQ-CIPN20 results relate to the traditional CTCAE during the serial assessment of patients undergoing chemotherapy. METHODS: Two large CIPN clinical trial datasets (538 patients) pairing QLQ-CIPN20 and CTCAE outcomes were analyzed using a multivariable linear mixed model with QLQ-CIPN20 score as the outcome variable, CTCAE grade as the main effect, and patient as random effect (accounting for internal correlation of serial measures). RESULTS: The association between QLQ-CIPN20 scores and CTCAE grades was strong (p < 0.0001), whereby patients with higher CTCAE grade had worse QLQ-CIPN20 scores. Some variation of QLQ-CIPN20 scores was observed based on drug, treatment, and cycle. While there was a marked difference in the mean QLQ-CIPN20 scores between CTCAE grades, the ranges of QLQ-CIPN20 scores within each CTCAE grade were large, leading to large overlap in CIPN20 scores across CTCAE grades. CONCLUSIONS: A strong positive association of QLQ-CIPN20 scores and CTCAE grade provides evidence of convergent validity as well as practical guidance, as to how to quantitatively interpret QLQ-CIPN20 scores at the study level in terms of the traditional CTCAE. The present results also highlight an important clinical caveat, specifically, that conversion of a specific QLQ-CIPN20 score to a specific CTCAE score may not be reliable at the level of an individual patient.


Assuntos
Antineoplásicos/efeitos adversos , Carboplatina/efeitos adversos , Compostos Organoplatínicos/efeitos adversos , Paclitaxel/efeitos adversos , Medidas de Resultados Relatados pelo Paciente , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias/tratamento farmacológico , Oxaliplatina , Doenças do Sistema Nervoso Periférico/patologia , Médicos
5.
Oncotarget ; 8(16): 27145-27154, 2017 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-28423702

RESUMO

BACKGROUND: The ability to analyze the genomics of malignancies has opened up new possibilities for off-label targeted therapy in cancers that are refractory to standard therapy. At Mayo Clinic these efforts are organized through the Center for Individualized Medicine (CIM). RESULTS: Prior to GTB, datasets were analyzed and integrated by a team of bioinformaticians and cancer biologists. Therapeutically actionable mutations were identified in 65% (92/141) of the patients tested with 32% (29/92) receiving genomically targeted therapy with FDA approved drugs or in an independent clinical trial with 45% (13/29) responding. Standard of care (SOC) options were continued by 15% (14/92) of patients tested before exhausting SOC options, with 71% (10/14) responding to treatment. Over 35% (34/92) of patients with actionable targets were not treated with 65% (22/34) choosing comfort measures or passing away. MATERIALS AND METHODS: Patients (N = 165) were referred to the CIM Clinic between October 2012 and December 2015. All patients received clinical genomic panel testing with selected subsets receiving array comparative genomic hybridization and clinical whole exome sequencing to complement and validate panel findings. A genomic tumor board (GTB) reviewed results and, when possible, developed treatment recommendations. CONCLUSIONS: Treatment decisions driven by tumor genomic analysis can lead to significant clinical benefit in a minority of patients. The success of genomically driven therapy depends both on access to drugs and robustness of bioinformatics analysis. While novel clinical trial designs are increasing the utility of genomic testing, robust data sharing of outcomes is needed to optimize clinical benefit for all patients.


Assuntos
Biomarcadores Tumorais , Genômica , Neoplasias/genética , Medicina de Precisão , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Criança , Pré-Escolar , Biologia Computacional/métodos , Feminino , Predisposição Genética para Doença , Genômica/métodos , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Terapia de Alvo Molecular , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Medicina de Precisão/métodos , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento , Adulto Jovem
6.
JCO Precis Oncol ; 20172017.
Artigo em Inglês | MEDLINE | ID: mdl-30761385

