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Our purpose is to investigate the feasibility of imaging tumor metabolism in breast cancer patients using 13C magnetic resonance spectroscopic imaging (MRSI) of hyperpolarized 13C label exchange between injected [1-13C]pyruvate and the endogenous tumor lactate pool. Treatment-naïve breast cancer patients were recruited: four triple-negative grade 3 cancers; two invasive ductal carcinomas that were estrogen and progesterone receptor-positive (ER/PR+) and HER2/neu-negative (HER2-), one grade 2 and one grade 3; and one grade 2 ER/PR+ HER2- invasive lobular carcinoma (ILC). Dynamic 13C MRSI was performed following injection of hyperpolarized [1-13C]pyruvate. Expression of lactate dehydrogenase A (LDHA), which catalyzes 13C label exchange between pyruvate and lactate, hypoxia-inducible factor-1 (HIF1α), and the monocarboxylate transporters MCT1 and MCT4 were quantified using immunohistochemistry and RNA sequencing. We have demonstrated the feasibility and safety of hyperpolarized 13C MRI in early breast cancer. Both intertumoral and intratumoral heterogeneity of the hyperpolarized pyruvate and lactate signals were observed. The lactate-to-pyruvate signal ratio (LAC/PYR) ranged from 0.021 to 0.473 across the tumor subtypes (mean ± SD: 0.145 ± 0.164), and a lactate signal was observed in all of the grade 3 tumors. The LAC/PYR was significantly correlated with tumor volume (R = 0.903, P = 0.005) and MCT 1 (R = 0.85, P = 0.032) and HIF1α expression (R = 0.83, P = 0.043). Imaging of hyperpolarized [1-13C]pyruvate metabolism in breast cancer is feasible and demonstrated significant intertumoral and intratumoral metabolic heterogeneity, where lactate labeling correlated with MCT1 expression and hypoxia.
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Neoplasias de la Mama/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Isótopos de Carbono/química , Isótopos de Carbono/metabolismo , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , L-Lactato Deshidrogenasa/genética , L-Lactato Deshidrogenasa/metabolismo , Imagen por Resonancia Magnética/instrumentación , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Ácido Pirúvico/química , Ácido Pirúvico/metabolismo , Simportadores/genética , Simportadores/metabolismoRESUMEN
BACKGROUND: Circulating tumour DNA (ctDNA) carrying tumour-specific sequence alterations may provide a minimally invasive means to dynamically assess tumour burden and response to treatment in cancer patients. Somatic TP53 mutations are a defining feature of high-grade serous ovarian carcinoma (HGSOC). We tested whether these mutations could be used as personalised markers to monitor tumour burden and early changes as a predictor of response and time to progression (TTP). METHODS AND FINDINGS: We performed a retrospective analysis of serial plasma samples collected during routine clinical visits from 40 patients with HGSOC undergoing heterogeneous standard of care treatment. Patient-specific TP53 assays were developed for 31 unique mutations identified in formalin-fixed paraffin-embedded tumour DNA from these patients. These assays were used to quantify ctDNA in 318 plasma samples using microfluidic digital PCR. The TP53 mutant allele fraction (TP53MAF) was compared to serum CA-125, the current gold-standard response marker for HGSOC in blood, as well as to disease volume on computed tomography scans by volumetric analysis. Changes after one cycle of treatment were compared with TTP. The median TP53MAF prior to treatment in 51 relapsed treatment courses was 8% (interquartile range [IQR] 1.2%-22%) compared to 0.7% (IQR 0.3%-2.0%) for seven untreated newly diagnosed stage IIIC/IV patients. TP53MAF correlated with volumetric measurements (Pearson r = 0.59, p < 0.001), and this correlation improved when patients with ascites were excluded (r = 0.82). The ratio of TP53MAF to volume of disease was higher in relapsed patients (0.04% per cm3) than in untreated patients (0.0008% per cm3, p = 0.004). In nearly all relapsed patients with disease volume > 32 cm3, ctDNA was detected at ≥20 amplifiable copies per millilitre of plasma. In 49 treatment courses for relapsed disease, pre-treatment TP53MAF concentration, but not CA-125, was associated with TTP. Response to chemotherapy was seen earlier with ctDNA, with a median time to nadir of 37 d (IQR 28-54) compared with a median time to nadir of 84 d (IQR 42-116) for CA-125. In 32 relapsed treatment courses evaluable for response after one