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BACKGROUND: We have previously reported the development of an outpatient palliative care practice under pharmacist-physician collaboration. The Doris A. Howell Service at the University of California, San Diego Moores Cancer Center includes two pharmacists who participate in a transdisciplinary clinic and provide follow-up care to patients. OBJECTIVE: This study evaluated pharmacist interventions and patient outcomes of a pharmacist-led outpatient palliative care practice. METHODS: This was a retrospective data analysis conducted at a single, academic, comprehensive cancer center. New (first visit) patient consultations were referred by an oncologist or hematologist to an outpatient palliative care practice. A pharmacist evaluated the patient at the first visit and at follow-up (second, third, and fourth visits). Medication problems identified, medication changes made, and changes in pain scores were assessed. RESULTS: Eighty-four new and 135 follow-up patient visits with the pharmacist occurred from March 2011 to March 2012. All new patients (n = 80) were mostly women (n = 44), had localized disease (n = 42), a gastrointestinal cancer type (n = 21), and were on a long-acting (n = 61) and short-acting (n = 70) opioid. A lack of medication efficacy was the most common problem for symptoms of pain, constipation, and nausea/vomiting that was identified by the pharmacist at all visits. A change in pain medication dose and initiation of a new medication for constipation and nausea/vomiting were the most common interventions by the pharmacist. A statistically significant change in pain score was observed for the third visit, but not for the second and fourth visits. CONCLUSIONS: A pharmacist-led outpatient palliative care practice identified medication problems for management of pain, constipation, and nausea/vomiting. Medication changes involved a change in dose and/or initiating a new medication. Trends were observed in improvement and stabilization of pain over subsequent clinic visits.
Assuntos
Assistência Ambulatorial/métodos , Manejo da Dor/métodos , Cuidados Paliativos/métodos , Farmacêuticos , Papel Profissional , Adulto , Idoso , Analgésicos/uso terapêutico , Analgésicos Opioides/uso terapêutico , Comportamento Cooperativo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Médicos , Estudos RetrospectivosRESUMO
BACKGROUND: Genomic instability resulting in copy number variation is a hallmark of malignant transformation and may be identified through massive parallel sequencing. Tumor-specific cell free DNA (cfDNA) present in serum and plasma provides a real-time, easily accessible surrogate. METHODS: DNA was extracted from serum of 204 patients with prostate cancer (Gleason score 2-10), 207 male controls, and patients with benign hyperplasia (n = 10) and prostatitis (n = 10). DNA was amplified by use of random primers, tagged with molecular identifiers, sequenced on a SOLID system, and aligned to the human genome. We evaluated the number of sequence reads of cfDNA in sliding 100-kbp intervals for variation from controls. We used chromosomal regions with significant variations in alignment hits for their ability to segregate patients and matched controls. RESULTS: Using ROC curves to assess diagnostic performance, we evaluated the number of regions in a first subset (n = 177), with variations in alignment hits alone, provided an area under the curve (AUC) of 0.81 (95% CI 0.7-0.9, P < 0.001). Using 5 rounds of 10-fold cross-validation with the full data set, we established a final model that discriminated prostate cancer from controls with an AUC of 0.92 (0.87-0.95), reaching a diagnostic accuracy of 83%. Both benign prostatic hypertrophy and prostatitis could be distinguished from prostate cancer by use of cfDNA, with an accuracy of 90%. CONCLUSIONS: Assessment of a limited number of chromosomal structural instabilities by use of massive parallel sequencing of cfDNA was sufficient to distinguish between prostate cancer and controls. This large cohort demonstrates the utility of cfDNA in prostate cancer recently established in other malignant neoplasms.