RESUMO

PURPOSE: Genomic testing has increased the quantity of information available to oncologists. Unfortunately, many identified sequence alterations are variants of unknown significance (VUSs), which thus limit the clinician's ability to use these findings to inform treatment. We applied a combination of in silico prediction and molecular modeling tools and laboratory techniques to rapidly define actionable VUSs. MATERIALS AND METHODS: Exome sequencing was conducted on 308 tumors from various origins. Most single nucleotide alterations within gene coding regions were VUSs. These VUSs were filtered to identify a subset of therapeutically targetable genes that were predicted with in silico tools to be altered in function by their variant sequence. A subset of receptor tyrosine kinase VUSs was characterized by laboratory comparison of each VUS versus its wild-type counterpart in terms of expression and signaling activity. RESULTS: The study identified 4,327 point mutations of which 3,833 were VUSs. Filtering for mutations in genes that were therapeutically targetable and predicted to affect protein function reduced these to 522VUSs of interest, including a large number of kinases. Ten receptortyrosine kinase VUSs were selected to explore in the laboratory. Of these, seven were found to be functionally altered. Three VUSs (FGFR2 F276C, FGFR4 R78H, and KDR G539R) showed increased basal or ligand-stimulated ERK phosphorylation compared with their wild-type counterparts, which suggests that they support transformation. Treatment of a patient who carried FGFR2 F276C with an FGFR inhibitor resulted in significant and sustained tumor response with clinical benefit. CONCLUSION: The findings demonstrate the feasibility of rapid identification of the biologic relevance of somatic mutations, which thus advances clinicians' ability to make informed treatment decisions.

7.
Cancer Med ; 5(4): 631-9, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26763541

RESUMO

Paclitaxel-induced peripheral neuropathy (PIPN) cannot be predicted from clinical parameters and might have a pharmacogenomic basis. Previous studies identified single nucleotide variants (SNV) associated with PIPN. However, only a subset of findings has been confirmed to date in more than one study, suggesting a need for further re-testing and validation in additional clinical cohorts. Candidate PIPN-associated SNVs were identified from the literature. SNVs were retested in 119 patients selected by extreme phenotyping from 269 in NCCTG N08C1 (Alliance) as previously reported. SNV genotyping was performed by a combination of short-read sequencing analysis and Taqman PCR. These 22 candidate PIPN SNVs were genotyped. Two of these, rs7349683 in the EPHA5 and rs3213619 in ABCB1 were found to be significantly associated with PIPN with an Odds ratios OR = 2.07 (P = 0.02) and OR = 0.12 (P = 0.03), respectively. In addition, three SNVs showed a trend toward a risk- or protective effect that was consistent with previous reports. The rs10509681 and rs11572080 in the gene CYP2C8*3 showed risk effect with an OR = 1.49 and rs1056836 in CYP1B1 showed a protective effect with an OR = 0.66. None of the other results supported the previously reported associations, including some SNVs displaying an opposite direction of effect from previous reports, including rs1058930 in CYP2C8, rs17222723 and rs8187710 in ABCC2, rs10771973 in FGD4, rs16916932 in CACNB2 and rs16948748 in PITPNA. Alliance N08C1 validated or supported a minority of previously reported SNV-PIPN associations. Associations previously reported by multiple studies appeared to have a higher likelihood to be validated by Alliance N08C1.


Assuntos
Antineoplásicos Fitogênicos/efeitos adversos , Predisposição Genética para Doença , Neoplasias/complicações , Paclitaxel/efeitos adversos , Doenças do Sistema Nervoso Periférico/etiologia , Polimorfismo de Nucleotídeo Único , Alelos , Ensaios Clínicos como Assunto , Feminino , Frequência do Gene , Genótipo , Humanos , Masculino , Proteína 2 Associada à Farmacorresistência Múltipla , Neoplasias/tratamento farmacológico , Razão de Chances
8.
J Neurol Sci ; 357(1-2): 35-40, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26143528

RESUMO

The predisposition of patients to develop polyneuropathy in response to toxic exposure may have a genetic basis. The previous study Alliance N08C1 found an association of the Charcot-Marie-Tooth disease (CMT) gene ARHGEF10 with paclitaxel chemotherapy induced peripheral neuropathy (CIPN) related to the three non-synonymous, recurrent single nucleotide variants (SNV), whereby rs9657362 had the strongest effect, and rs2294039 and rs17683288 contributed only weakly. In the present report, Alliance N08CA was chosen to attempt to replicate the above finding. N08CA was chosen because it is the methodologically most similar study (to N08C1) performed in the CIPN field to date. N08CA enrolled patients receiving the neurotoxic chemotherapy agent paclitaxel. Polyneuropathy was assessed by serial repeat administration of the previously validated patient reported outcome instrument CIPN20. A study-wide, Rasch type model was used to perform extreme phenotyping in n=138 eligible patients from which "cases" and "controls" were selected for genetic analysis of SNV performed by TaqMan PCR. A significant association of ARHGEF10 with CIPN was found under the pre-specified primary endpoint, with a significance level of p=0.024. As in the original study, the strongest association of a single SNV was seen for rs9657362 (odds ratio=3.56, p=0.018). To further compare results across the new and the previous study, a statistical "classifier" was tested, which achieved a ROC area under the curve of 0.60 for N08CA and 0.66 for N08C1, demonstrating good agreement. Retesting of the primary endpoint of N08C1 in the replication study N08CA validated the association of ARHGEF10 with CIPN.


