Efficacy of antiviral therapy and host-virus interactions visualized using serial liver sampling with fine-needle aspirates
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ABSTRACT:
ORGANISM(S): Hepatitis B virus
PROVIDER: GSE234015 | GEO | 2023/06/07
REPOSITORIES: GEO
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Project description:An ultra-performance liquid chromatography with triple quadrupole mass spectrometry method was developed and validated for the determination of direct acting antiviral drug concentrations in human liver fine needle aspirates. Liver fine needle aspirate (FNA) biopsy samples were homogenized in acetonitrile to stabilize the analytes and precipitate protein. The acetonitrile supernatants were diluted with internal standards and mobile phase. Separation was achieved with a Waters Acquity BEH C18 column (50×2.1mm, 1.7um) with a gradient elution of 0.1% formic acid in water and acetonitrile. The total run time was 4.25min. Detection of analytes was achieved using electrospray ionization (positive mode) and triple quadrupole selected reaction monitoring. Standard curve concentrations ranged from 12.5 to 5000ng/mL for dasabuvir and the m1 metabolite of dasabuvir, 1.25 to 2500ng/mL for ombitasvir and ritonavir, and 5.00 to 5000ng/mL for paritaprevir. The intra- and inter-day accuracy and precision were less than 13.7% in low, medium, and high quality control samples. The validated method was applied to the analysis of a liver fine needle aspirate of a patient undergoing direct acting antiviral therapy for hepatitis C virus.
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Project description:Immunohistochemistry-based clinical diagnoses require invasive core biopsies and use a limited number of protein stains to identify and classify cancers. We introduce a technology that allows analysis of hundreds of proteins from minimally invasive fine-needle aspirates (FNAs), which contain much smaller numbers of cells than core biopsies. The method capitalizes on DNA-barcoded antibody sensing, where barcodes can be photocleaved and digitally detected without any amplification steps. After extensive benchmarking in cell lines, this method showed high reproducibility and achieved single-cell sensitivity. We used this approach to profile ~90 proteins in cells from FNAs and subsequently map patient heterogeneity at the protein level. Additionally, we demonstrate how the method could be used as a clinical tool to identify pathway responses to molecularly targeted drugs and to predict drug response in patient samples. This technique combines specificity with ease of use to offer a new tool for understanding human cancers and designing future clinical trials.
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Project description:BackgroundTo compare the sampling adequacy and diagnostic efficiency of ultrasound-guided fine-needle aspiration with 22-, 25-gauge needles and capillary sampling with 22-gauge needle in the biopsy of cervical lymph node.MethodsA total of 130 cervical lymph nodes from 103 patients were consecutively included in the prospective study. Each suspected lymph node was aspirated with a 22-gauge needle, capillary sampled with a 22-gauge needle and aspirated with a 25-gauge needle. The adequacy rates and nondiagnostic rates of obtained specimen were calculated.ResultsOf the 130 suspected lymph nodes, there were 77 lymph nodes<6.0 mm and 53 lymph nodes≥6.0mm in the smallest dimension. Both FNA22G and FNC22G got significantly higher sampling adequacy than FNA25G for the total lymph nodes. For lymph nodes<6.0 mm, the sampling adequacy was significantly higher with FNA22G than with FNA25G for each parameter and the cumulative score (all P<0.05), while no difference were seen between FNA22G and FNC22G, and between FNC22G and FNA25G. There were higher nondiagnostic rates for FNA25G compared with FNA22G and FNC22G in all lymph nodes and in each size subgroups. FNA25G yielded more diagnostically inadequate specimens than FNA22G and FNC22G did in the total lymph nodes (P=0.002), in lymph nodes<6.0 mm (P=0.014), and in those ≥ 6.0 mm (P=0.000).ConclusionsFNA22G and FNC22G obtained more diagnostically adequate specimens than FNA25G in cervical lymph nodes. FNA22G and FNC22G may be more suitable than FNA25G in diagnosing cervical lymph nodes. FNA22G and FNC22G may yield specimens with similar quality.