Assuntos
Biomarcadores Tumorais/sangue , Instabilidade Cromossômica , DNA de Neoplasias/sangue , Neoplasias da Próstata/sangue , Neoplasias da Próstata/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Estudos de Casos e Controles , Variações do Número de Cópias de DNA , DNA de Neoplasias/genética , Heterogeneidade Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Calicreínas/sangue , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Antígeno Prostático Específico/sangue , Neoplasias da Próstata/patologiaRESUMO
Toll-like receptors (TLRs) are highly conserved type 1 membrane proteins that initiate a multiplicity of transient gene transcriptions, resulting in innate and adaptive immune responses. These essential immune responses are triggered by common TLR pattern recognition receptors of microbial products expressed through the cytoplasmic carboxy-terminal Toll/IL-1 domain. Toll/IL-1 adapter protein cascades are induced by an activated Toll/IL-1 to induce transient transcription responses. All TLRs, with the exception of TLR3, use an MyD88 adapter to Toll/IL-1 to initiate a proinflammatory cascade. TLR3 uses the toll receptor 3/4 induction factor adapter to initiate a different cytosolic adapter cascade with double-stranded RNA agonists. This non-MyD88 pathway induces both NF-κB and type 1 interferon responses. By using a TLR3-restricted double-stranded RNA agonist, rintatolimod, we demonstrate significant unexpected differences in toxic responses between rats and primates. The mechanism of this differential response is consistent with a relative down-regulation of the NF-κB inflammatory cytokine induction pathway in the cynomolgus monkey and humans, but not observed systemically in rat. Our findings suggest evaluation of TLR3 agonists in drug therapy.
Assuntos
Inflamação/metabolismo , Poli I-C/farmacologia , Poli U/farmacologia , Especificidade da Espécie , Receptor 3 Toll-Like/antagonistas & inibidores , Sequência de Aminoácidos , Animais , Cães , Síndrome de Fadiga Crônica/tratamento farmacológico , Humanos , Inflamação/imunologia , Macaca fascicularis , Dose Máxima Tolerável , Dados de Sequência Molecular , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
Ebola virus (EBOV) is a highly infectious and lethal pathogen responsible for sporadic self-limiting clusters of Ebola virus disease (EVD) in Central Africa capable of reaching epidemic status. 100% protection from lethal EBOV-Zaire in Balb/c mice was achieved by rintatolimod (Ampligen) at the well tolerated human clinical dose of 6 mg/kg. The data indicate that the mechanism of action is rintatolimod's dual ability to act as both a competitive decoy for the IID domain of VP35 blocking viral dsRNA sequestration and as a pathogen-associated molecular pattern (PAMP) restricted agonist for direct TLR3 activation but lacking RIG-1-like cytosolic helicase agonist properties. These data show promise for rintatolimod as a prophylactic therapy against human Ebola outbreaks.
Assuntos
Ebolavirus , Doença pelo Vírus Ebola , Animais , Camundongos , Humanos , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/prevenção & controle , Receptor 3 Toll-Like , Proteínas Virais Reguladoras e Acessórias , Poli I-C , Ebolavirus/genéticaRESUMO
To explore the effect of lingual artery ligation on tongue vascularity, we performed an analysis of 25 patients who underwent transoral robotic surgery for base of tongue cancers (May 2011 to December 2019). Hounsfield units of the intrinsic muscles (IMs) and genioglossus muscles (GGs) were measured in postoperative imaging (mean 4 months) as a surrogate for vascularity. In ligated patients (n = 15), the values from the ligated/resected side of the tongue were compared with the contralateral side and the nonligated side of resection. Individually, IMs and GGs on the ligated side demonstrated no significant difference to the contralateral side (P = .662 and .618, respectively). Ligation produced a significant decrease in IM measurements but no difference between GG values vs nonligated patients (P = .050 and .818, respectively). No difference was appreciated in mean values for combined IMs and GGs between cohorts (P = .212). No gross tongue atrophy or complications were incurred. Future studies are warranted to delineate long-term effects.
Assuntos
Procedimentos Cirúrgicos Robóticos , Robótica , Artérias/cirurgia , Glossectomia/métodos , Humanos , Procedimentos Cirúrgicos Robóticos/métodos , Língua/cirurgiaRESUMO
Coronary sinus ostial obstruction is an exceedingly rare anomaly that is particularly important to diagnose in patients with single-ventricle heart disease before surgical palliation. We present 2 cases, an infant and an adult, diagnosed with coronary sinus ostial obstruction, with different clinical outcomes due to timing of diagnosis. (Level of Difficulty: Intermediate.).