Assuntos
Doença de Charcot-Marie-Tooth/genética , Paclitaxel/efeitos adversos , Doenças do Sistema Nervoso Periférico/induzido quimicamente , Doenças do Sistema Nervoso Periférico/genética , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Antineoplásicos Fitogênicos/efeitos adversos , Estudos de Casos e Controles , Feminino , Predisposição Genética para Doença/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Genéticos , Mutação , Farmacogenética , Polimorfismo de Nucleotídeo Único/genética
9.
Cancer Genet ; 208(7-8): 374-81, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26087898

RESUMO

Neuroendocrine cancer cell lines are used to investigate therapeutic targets in neuroendocrine tumors (NET) and have been instrumental in the design of clinical trials targeting the PI3K/AKT/mTOR pathways, VEGF inhibitors, and somatostatin analogues. It remains unknown, however, whether the genomic makeup of NET cell lines reflect that of primary NET since comprehensive unbiased genome sequencing has not been performed on the cell lines. Four bronchopulmonary NET (BP-NET)-NCI-H720, NCI-H727, NCI-H835, and UMC11-and two pancreatic neuroendocrine tumors (panNET)-BON-1 and QGP1-were cultured. DNA was isolated, and exome sequencing was done. GATK and EXCAVATOR were used for bioinformatic analysis. We detected a total of 1,764 nonsynonymous single nucleotide variants at a rate of 8 per Mb in BP-NET and 4.3 per Mb in panNET cell lines, including 52 mutated COSMIC cancer genes in these cell lines, such as TP53, BRCA1, RB1, TSC2, NOTCH1, EP300, GNAS, KDR, STK11, and APC but not ATRX, DAXX, nor MEN1. Our data suggest that mutation rate, the pattern of copy number variations, and the mutational spectra in the BP-NET cell lines are more similar to the changes observed in small cell lung cancer than those found in primary BP-NET. Likewise, mutation rate and pattern including the absence of mutations in ATRX/DAXX, MEN1, and YY1 in the panNET cell lines BON1 and QGP1 suggest that these cell lines do not have the genetic signatures of a primary panNET. These results suggest that results from experiments with BP-NET and panNET cell lines need to be interpreted with caution.


Assuntos
Exoma/genética , Predisposição Genética para Doença/genética , Mutação , Tumores Neuroendócrinos/genética , Neoplasias Brônquicas/genética , Neoplasias Brônquicas/patologia , Linhagem Celular Tumoral , Aberrações Cromossômicas , Variações do Número de Cópias de DNA , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Tumores Neuroendócrinos/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia
11.
Epigenetics ; 10(3): 200-12, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25621511

RESUMO

The response of the peripheral nervous system (PNS) to injury may go together with alterations in epigenetics, a conjecture that has not been subjected to a comprehensive, genome-wide test. Using reduced representation bisulfite sequencing, we report widespread remodeling of DNA methylation in the rat dorsal root ganglion (DRG) occurring within 24 h of peripheral nerve ligation, a neuropathy model of allodynia. Significant (P < 10(-4)) cytosine hyper- and hypo-methylation was found at thousands of CpG sites. Remodeling occurred outside of CpG islands. Changes affected genes with known roles in the PNS, yet methylome remodeling also involved genes that were not linked to neuroplasticity by prior evidence. Consistent with emerging models relying on genome-wide methylation and RNA-seq analysis of promoter regions and gene bodies, variation of methylation was not tightly linked with variation of gene expression. Furthermore, approximately 44% of the dynamically changed CpGs were located outside of genes. We compared their positions with the intergenic, tissue-specific differentially methylated CpGs (tDMCs) of an independent experimental set consisting of liver, spleen, L4 control DRG, and muscle. Dynamic changes affected those intergenic CpGs that were different between tissues (P < 10(-15)) and almost never the invariant portion of the methylome (those CpGs that were identical across all tissues). Our findings-obtained in mixed tissue-show that peripheral nerve injury leads to methylome remodeling in the DRG. Future studies may address which of the cell types found in the DRG, such as specific groups of neurons or non-neuronal cells are affected by which aspect of the observed methylome remodeling.