cycle of chemotherapy, a decrease in TP53MAF of >60% was an independent predictor of TTP in multivariable analysis (hazard ratio 0.22, 95% CI 0.07-0.67, p = 0.008). Conversely, a decrease in TP53MAF of ≤60% was associated with poor response and identified cases with TTP < 6 mo with 71% sensitivity (95% CI 42%-92%) and 88% specificity (95% CI 64%-99%). Specificity was improved when patients with recent drainage of ascites were excluded. Ascites drainage led to a reduction of TP53MAF concentration. The limitations of this study include retrospective design, small sample size, and heterogeneity of treatment within the cohort. CONCLUSIONS: In this retrospective study, we demonstrated that ctDNA is correlated with volume of disease at the start of treatment in women with HGSOC and that a decrease of ≤60% in TP53MAF after one cycle of chemotherapy was associated with shorter TTP. These results provide evidence that ctDNA has the potential to be a highly specific early molecular response marker in HGSOC and warrants further investigation in larger cohorts receiving uniform treatment.
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Carcinoma/sangre , Carcinoma/genética , ADN de Neoplasias/sangre , ADN de Neoplasias/genética , Neoplasias Ováricas/sangre , Neoplasias Ováricas/genética , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/sangre , Carcinoma/metabolismo , Femenino , Humanos , Persona de Mediana Edad , Mutación , Células Neoplásicas Circulantes/metabolismo , Neoplasias Ováricas/metabolismo , Estudios Retrospectivos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: The management of metastatic breast cancer requires monitoring of the tumor burden to determine the response to treatment, and improved biomarkers are needed. Biomarkers such as cancer antigen 15-3 (CA 15-3) and circulating tumor cells have been widely studied. However, circulating cell-free DNA carrying tumor-specific alterations (circulating tumor DNA) has not been extensively investigated or compared with other circulating biomarkers in breast cancer. METHODS: We compared the radiographic imaging of tumors with the assay of circulating tumor DNA, CA 15-3, and circulating tumor cells in 30 women with metastatic breast cancer who were receiving systemic therapy. We used targeted or whole-genome sequencing to identify somatic genomic alterations and designed personalized assays to quantify circulating tumor DNA in serially collected plasma specimens. CA 15-3 levels and numbers of circulating tumor cells were measured at identical time points. RESULTS: Circulating tumor DNA was successfully detected in 29 of the 30 women (97%) in whom somatic genomic alterations were identified; CA 15-3 and circulating tumor cells were detected in 21 of 27 women (78%) and 26 of 30 women (87%), respectively. Circulating tumor DNA levels showed a greater dynamic range, and greater correlation with changes in tumor burden, than did CA 15-3 or circulating tumor cells. Among the measures tested, circulating tumor DNA provided the earliest measure of treatment response in 10 of 19 women (53%). CONCLUSIONS: This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer. (Funded by Cancer Research UK and others.).
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Biomarcadores de Tumor/sangre , Neoplasias de la Mama/secundario , ADN de Neoplasias/sangre , Mucina-1/sangre , Metástasis de la Neoplasia/diagnóstico , Neoplasias de la Mama/sangre , Neoplasias de la Mama/genética , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Mutación , Metástasis de la Neoplasia/diagnóstico por imagen , Metástasis de la Neoplasia/genética , Pronóstico , Radiografía , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodos , Carga TumoralRESUMEN
Neuroferritinopathy is a disorder of neurodegeneration with brain iron accumulation that has no proven disease-modifying treatments. Clinical trials require biomarkers of iron deposition. We examined brain iron accumulation in one presymptomatic FTL mutation carrier, two individuals with neuroferritinopathy and one healthy control using ultra-high-field 7T MRI. There was increased magnetic susceptibility, suggestive of iron deposition, in superficial and deep gray matter in both presymptomatic and symptomatic neuroferritinopathy. Cavitation of the putamen and globus pallidus increased with disease stage and at follow up. The widespread brain iron deposition in presymptomatic and early disease provides an opportunity for monitoring disease-modifying intervention.