RESUMO
BACKGROUND: Rintatolimod is a selective TLR3 agonist, which has demonstrated clinical activity for ME/CFS in Phase II and Phase III double-blind, placebo-controlled, randomized, multi-site clinical trials. METHODS AND FINDINGS: A hypothesis-based post-hoc analysis of the Intent to Treat (ITT) population diagnosed with ME/CFS from 12 independent clinical sites of a Phase III trial was performed to evaluate the effect of rintatolimod therapy based on disease duration. The clinical activity of rintatolimod was evaluated by exercise treadmill tolerance (ETT) using a modified Bruce protocol. The ITT population (n = 208) was divided into two subsets of symptom duration. Patients with symptom duration of 2-8 years were identified as the Target Subset (n = 75); the remainder (<2 year plus >8 year) were identified as the Non-Target Subset (n = 133). Placebo-adjusted percentage improvements in exercise duration and the vertical rise for the Target Subset (n = 75) were more than twice that of the ITT population. The Non-Target Subset (n = 133) failed to show any clinically significant ETT response to rintatolimod when compared to placebo. Within the Target Subset, 51.2% of rintatolimod-treated patients improved their exercise duration by ≥25% (p = 0.003) despite reduced statistical power from division of the original ITT population into two subsets. CONCLUSION/SIGNIFICANCE: Analysis of ETT from a Phase III trial has identified within the ITT population, a subset of ME/CFS patients with ≥2 fold increased exercise response to rintatolimod. Substantial improvement in physical performance was seen for the majority (51.2%) of these severely debilitated patients who improved exercise duration by ≥25%. This magnitude of exercise improvement was associated with clinically significant enhancements in quality of life. The data indicate that ME/CFS patients have a relatively short disease duration window (<8 years) to expect a significant response to rintatolimod under the dosing conditions utilized in this Phase III clinical trial. These results may have direct relevance to the cognitive impairment and fatigue being experienced by patients clinically recovered from COVID-19 and free of detectable SARS-CoV-2. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00215800.
Assuntos
Tolerância ao Exercício , Síndrome de Fadiga Crônica/tratamento farmacológico , Poli I-C/uso terapêutico , Poli U/uso terapêutico , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de TempoRESUMO
Visual deficits after traumatic brain injury (TBI) are common, but interventions that limit the post-trauma impairments have not been identified. We have found that treatment with the cannabinoid type-2 receptor (CB2) inverse agonist SMM-189 for 2â¯weeks after closed-head blast TBI greatly attenuates the visual deficits and retinal pathology this otherwise produces in mice, by modulating the deleterious role of microglia in the injury process after trauma. SMM-189, however, has not yet been approved for human use. Raloxifene is an FDA-approved estrogen receptor drug that is used to treat osteoporosis, but it was recently found to also show noteworthy CB2 inverse agonism. In the current studies, we found that a high pressure air blast in the absence of raloxifene treatment yields deficits in visual acuity and contrast sensitivity, reductions in the A-wave and B-wave of the scotopic electroretinogram (ERG), light aversion, and increased pupil constriction to light. Raloxifene delivered daily for two weeks after blast at 5-10â¯mg/kg mitigates or eliminates these abnormalities (with the higher dose generally more effective). This functional rescue with raloxifene is accompanied by a biasing of microglia from the harmful M1 to the helpful M2 state, and reductions in retinal, optic nerve, and oculomotor nucleus pathology. We also found that raloxifene treatment is still effective even when delayed until 48â¯h after TBI, and that raloxifene benefit appears attributable to its CB2 inverse agonism rather than its estrogenic actions. Our studies show raloxifene is effective in treating visual injury after brain and/or eye trauma, and they provide basis for phase-2 efficacy testing in human clinical trials.