Assuntos
Metilação de DNA , Neurônios/patologia , Dor/metabolismo , Nervos Espinhais/patologia , Animais , Ilhas de CpG , Modelos Animais de Doenças , Gânglios Espinais/metabolismo , Masculino , Ratos , Transcriptoma
12.
Ann Neurol ; 76(5): 727-37, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25164601

RESUMO

OBJECTIVE: Mutations in Charcot-Marie-Tooth disease (CMT) genes are the cause of rare familial forms of polyneuropathy. Whether allelic variability in CMT genes is also associated with common forms of polyneuropathy-considered "acquired" in medical parlance-is unknown. Chemotherapy-induced peripheral neuropathy (CIPN) occurs commonly in cancer patients and is individually unpredictable. We used CIPN as a clinical model to investigate the association of non-CMT polyneuropathy with CMT genes. METHODS: A total of 269 neurologically asymptomatic cancer patients were enrolled in the clinical trial Alliance N08C1 to receive the neurotoxic drug paclitaxel, while undergoing prospective assessments for polyneuropathy. Forty-nine CMT genes were analyzed by targeted massively parallel sequencing of genomic DNA from patient blood. RESULTS: A total of 119 (of 269) patients were identified from the 2 ends of the polyneuropathy phenotype distribution: patients that were most and least susceptible to paclitaxel polyneuropathy. The CMT gene PRX was found to be deleteriously mutated in patients who were susceptible to CIPN but not in controls (p = 8 × 10(-3)). Genetic variation in another CMT gene, ARHGEF10, was highly significantly associated with CIPN (p = 5 × 10(-4)). Three nonsynonymous recurrent single nucleotide variants contributed to the ARHGEF10 signal: rs9657362, rs2294039, and rs17683288. Of these, rs9657362 had the strongest effect (odds ratio = 4.8, p = 4 × 10(-4)). INTERPRETATION: The results reveal an association of CMT gene allelic variability with susceptibility to CIPN. The findings raise the possibility that other acquired polyneuropathies may also be codetermined by genetic etiological factors, of which some may be related to genes already known to cause the phenotypically related Mendelian disorders of CMT.


Assuntos
Antineoplásicos/efeitos adversos , Doença de Charcot-Marie-Tooth/genética , Polineuropatias/induzido quimicamente , Polineuropatias/genética , Alelos , Antineoplásicos Fitogênicos/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mutação/genética , Neoplasias/complicações , Paclitaxel/efeitos adversos , Estudos Prospectivos , Fatores de Troca de Nucleotídeo Guanina Rho/genética
13.
Curr Opin Gastroenterol ; 30(2): 163-7, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24441281

RESUMO

PURPOSE OF REVIEW: This review aims at summarizing progress in clinical trials and basic science redefining the diagnosis and treatment of well differentiated small intestine neuroendocrine tumors (SI-NET). RECENT FINDINGS: Two clinical trials demonstrated antitumor activity of the long-acting somatostatin analogues octreotide long-acting release and lanreotide for advanced SI-NET. The mammalian target of rapamycin (mTOR) inhibitor everolimus is another treatment option for patients with SI-NET, but awaits definitive proof of benefit in the ongoing RAD001 In Advanced Neuroendocrine Tumors study (RADIANT-4). Two whole exome/genome-sequencing studies reported in the past year provided the first genome-wide analysis of large sets of SI-NET at nucleotide resolution. Candidate therapeutically relevant alterations were found to affect SRC, SMAD genes, aurora kinase A, epidermal growth factor receptor, heat shock protein 90, and platelet-derived growth factor receptor as well as mutually exclusive amplification of RAC-alpha serine/threonine-protein kinase (AKT1) or AKT2 and other alterations of PI3K/Akt/mTOR signaling genes. The gene CDKN1B is inactivated by small insertions/deletions in 8% of patients with SI-NET suggesting cell cycle inhibitors as new candidate drugs for SI-NET. Circulating tumor cells and tumor-derived RNA in the blood are promising clinical tests for SI-NET. SUMMARY: Clinical and genomic research may merge in the near future to re-shape clinical trials and to define the 'personalized' treatment options for patients with SI-NET.