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Trastornos del Metabolismo del Hierro , Hierro , Imagen por Resonancia Magnética , Distrofias Neuroaxonales , Humanos , Distrofias Neuroaxonales/diagnóstico por imagen , Distrofias Neuroaxonales/genética , Distrofias Neuroaxonales/metabolismo , Distrofias Neuroaxonales/patología , Trastornos del Metabolismo del Hierro/diagnóstico por imagen , Trastornos del Metabolismo del Hierro/metabolismo , Trastornos del Metabolismo del Hierro/genética , Hierro/metabolismo , Adulto , Masculino , Femenino , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Persona de Mediana Edad , Apoferritinas/metabolismo , Apoferritinas/genéticaRESUMEN
Aim: To investigate the influence of fluorine in reducing the adsorption of immune-reactive proteins onto PEGylated gold nanoparticles. Methods: Reversible addition fragmentation chain transfer polymerization, the Turkevich method and ligand exchange were used to prepare polymer-coated gold nanoparticles. Subsequent in vitro physicochemical and biological characterizations and proteomic analysis were performed. Results: Fluorine-modified polymers reduced the adsorption of complement and other immune-reactive proteins while potentially improving circulatory times and modulating liver toxicity by reducing apolipoprotein E adsorption. Fluorine actively discouraged phagocytosis while encouraging the adsorption of therapeutic targets, CD209 and signaling molecule calreticulin. Conclusion: This study suggests that the addition of fluorine in the surface coating of nanoparticles could lead to improved performance in nanomedicine designed for the intravenous delivery of cargos.
Nanomedicines are based around the delivery of therapies by tiny, nanosized delivery vehicles. This method offers a much better way of specifically targeting life-threatening diseases. For fast delivery, nanomedicines can be injected into the blood (intravenously); however, this often leads to an unwanted and exaggerated immune response. The immune system is activated by proteins in the blood that attach themselves to nanoparticles through various chemical interactions (the protein corona effect). Fluorine is a chemical routinely used in surfactants such as firefighting foam and more recently in molecular imaging and nanoparticles designed for the delivery of therapies aimed at cancer. While fluorine has great potential to improve the cellular uptake of therapies, little is known about whether it can also help camouflage the nanoparticles against the immune system responses. Here, using fluorinated polymer-coated gold nanoparticles, the authors demonstrate that fluorine reduces uptake by immune cells and is highly effective at reducing the binding of immune system-initiating proteins. This work successfully illustrates the rationale for more widespread investigation of fluorine during the development of polymer-coated nanoparticles designed for the intravenous delivery of nanomedicines.
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Flúor , Oro , Nanopartículas del Metal , Polietilenglicoles , Oro/química , Nanopartículas del Metal/química , Flúor/química , Adsorción , Polietilenglicoles/química , Humanos , Polímeros/química , Fagocitosis/efectos de los fármacos , Animales , Propiedades de Superficie , Proteínas del Sistema Complemento/inmunología , Proteínas del Sistema Complemento/metabolismo , RatonesRESUMEN
A leading explanation for translational failure in neurodegenerative disease is that new drugs are evaluated late in the disease course when clinical features have become irreversible. Here, to address this gap, we cognitively profiled 21,051 people aged 17-85 years as part of the Genes and Cognition cohort within the National Institute for Health and Care Research BioResource across England. We describe the cohort, present cognitive trajectories and show the potential utility. Surprisingly, when studied at scale, the APOE genotype had negligible impact on cognitive performance. Different cognitive domains had distinct genetic architectures, with one indicating brain region-specific activation of microglia and another with glycogen metabolism. Thus, the molecular and cellular mechanisms underpinning cognition are distinct from dementia risk loci, presenting different targets to slow down age-related cognitive decline. Participants can now be recalled stratified by genotype and cognitive phenotype for natural history and interventional studies of neurodegenerative and other disorders.