Assuntos
Concussão Encefálica/complicações , Fármacos Neuroprotetores/farmacologia , Cloridrato de Raloxifeno/farmacologia , Receptor CB2 de Canabinoide/efeitos dos fármacos , Transtornos da Visão/etiologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/patologia , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/patologia , Receptor CB2 de Canabinoide/agonistas , Retina/patologia , Transtornos da Visão/patologiaRESUMO
Purpose: Chromosomal instability is a fundamental property of cancer, which can be quantified by next-generation sequencing (NGS) from plasma/serum-derived cell-free DNA (cfDNA). We hypothesized that cfDNA could be used as a real-time surrogate for imaging analysis of disease status as a function of response to immunotherapy and as a more reliable tool than tumor biomarkers.Experimental Design: Plasma cfDNA sequences from 56 patients with diverse advanced cancers were prospectively collected and analyzed in a single-blind study for copy number variations, expressed as a quantitative chromosomal number instability (CNI) score versus 126 noncancer controls in a training set of 23 and a blinded validation set of 33. Tumor biomarker concentrations and a surrogate marker for T regulatory cells (Tregs) were comparatively analyzed.Results: Elevated CNI scores were observed in 51 of 56 patients prior to therapy. The blinded validation cohort provided an overall prediction accuracy of 83% (25/30) and a positive predictive value of CNI score for progression of 92% (11/12). The combination of CNI score before cycle (Cy) 2 and 3 yielded a correct prediction for progression in all 13 patients. The CNI score also correctly identified cases of pseudo-tumor progression from hyperprogression. Before Cy2 and Cy3, there was no significant correlation for protein tumor markers, total cfDNA, or surrogate Tregs.Conclusions: Chromosomal instability quantification in plasma cfDNA can serve as an early indicator of response to immunotherapy. The method has the potential to reduce health care costs and disease burden for cancer patients following further validation. Clin Cancer Res; 23(17); 5074-81. ©2017 AACR.
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Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Imunoterapia , Neoplasias/sangue , Adulto , Idoso , Biomarcadores Tumorais/imunologia , Instabilidade Cromossômica/imunologia , Variações do Número de Cópias de DNA/genética , Progressão da Doença , Feminino , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
Chronic fatigue syndrome/ Myalgic encephalomyelitis (CFS/ME) is a poorly understood seriously debilitating disorder in which disabling fatigue is an universal symptom in combination with a variety of variable symptoms. The only drug in advanced clinical development is rintatolimod, a mismatched double stranded polymer of RNA (dsRNA). Rintatolimod is a restricted Toll-Like Receptor 3 (TLR3) agonist lacking activation of other primary cellular inducers of innate immunity (e.g.- cytosolic helicases). Rintatolimod also activates interferon induced proteins that require dsRNA for activity (e.g.- 2'-5' adenylate synthetase, protein kinase R). Rintatolimod has achieved statistically significant improvements in primary endpoints in Phase II and Phase III double-blind, randomized, placebo-controlled clinical trials with a generally well tolerated safety profile and supported by open-label trials in the United States and Europe. The chemistry, mechanism of action, clinical trial data, and current regulatory status of rintatolimod for CFS/ME including current evidence for etiology of the syndrome are reviewed.
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Síndrome de Fadiga Crônica/tratamento farmacológico , Poli I-C/uso terapêutico , Poli U/uso terapêutico , Receptor 3 Toll-Like/agonistas , Animais , Síndrome de Fadiga Crônica/fisiopatologia , Humanos , Imunidade Inata/imunologia , Poli I-C/efeitos adversos , Poli I-C/farmacologia , Poli U/efeitos adversos , Poli U/farmacologia , RNA de Cadeia Dupla/metabolismo , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
Using an established nonhuman primate model for H5N1 highly pathogenic influenza virus infection in humans, we have been able to demonstrate the prophylactic mitigation of the pulmonary damage characteristic of human fatal cases from primary influenza virus pneumonia with a low dose oral formulation of a commercially available parenteral natural human interferon alpha (Alferon N Injection®). At the highest oral dose (62.5IU/kg body weight) used there was a marked reduction in the alveolar inflammatory response with minor evidence of alveolar and interstitial edema in contrast to the hemorrhage and inflammatory response observed in the alveoli of control animals. The mitigation of severe damage to the lower pulmonary airway was observed without a parallel reduction in viral titers. Clinical trial data will be necessary to establish its prophylactic human efficacy for highly pathogenic influenza viruses.