Assuntos
Neoplasias Intestinais/tratamento farmacológico , Intestino Delgado , Tumores Neuroendócrinos/tratamento farmacológico , Antineoplásicos Hormonais/uso terapêutico , Genômica , Humanos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/genética , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/genética , Octreotida/uso terapêutico , Peptídeos Cíclicos/uso terapêutico , Somatostatina/análogos & derivados , Somatostatina/uso terapêutico
14.
Nat Genet ; 45(12): 1483-6, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24185511

RESUMO

The diagnosed incidence of small intestine neuroendocrine tumors (SI-NETs) is increasing, and the underlying genomic mechanisms have not yet been defined. Using exome- and genome-sequence analysis of SI-NETs, we identified recurrent somatic mutations and deletions in CDKN1B, the cyclin-dependent kinase inhibitor gene, which encodes p27. We observed frameshift mutations of CDKN1B in 14 of 180 SI-NETs, and we detected hemizygous deletions encompassing CDKN1B in 7 out of 50 SI-NETs, nominating p27 as a tumor suppressor and implicating cell cycle dysregulation in the etiology of SI-NETs.


Assuntos
Inibidor de Quinase Dependente de Ciclina p27/genética , Neoplasias Intestinais/genética , Mutação , Tumores Neuroendócrinos/genética , Ciclo Celular/genética , Estudos de Coortes , Genes Supressores de Tumor , Predisposição Genética para Doença , Humanos , Neoplasias Intestinais/epidemiologia , Neoplasias Intestinais/patologia , Intestino Delgado/patologia , Tumores Neuroendócrinos/epidemiologia , Tumores Neuroendócrinos/patologia , Análise de Sequência de DNA
15.
J Clin Invest ; 123(6): 2502-8, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23676460

RESUMO

Small intestine neuroendocrine tumors (SI-NETs) are the most common malignancy of the small bowel. Several clinical trials target PI3K/Akt/mTOR signaling; however, it is unknown whether these or other genes are genetically altered in these tumors. To address the underlying genetics, we analyzed 48 SI-NETs by massively parallel exome sequencing. We detected an average of 0.1 somatic single nucleotide variants (SNVs) per 106 nucleotides (range, 0-0.59), mostly transitions (C>T and A>G), which suggests that SI-NETs are stable cancers. 197 protein-altering somatic SNVs affected a preponderance of cancer genes, including FGFR2, MEN1, HOOK3, EZH2, MLF1, CARD11, VHL, NONO, and SMAD1. Integrative analysis of SNVs and somatic copy number variations identified recurrently altered mechanisms of carcinogenesis: chromatin remodeling, DNA damage, apoptosis, RAS signaling, and axon guidance. Candidate therapeutically relevant alterations were found in 35 patients, including SRC, SMAD family genes, AURKA, EGFR, HSP90, and PDGFR. Mutually exclusive amplification of AKT1 or AKT2 was the most common event in the 16 patients with alterations of PI3K/Akt/mTOR signaling. We conclude that sequencing-based analysis may provide provisional grouping of SI-NETs by therapeutic targets or deregulated pathways.


Assuntos
Neoplasias Intestinais/genética , Tumores Neuroendócrinos/genética , Sequência de Bases , Exoma , Genes Neoplásicos , Genômica , Humanos , Neoplasias Intestinais/mortalidade , Mutação , Tumores Neuroendócrinos/mortalidade , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-akt/genética , Sítios de Splice de RNA , Análise de Sequência de DNA , Transdução de Sinais/genética
16.
Epigenetics ; 7(5): 421-8, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22415013