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Project description:Methods to determine individualized breast cancer risk lack sufficient sensitivity to select women most likely to benefit from preventive strategies. Alterations in DNA methylation occur early in breast cancer. We hypothesized that cancer-specific methylation markers could enhance breast cancer risk assessment. We evaluated 380 women without a history of breast cancer. We determined their menopausal status or menstrual cycle phase, risk of developing breast cancer (Gail model), and breast density and obtained random fine-needle aspiration (rFNA) samples for assessment of cytopathology and cumulative methylation index (CMI). Eight methylated gene markers were identified through whole-genome methylation analysis and included novel and previously established breast cancer detection genes. We performed correlative and multivariate linear regression analyses to evaluate DNA methylation of a gene panel as a function of clinical factors associated with breast cancer risk. CMI and individual gene methylation were independent of age, menopausal status or menstrual phase, lifetime Gail risk score, and breast density. CMI and individual gene methylation for the eight genes increased significantly (P < 0.001) with increasing cytological atypia. The findings were verified with multivariate analyses correcting for age, log (Gail), log (percent density), rFNA cell number, and body mass index. Our results demonstrate a significant association between cytological atypia and high CMI, which does not vary with menstrual phase or menopause and is independent of Gail risk and mammographic density. Thus, CMI is an excellent candidate breast cancer risk biomarker, warranting larger prospective studies to establish its utility for cancer risk assessment. Cancer Prev Res; 9(8); 673-82. ©2016 AACR.
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Project description:BackgroundDespite substantial efforts, reliable preoperative diagnostic for human thyroid malignancies in case of cytologically indeterminate nodules is still missing, resulting in high number of unnecessary thyroidectomies. In an attempt to increase precision of existing preoperative diagnostics, we aimed at validating the panel of molecular biomarkers predictive for papillary thyroid carcinoma (PTC) in preoperative fine needle aspirate (FNA) samples.MethodsIn this prospective study conducted in preoperative thyroid FNA from 44 thyroid nodules, expression levels of 11 molecular biomarkers previously validated on the postoperative samples of PTCs were measured by Cell-to-CT and QuantiGene Plex methods and correlated with final diagnosis.ResultsThe QuantiGene Plex resulted in reliable gene expression measurements for FNA and core-needle biopsy (CNB) samples, however this method was less sensitive than pre-amplification based Cell-to-CT. Measurements conducted on the same samples by the two methods significantly correlated for most of the genes. Expression levels of TIMP1, c-MET and ARNTL were upregulated in PTC nodules as compared to benign counterparts, supporting previous post-operative studies. Strong correlation was observed between these biomarker alterations in the same samples. Within the sub-group of 15 indeterminate nodules (Bethesda II-V), TIMP1 had 100% specificity and 83% sensitivity for PTC cases.ConclusionsAssessment of TIMP1, c-MET and core-clock gene ARNTL expression levels by QuantiGene Plex assay in FNA samples holds promise as an ancillary method to the cytological preoperative diagnostics.
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Project description:Background and aimsBiomarker use for pancreatic cancer diagnosis has been impaired by a lack of samples suitable for reliable quantitative RT-PCR (qRT-PCR). Fine needle aspirates (FNAs) from pancreatic masses were studied to define potential causes of RNA degradation and develop methods for accurately measuring gene expression.MethodsSamples from 32 patients were studied. RNA degradation was assessed by using a multiplex PCR assay for varying lengths of glyceraldehyde-3-phosphate dehydrogenase, and effects on qRT-PCR were determined by using a 150-bp and a 80-bp amplicon for RPS6. Potential causes of and methods to circumvent RNA degradation were studied by using FNAs from a pancreatic cancer xenograft.ResultsRNA extracted from pancreatic mass FNAs was extensively degraded. Fragmentation was related to needle bore diameter and could not be overcome by alterations in aspiration technique. Multiplex PCR for glyceraldehyde-3-phosphate dehydrogenase could distinguish samples that were suitable for qRT-PCR. The use of short PCR amplicons (<100 bp) provided reliable gene expression analysis from FNAs. When appropriate samples were used, the assay was highly reproducible for gene copy number with minimal (0.0003 or about 0.7% of total) variance.ConclusionsThe degraded properties of endoscopic FNAs markedly affect the accuracy of gene expression measurements. Our novel approach to designate specimens "informative" for qRT-PCR allowed accurate molecular assessment for the diagnosis of pancreatic diseases.
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