Assuntos
Interferon-alfa/farmacologia , Lesão Pulmonar/prevenção & controle , Doenças dos Macacos/tratamento farmacológico , Infecções por Orthomyxoviridae/tratamento farmacológico , Administração Oral , Animais , Modelos Animais de Doenças , Hemorragia/prevenção & controle , Humanos , Inflamação/prevenção & controle , Virus da Influenza A Subtipo H5N1/patogenicidade , Interferon-alfa/administração & dosagem , Macaca fascicularis/virologia , Doenças dos Macacos/virologia , Mucosa Bucal , Infecções por Orthomyxoviridae/prevenção & controle , Alvéolos Pulmonares/patologia , Alvéolos Pulmonares/virologia , Replicação Viral/efeitos dos fármacosRESUMO
CONTEXT: Randomized data support shorter radiotherapy courses for management of cancer-related symptoms in the palliative setting. OBJECTIVES: The purpose of this study was to evaluate the length of palliative radiotherapy before hospice enrollment among the elderly U.S. population, with a further focus on factors that influence the duration of radiation and the length of survival on hospice, including whether the duration of radiation was associated with length of survival on hospice. METHODS: A total of 6982 patients with breast, prostate, lung, or colorectal cancer who received a course of radiotherapy within 30 days before hospice enrollment were identified within the Surveillance, Epidemiology, and End Results-Medicare linked database. The primary end points included the duration of palliative radiotherapy and the time from hospice enrollment through death (hospice duration). Multivariate linear regression and multivariate Cox models evaluated factors associated with the length of radiotherapy course and hospice duration. RESULTS: The median length of palliative radiotherapy was 14 days, and the median hospice duration was 13 days. The course of palliative radiotherapy was longer than hospice duration in 48% of the patients. Breast and lung cancer were associated with longer courses of radiotherapy and shorter stays on hospice. Patients treated in freestanding radiation centers had longer courses of radiotherapy. For these groups, a longer radiotherapy course was not associated with longer hospice duration. CONCLUSION: This study found relatively long courses of radiotherapy before short lengths of survival on hospice. Future research is needed to identify barriers to shorter radiotherapy courses.
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Neoplasias da Mama/terapia , Neoplasias Colorretais/terapia , Cuidados Paliativos na Terminalidade da Vida/métodos , Neoplasias Pulmonares/terapia , Cuidados Paliativos/métodos , Radioterapia/métodos , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Bases de Dados Factuais , Feminino , Hospitais para Doentes Terminais , Humanos , Modelos Lineares , Masculino , Medicare , Análise Multivariada , Modelos de Riscos Proporcionais , Fatores de Tempo , Estados UnidosRESUMO
The novel avian H7N9 influenza virus has caused more than 130 human infections with 43 deaths (as of September, 2013) in China. Because of the lack of existing immunity against H7 subtype influenza viruses in the human population and the absence of a licensed commercial vaccine, antiviral drugs are critical tools for the treatment of infection with this novel H7N9. Both M2-ion channel blockers and neuraminidase inhibitors are used as antiviral drugs for influenza infections of humans. The emerging H7N9 viruses are resistant to the M2-ion channel blockers because of a S31N mutation in the M2 protein; additionally, some H7N9 isolates have gained neuraminidase R292K substitution resulting in broad resistance to neuraminidase inhibitors. In this study we report that Alferon N can inhibit wild type and 292K H7N9 viruses replication in vitro. Since Alferon N is approved for clinical use, this would allow a rapid regulatory approval process for this drug under pandemic threat.