RESUMO

DNA methylation can control some CpG-poor genes but unbiased studies have not found a consistent genome-wide association with gene activity outside of CpG islands or shores possibly due to use of cell lines or limited bioinformatics analyses. We performed reduced representation bisulfite sequencing (RRBS) of rat dorsal root ganglia encompassing postmitotic primary sensory neurons (n = 5, r > 0.99; orthogonal validation p < 10(-19)). The rat genome suggested a dichotomy of genes previously reported in other mammals: low CpG content (< 3.2%) promoter (LCP) genes and high CpG content (≥ 3.2%) promoter (HCP) genes. A genome-wide integrated methylome-transcriptome analysis showed that LCP genes were markedly hypermethylated when repressed, and hypomethylated when active with a 40% difference in a broad region at the 5' of the transcription start site (p < 10(-87) for -6000 bp to -2000 bp, p < 10(-73) for -2000 bp to +2000 bp, no difference in gene body p = 0.42). HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes. Therefore, diametrically opposite methylome-transcriptome associations characterize LCP and HCP genes in postmitotic neural tissue in vivo.


Assuntos
Ilhas de CpG , Metilação de DNA , Gânglios Espinais/citologia , Regiões Promotoras Genéticas , Células Receptoras Sensoriais/citologia , Transcriptoma , Animais , Citosina/metabolismo , Bases de Dados de Ácidos Nucleicos , Gânglios Espinais/metabolismo , Perfilação da Expressão Gênica , Genoma , Masculino , Mitose , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/metabolismo , Análise de Sequência de DNA , Sítio de Iniciação de Transcrição , Ativação Transcricional
17.
Genome Res ; 20(6): 847-60, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20452967

RESUMO

mRNA-seq is a paradigm-shifting technology because of its superior sensitivity and dynamic range and its potential to capture transcriptomes in an agnostic fashion, i.e., independently of existing genome annotations. Implementation of the agnostic approach, however, has not yet been fully achieved. In particular, agnostic mapping of pre-mRNA splice sites has not been demonstrated. The present study pursued dual goals: (1) to advance mRNA-seq bioinformatics toward unbiased transcriptome capture and (2) to demonstrate its potential for discovery in neuroscience by applying the approach to an in vivo model of neurological disease. We have performed mRNA-seq on the L4 dorsal root ganglion (DRG) of rats with chronic neuropathic pain induced by spinal nerve ligation (SNL) of the neighboring (L5) spinal nerve. We found that 12.4% of known genes were induced and 7% were suppressed in the dysfunctional (but anatomically intact) L4 DRG 2 wk after SNL. These alterations persisted chronically (2 mo). Using a read cluster classifier with strong test characteristics (ROC area 97%), we discovered 10,464 novel exons. A new algorithm for agnostic mapping of pre-mRNA splice junctions (SJs) achieved a precision of 97%. Integration of information from all mRNA-seq read classes including SJs led to genome reannotations specifically relevant for the species used (rat), the anatomical site studied (DRG), and the neurological disease considered (pain); for example, a 64-exon coreceptor for the nociceptive transmitter substance P was identified, and 21.9% of newly discovered exons were shown to be dysregulated. Thus, mRNA-seq with agnostic analysis methods appears to provide a highly productive approach for in vivo transcriptomics in the nervous system.


Assuntos
Gânglios Espinais/metabolismo , Perfilação da Expressão Gênica , Dor/genética , Splicing de RNA , RNA Mensageiro/genética , Animais , Doença Crônica , Ratos
18.
Clin Cancer Res ; 15(24): 7492-7501, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-20008849

RESUMO

Genomics and proteomics have held out the promise of individualized medicine for the last 10 or 20 years, but clinical medicine has not yet delivered on this promise. Some cancers, such as breast cancer and some hematologic malignancies, have been at the forefront of individualized therapeutic approaches by integrating molecular biomarkers into treatment decision algorithms. Until recently, the treatment of colorectal cancer (CRC) has lagged behind these other cancers in this regard and therapeutic decisions have been solely empirical. Data from various clinical trials and translational studies have now opened the door for individualized treatment approaches by identifying patients with metastatic CRC who are most likely to benefit from antibodies against the epidermal growth factor receptor (EGFR), cetuximab and panitumumab. Activating mutations of KRAS, a downstream mediator of EGFR signaling, has been shown to render EGFR antibodies ineffective, such that analyzing tumors for these mutations has become mandatory before the use of EGFR antibodies is considered in CRC. Beyond KRAS, several additional biomarkers are currently being investigated as potential positive or negative predictors for the efficacy of EGFR-targeted therapy. Most of these markers are alterations of molecules integrated in the EGFR pathway. This review will focus on the type and quality of evidence that has been gathered to date to predict resistance to monoclonal antibodies against EGFR in CRC. (Clin Cancer Res 2009;15(24):7492-501).