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Cognición , Genotipo , Humanos , Anciano , Persona de Mediana Edad , Anciano de 80 o más Años , Adolescente , Adulto , Adulto Joven , Femenino , Estudios de Cohortes , Masculino , Apolipoproteínas E/genética , Envejecimiento/genética , InglaterraRESUMEN
Patients with brain cancers including medulloblastoma lack treatments that are effective long-term and without side effects. In this study, a multifunctional fluoropolymer-engineered iron oxide nanoparticle gene-therapeutic platform is presented to overcome these challenges. The fluoropolymers are designed and synthesized to incorporate various properties including robust anchoring moieties for efficient surface coating, cationic components to facilitate short interference RNA (siRNA) binding, and a fluorinated tail to ensure stability in serum. The blood-brain barrier (BBB) tailored system demonstrates enhanced BBB penetration, facilitates delivery of functionally active siRNA to medulloblastoma cells, and delivers a significant, almost complete block in protein expression within an in vitro extracellular acidic environment (pH 6.7) - as favored by most cancer cells. In vivo, it effectively crosses an intact BBB, provides contrast for magnetic resonance imaging (MRI), and delivers siRNA capable of slowing tumor growth without causing signs of toxicity - meaning it possesses a safe theranostic function. The pioneering methodology applied shows significant promise in the advancement of brain and tumor microenvironment-focused MRI-siRNA theranostics for the better treatment and diagnosis of medulloblastoma.
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Barrera Hematoencefálica , Silenciador del Gen , Meduloblastoma , ARN Interferente Pequeño , Meduloblastoma/genética , Meduloblastoma/metabolismo , Meduloblastoma/terapia , Barrera Hematoencefálica/metabolismo , Animales , Ratones , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/administración & dosificación , Humanos , Modelos Animales de Enfermedad , Nanopartículas de Magnetita/química , Imagen por Resonancia Magnética/métodos , Línea Celular Tumoral , Polímeros/química , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/terapiaRESUMEN
We aimed to provide a detailed phenotypic description of status epilepticus (SE) in a large cohort of patients with POLG disease and identify prognostic biomarkers to improve the management of this life-threatening condition. In a multinational, retrospective study with data on patients with POLG disease from seven European countries, we identified those who had SE. The age of SE onset, accompanying clinical, laboratory, imaging and genetic findings were analysed. One hundred and ninety-five patients with genetically confirmed POLG disease were recruited, of whom 67% (130/194) had epilepsy. SE was identified in 77% (97/126), with a median age of SE onset of 7 years. SE was the presenting symptom of the disease in 43% (40/93) of those with SE, while 57% (53/93) developed SE during the disease course. Convulsive SE was reported in 97% (91/94) followed by epilepsia partialis continua in 67% (56/84). Liver impairment 78% (74/95), ataxia 69% (60/87), stroke-like episodes 57% (50/88), were the major comorbidities. In the majority (66%; 57/86) with SE this became refractory or super-refractory. The presence of seizures was associated with significantly higher mortality compared to those without (P ≤ 0.001). The median time from SE debut to death was 5 months. SE is a major clinical feature of POLG disease in early and juvenile to adult-onset disease and can be the presenting feature or arise as part of a multisystem disease. It is associated with high morbidity and mortality, with the majority of patients with SE going on to develop refractory or super-refractory SE.