Assuntos
Antivirais/farmacologia , Farmacorresistência Viral/efeitos dos fármacos , Farmacorresistência Viral/genética , Fatores Imunológicos/farmacologia , Subtipo H7N9 do Vírus da Influenza A/efeitos dos fármacos , Interferon-alfa/farmacologia , Animais , Linhagem Celular , Cães , Inibidores Enzimáticos/farmacologia , Humanos , Subtipo H7N9 do Vírus da Influenza A/genética , Influenza Humana/tratamento farmacológico , Influenza Humana/imunologia , Influenza Humana/virologia , Células Madin Darby de Rim Canino , Moduladores de Transporte de Membrana/farmacologia , Mutação , Neuraminidase/antagonistas & inibidores , Neuraminidase/metabolismo , Oseltamivir/farmacologia , Proteínas da Matriz Viral/antagonistas & inibidores , Proteínas da Matriz Viral/metabolismo , Replicação Viral/efeitos dos fármacosAssuntos
Antibacterianos/uso terapêutico , Arteriosclerose/tratamento farmacológico , Infecções por Chlamydophila/tratamento farmacológico , Chlamydophila pneumoniae/efeitos dos fármacos , Animais , Arteriosclerose/microbiologia , Azitromicina/uso terapêutico , Doença das Coronárias/tratamento farmacológico , Farmacorresistência Bacteriana , Quimioterapia Combinada , Fluoroquinolonas/uso terapêutico , Gatifloxacina , HumanosRESUMO
BACKGROUND: Chronic fatigue syndrome/myalgic encephalomyelitis (CFS/ME) is a severely debilitating disease of unknown pathogenesis consisting of a variety of symptoms including severe fatigue. The objective of the study was to examine the efficacy and safety of a TLR-3 agonist, rintatolimod (Poly I: C(12)U), in patients with debilitating CFS/ME. METHODS AND FINDINGS: A Phase III prospective, double-blind, randomized, placebo-controlled trial comparing twice weekly IV rintatolimod versus placebo was conducted in 234 subjects with long-standing, debilitating CFS/ME at 12 sites. The primary endpoint was the intra-patient change from baseline at Week 40 in exercise tolerance (ET). Secondary endpoints included concomitant drug usage, the Karnofsky Performance Score (KPS), Activities of Daily Living (ADL), and Vitality Score (SF 36). Subjects receiving rintatolimod for 40 weeks improved intra-patient placebo-adjusted ET 21.3% (pâ=â0.047) from baseline in an intention-to-treat analysis. Correction for subjects with reduced dosing compliance increased placebo-adjusted ET improvement to 28% (pâ=â0.022). The improvement observed represents approximately twice the minimum considered medically significant by regulatory agencies. The rintatolimod cohort vs. placebo also reduced dependence on drugs commonly used by patients in an attempt to alleviate the symptoms of CFS/ME (pâ=â0.048). Placebo subjects crossed-over to receive rintatolimod demonstrated an intra-patient improvement in ET performance at 24 weeks of 39% (pâ=â0.04). Rintatolimod at 400 mg twice weekly was generally well-tolerated. CONCLUSIONS/SIGNIFICANCE: Rintatolimod produced objective improvement in ET and a reduction in CFS/ME related concomitant medication usage as well as other secondary outcomes. TRIAL REGISTRATION: ClinicalTrials.gov NCT00215800.
Assuntos
Síndrome de Fadiga Crônica/tratamento farmacológico , Poli I-C/farmacologia , Poli I-C/uso terapêutico , Poli U/farmacologia , Poli U/uso terapêutico , Receptor 3 Toll-Like/agonistas , Adulto , Demografia , Método Duplo-Cego , Quimioterapia Combinada , Determinação de Ponto Final , Teste de Esforço , Tolerância ao Exercício , Feminino , Humanos , Masculino , Placebos , Poli I-C/efeitos adversos , Poli U/efeitos adversos , Receptor 3 Toll-Like/metabolismoRESUMO
Circulating nucleic acids (CNA) isolated from serum or plasma are increasingly recognized as biomarkers for cancers. Recently developed next generation sequencing provides high numbers of DNA sequences to detect the trace amounts of unique serum biomarkers associated with breast carcinoma. Serum CNA of 38 women with ductal carcinoma was extracted and sequenced on a 454/Roche high-throughput GS-FLX platform and compared with healthy controls and patients with other medical conditions. Repetitive elements present in CNA were detected and classified, and each repetitive element was normalized based on total sequence count or repeat count. Multivariate regression models were calculated using an information-theoretical approach and multimodel inference. A total of 423,150 and 953,545 sequences for the cancer patients and controls, respectively, were obtained. Data from 26 patients with stages II to IV tumors and from 67 apparently healthy female controls were used as the training data set. Using a bootstrap method to avoid sampling bias, a five-parameter model was developed. When this model was applied to a validation data set consisting of patients with tumor stage I (n = 10) compared with healthy and nonmalignant disease controls (n = 87; 1,261,561 sequences) a sensitivity of 70% at a specificity of 100% was obtained. At a diagnostic specificity level of 95%, a sensitivity of 90% was calculated. Identification of specific breast cancer-related CNA sequences provides the basis for the development of a serum-based routine laboratory test for breast cancer screening and monitoring.