19.
Epigenetics ; 4(2): 100-6, 2009 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-19242095

RESUMO

The zinc finger protein 217 (ZNF217) is an important oncogene based on the high frequency of amplification and overexpression in many cancer types, but its molecular mode of gene regulation is poorly understood. We purified a complex of nuclear ZNF217-binding proteins by affinity chromatography and identified its components by mass spectrometry as Jarid1b/Plu-1, G9a, LSD1, CoREST and CtBP1. Individual binding of these with ZNF217 was confirmed by co-immunoprecipiation (IP). Known activities of these proteins suggested a role of the ZNF217 complex in histone modification. Using in vitro assays the following activities were demonstrated: Histone H3 lysine 4 trimethyl (H3K4me3) demethylase activity, which co-fractionated with Jarid1b/Plu-1 in anion-exchange chromatography; H3K9 methylation, the known principal activity of G9a; and H3K27 methylation. The latter suggested EZH2 as another ZNF217 binding candidate, which could be confirmed by co-IP. Taken together, these findings suggest that ZNF217 assembles a distinct set of histone modifying proteins at target DNA sites that act synergistically in transcriptional repression.


Assuntos
Regulação da Expressão Gênica , Histonas/metabolismo , Transativadores/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo/genética , Proteína Potenciadora do Homólogo 2 de Zeste , Humanos , Histona Desmetilases com o Domínio Jumonji , Proteínas de Neoplasias , Proteínas Nucleares/metabolismo , Oncogenes , Oxirredutases N-Desmetilantes/metabolismo , Complexo Repressor Polycomb 2 , Ligação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Repressoras/metabolismo , Transativadores/metabolismo , Transativadores/fisiologia , Fatores de Transcrição/metabolismo
20.
Proc Natl Acad Sci U S A ; 105(3): 1055-60, 2008 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-18215993

RESUMO

Lumbar puncture (LP) is an attractive route to deliver drugs to the nervous system because it is a safe bedside procedure. Its use for gene therapy has been complicated by poor vector performance and failure to target neurons. Here we report highly effective gene transfer to the primary sensory neurons of the dorsal root ganglia (DRGs) with self-complementary recombinant adeno-associated virus serotype 8 (sc-rAAV8) modeling an LP. Transgene expression was selective for these neurons outlining their cell bodies in the DRGs and their axons projecting into the spinal cord. Immunohistochemical studies demonstrated transduction of cells positive for the nociceptive neuron marker vanilloid receptor subtype 1, the small peptidergic neuron markers substance P and calcitonin gene-related peptide, and the nonpeptidergic neuron marker griffonia simplicifolia isolectin B4. We tested the efficacy of the approach in a rat model of chronic neuropathic pain. A single administration of sc-rAAV8 expressing the analgesic gene prepro-beta-endorphin (ppbetaEP) led to significant (P < 0.0001) reversal of mechanical allodynia for >/=3 months. The antiallodynic effect could be reversed by the mu-opioid antagonist naloxone 4 months after gene transfer (P < 0.001). Testing of an alternative nonopioid analgesic gene, IL-10, alone or in combination with ppbetaEP was equally effective (P < 0.0001). All aspects of the procedure, such as the use of an atraumatic injection technique, isotonic diluent, a low-infusion pressure, and a small injection volume, are consistent with clinical practice of intrathecal drug use. Therefore, gene transfer by LP may be suitable for developing gene therapy-based treatments for chronic pain.


Assuntos
Dependovirus/genética , Neurônios Aferentes/metabolismo , Animais , Doença Crônica , Modelos Animais de Doenças , Terapia Genética , Vetores Genéticos/genética , Imuno-Histoquímica , Cinética , Masculino , Dor/genética , Dor/metabolismo , Dor/patologia , Manejo da Dor , Doenças do Sistema Nervoso Periférico/genética , Doenças do Sistema Nervoso Periférico/metabolismo , Doenças do Sistema Nervoso Periférico/patologia , Doenças do Sistema Nervoso Periférico/terapia , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Opioides/metabolismo , Punção Espinal , beta-Endorfina/genética , beta-Endorfina/metabolismo
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