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ADN Polimerasa gamma , Estado Epiléptico , Humanos , Estado Epiléptico/etiología , Estado Epiléptico/genética , Masculino , Femenino , Adulto , Adolescente , Adulto Joven , Estudios Retrospectivos , Niño , Europa (Continente)/epidemiología , ADN Polimerasa gamma/genética , Preescolar , Persona de Mediana Edad , Lactante , Enfermedades Mitocondriales/genética , Enfermedades Mitocondriales/complicaciones , Enfermedades Mitocondriales/epidemiología , Edad de InicioRESUMEN
Analysis of circulating tumor DNA (ctDNA) to monitor cancer dynamics and detect minimal residual disease has been an area of increasing interest. Multiple methods have been proposed but few studies have compared the performance of different approaches. Here, we compare detection of ctDNA in serial plasma samples from patients with breast cancer using different tumor-informed and tumor-naïve assays designed to detect structural variants (SVs), single nucleotide variants (SNVs), and/or somatic copy-number aberrations, by multiplex PCR, hybrid capture, and different depths of whole-genome sequencing. Our results demonstrate that the ctDNA dynamics and allele fractions (AFs) were highly concordant when analyzing the same patient samples using different assays. Tumor-informed assays showed the highest sensitivity for detection of ctDNA at low concentrations. Hybrid capture sequencing targeting between 1,347 and 7,491 tumor-identified mutations at high depth was the most sensitive assay, detecting ctDNA down to an AF of 0.00024% (2.4 parts per million, ppm). Multiplex PCR targeting 21-47 tumor-identified SVs per patient detected ctDNA down to 0.00047% AF (4.7 ppm) and has potential as a clinical assay.
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Neoplasias de la Mama , ADN Tumoral Circulante , Humanos , Femenino , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/genética , Biomarcadores de Tumor/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ADN Tumoral Circulante/genética , MutaciónRESUMEN
OBJECTIVE: To explore the possibilities of radioligands against the mitochondrial outer membrane protein TSPO as biomarkers for mitochondrial disease, we performed PET (PET)-MR brain imaging with [11C]PK11195 in 14 patients with genetically confirmed mitochondrial disease and 33 matched controls. METHODS: A case-control study of PET-MR imaging with the TSPO radioligand [11C]PK11195. RESULTS: Forty-six percent of symptomatic patients had volumes of abnormal radiotracer binding greater than the 95th percentile in controls. [11C]PK11195 binding was generally greater in grey matter and significantly decreased in white matter. This was most striking in patients with nuclear TYMP or mitochondrial m.3243A>G MT-TL1 mutations, in keeping with differences in mitochondrial density seen post mortem. Some regional binding patterns corresponded to clinical presentation and underlying mutation, even in the absence of structural changes on MRI. This was most obvious for the cerebellum, where patients with ataxia had decreased binding in the cerebellar cortex, but not necessarily volume loss. Overall, there was a positive correlation between aberrant [11C]PK11195 binding and clinical severity. CONCLUSION: These findings endorse the use of PET imaging with TSPO radioligands as a non-invasive in vivo biomarker of mitochondrial pathology. CLASSIFICATION OF EVIDENCE: This study provides Class III evidence that PET-MR brain imaging with TSPO radioligands identifies mitochondrial pathology.
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Purpose: To compare hyperpolarized carbon 13 (13C) MRI with dynamic contrast material-enhanced (DCE) MRI in the detection of early treatment response in breast cancer. Materials and Methods: In this institutional review board-approved prospective study, a woman with triple-negative breast cancer (age, 49 years) underwent 13C MRI after injection of hyperpolarized [1-carbon 13 {13C}]-pyruvate and DCE MRI at 3 T at baseline and after one cycle of neoadjuvant therapy. The 13C-labeled lactate-to-pyruvate ratio derived from hyperpolarized 13C MRI and the pharmacokinetic parameters transfer constant (K trans) and washout parameter (k ep) derived from DCE MRI were compared before and after treatment. Results: Exchange of the 13C label between injected hyperpolarized [1-13C]-pyruvate and the endogenous lactate pool was observed, catalyzed by the enzyme lactate dehydrogenase. After one cycle of neoadjuvant chemotherapy, a 34% reduction in the 13C-labeled lactate-to-pyruvate ratio resulted in correct identification of the patient as a responder to therapy, which was subsequently confirmed via a complete pathologic response. However, DCE MRI showed an increase in mean K trans (132%) and mean k ep (31%), which could be incorrectly interpreted as a poor response to treatment. Conclusion: Hyperpolarized 13C MRI enabled successful identification of breast cancer response after one cycle of neoadjuvant chemotherapy and may improve response prediction when used in conjunction with multiparametric proton MRI.Published under a CC BY 4.0 license.