Assuntos
Neoplasias da Mama/sangue , Neoplasias da Mama/genética , Carcinoma Ductal/sangue , Carcinoma Ductal/genética , Ácidos Nucleicos/sangue , Ácidos Nucleicos/genética , Biomarcadores/análise , Biomarcadores/sangue , Neoplasias da Mama/diagnóstico , Carcinoma Ductal/diagnóstico , Interpretação Estatística de Dados , Feminino , Marcadores Genéticos/genética , Humanos , Sequências Repetitivas Dispersas/genética , Programas de Rastreamento , Modelos Estatísticos , Biologia Molecular/métodos , Monitorização Fisiológica , Ácidos Nucleicos/análise , Valores de Referência , Análise de Regressão , Análise de Sequência de DNA/métodos , SoftwareRESUMO
Using recently available mass sequencing and assembly technologies, we have been able to identify and quantify unique cell-free DNA motifs in the blood of patients with multiple sclerosis (MS). The most common MS clinical syndrome, relapsing-remitting MS (RRMS), is accompanied by a unique fingerprint of both inter- and intragenic cell-free circulating nucleic acids as specific DNA sequences that provide significant clinical sensitivity and specificity. Coding genes that are differentially represented in MS serum encode cytoskeletal proteins, brain-expressed regulators of growth, and receptors involved in nervous system signal transduction. Although coding genes distinguish RRMS and its clinical activity, several repeat sequences, such as the L1M family of LINE elements, are consistently different in all MS patients and clinical status versus the normal database. These data demonstrate that DNA motifs observed in serum are characteristic of RRMS and disease activity and are promising as a clinical tool in monitoring patient responses to treatment modalities.
Assuntos
DNA/sangue , Esclerose Múltipla/sangue , Esclerose Múltipla/genética , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Esclerose Múltipla Recidivante-Remitente/sangue , Esclerose Múltipla Recidivante-Remitente/genética , Análise de Sequência de DNARESUMO
A standardized molecular test for the detection of Chlamydophila pneumoniae DNA in cerebrospinal fluid (CSF) would assist the further assessment of the association of C. pneumoniae with multiple sclerosis (MS). We developed and validated a qualitative colorimetric microtiter plate-based PCR assay (PCR-EIA) and a real-time quantitative PCR assay (TaqMan) for detection of C. pneumoniae DNA in CSF specimens from MS patients and controls. Compared to a touchdown nested-PCR assay, the sensitivity, specificity, and concordance of the PCR-EIA assay were 88.5%, 93.2%, and 90.5%, respectively, on a total of 137 CSF specimens. PCR-EIA presented a significantly higher sensitivity in MS patients (p = 0.008) and a higher specificity in other neurological diseases (p = 0.018). Test reproducibility of the PCR-EIA assay was statistically related to the volumes of extract DNA included in the test (p = 0.033); a high volume, which was equivalent to 100 microl of CSF per reaction, yielded a concordance of 96.8% between two medical technologists running the test at different times. The TaqMan quantitative PCR assay detected 26 of 63 (41.3%) of positive CSF specimens that tested positive by both PCR-EIA and nested-PCR qualitative assays. None of the CSF specimens that were negative by the two qualitative PCR methods were detected by the TaqMan quantitative PCR. The PCR-EIA assay detected a minimum of 25 copies/ml C. pneumoniae DNA in plasmid-spiked CSF, which was at least 10 times more sensitive than TaqMan. These data indicated that the PCR-EIA assay possessed a sensitivity that was equal to the nested-PCR procedures for the detection of C. pneumoniae DNA in CSF. The TaqMan system may not be sensitive enough for diagnostic purposes due to the low C. pneumoniae copies existing in the majority of CSF specimens from MS patients.