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Neoplasias de la Mama , Terapia Neoadyuvante , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Medios de Contraste , Femenino , Humanos , Imagen por Resonancia Magnética , Persona de Mediana Edad , Estudios Prospectivos , Resultado del TratamientoRESUMEN
Circulating tumour DNA (ctDNA) detection and monitoring have enormous potential clinical utility in oncology. We describe here a fast, flexible and cost-effective method to profile multiple genes simultaneously in low input cell-free DNA (cfDNA): Next Generation-Targeted Amplicon Sequencing (NG-TAS). We designed a panel of 377 amplicons spanning 20 cancer genes and tested the NG-TAS pipeline using cell-free DNA from two HapMap lymphoblastoid cell lines. NG-TAS consistently detected mutations in cfDNA when mutation allele fraction was > 1%. We applied NG-TAS to a clinical cohort of metastatic breast cancer patients, demonstrating its potential in monitoring the disease. The computational pipeline is available at https://github.com/cclab-brca/NGTAS_pipeline .
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Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Biomarcadores de Tumor/sangre , Línea Celular Tumoral , ADN Tumoral Circulante/sangre , Costos y Análisis de Costo , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Humanos , Mutación , Análisis de Secuencia de ADN/economíaRESUMEN
A 73-year-old woman was admitted to the hospital via the emergency room with increasing pain and tenderness in the right axilla and high fevers. She had a long history of peripheral vascular disease with multiple graft failures and recent insertion of a right axillofemoral graft. A computed tomography study of the chest obtained at admission confirmed graft patency and a small fluid collection around the graft at the level below the right axilla. It also confirmed a saccular aneurysm of the aortic arch. A SPECT/CT study of the chest at 3 hours after reinjection of autologous leukocytes labeled with Tc-99m showed intense labeled leukocyte uptake around the vascular graft in the lateral chest wall. Culture of the infected tissue around the excised graft grew Staphylococcus aureus sensitive to all tested antibiotics. Vascular graft infections are rare, with few reported cases of axillofemoral graft infections utilizing Ga-67. Labeled leukocytes have been used successfully in the setting together with CT scanning. This is a rare case of labeled leukocyte uptake in an infected axillofemoral graft by SPECT/CT.
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Fémur/trasplante , Rechazo de Injerto/diagnóstico por imagen , Infecciones Relacionadas con Prótesis/diagnóstico por imagen , Tomografía Computarizada de Emisión de Fotón Único/métodos , Tomografía Computarizada por Rayos X/métodos , Anciano , Axila/diagnóstico por imagen , Femenino , Fémur/diagnóstico por imagen , Humanos , Leucocitos/diagnóstico por imagenRESUMEN
Circulating tumor DNA (ctDNA) offers new opportunities for noninvasive cancer management. Detecting ctDNA in plasma is challenging because it constitutes only a minor fraction of the total cell-free DNA (cfDNA). Pre-analytical factors affect cfDNA levels contributed from leukocyte lysis, hence the ability to detect low-frequency mutant alleles. This study investigates the effects of the delay in processing, storage temperatures, different blood collection tubes, centrifugation protocols, and sample shipment on cfDNA levels. Peripheral blood (n = 231) from cancer patients (n = 62) were collected into K3EDTA or Cell-free DNA BCT tubes and analyzed by digital PCR, targeted amplicon, or shallow whole-genome sequencing. To assess pre-analytic effects, plasma was processed under different conditions after 0, 6, 24, 48, 96 hours, and 1 week at room temperature or 4°C, or using different centrifugation protocols. Digital PCR showed that cfDNA levels increased gradually with time in K3EDTA tubes, but were stable in BCT tubes. K3EDTA samples stored at 4°C showed less variation than room temperature storage, but levels were elevated compared with BCT. A second centrifugation at 3000 × g gave similar cfDNA yields compared with higher-speed centrifugation. Next-generation sequencing showed negligible differences in background error or copy number changes between K3EDTA and BCT, or following shipment in BCT. This study provides insights into the effects of sample processing on ctDNA analysis.