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The spirochaete bacterium Borrelia burgdorferisensu lato is the aetiologic agent of Lyme disease. Borrelia is transmitted to mammals through tick bite and is adapted to survive at tick and mammalian physiological temperatures. We have previously shown that B. burgdorferi can exist in different morphological forms, including the antibiotic-resistant biofilm form, in vitro and in vivo. B. burgdorferi forms aggregates in ticks as well as in humans, indicating potential of biofilm formation at both 23 and 37 °C. However, the role of various environmental factors that influence Borrelia biofilm formation remains unknown. In this study, we investigated the effect of tick (23 °C), mammalian physiological (37 °C) and standard in vitro culture (33 °C) temperatures with the objective of elucidating the effect of temperature on Borrelia biofilm phenotypes invitro using two B. burgdorferisensu stricto strains (B31 and 297). Our findings show increased biofilm quantity, biofilm size, exopolysaccharide content and enhanced adherence as well as reduced free spirochaetes at 37 °C for both strains, when compared to growth at 23 and 33 °C. There were no significant variations in the biofilm nano-topography and the type of extracellular polymeric substance in Borrelia biofilms formed at all three temperatures. Significant variations in extracellular DNA content were observed in the biofilms of both strains cultured at the three temperatures. Our results indicate that temperature is an important regulator of Borrelia biofilm development, and that the mammalian physiological temperature favours increased biofilm formation in vitro compared to tick physiological temperature and in vitro culture temperature.
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Biofilmes , Borrelia burgdorferi/fisiologia , Doença de Lyme/microbiologia , Doença de Lyme/fisiopatologia , Carrapatos/microbiologia , Carrapatos/fisiologia , Animais , Temperatura Corporal , Borrelia burgdorferi/genética , HumanosRESUMO
Pancreatic cystic disease, including duct dilation, represents precursor states towards the development of pancreatic cancer, a form of malignancy with relatively low incidence but high mortality. While most of these cysts (>85%) are benign, the remainder can progress over time, leading to malignant transformation, invasion, and metastasis. Cytologic diagnosis is challenging, limited by the paucity or complete absence of cells representative of cystic lesions and fibrosis. Molecular analysis of fluids collected from endoscopic-guided fine-needle aspiration of pancreatic cysts and dilated duct lesions can be used to evaluate the risk of progression to malignancy. The basis for the enhanced diagnostic utility of molecular approaches is the ability to interrogate cell-free nucleic acid of the cyst/duct and/or extracellular fluid. The allelic imbalances at tumor suppressor loci and the selective oncogenic drivers are used clinically to help differentiate benign stable pancreatic cysts from those progressing toward high-grade dysplasia. Methods are discussed and used to determine the efficacy for diagnostic implementation. Here, we report the analytical validation of methods to detect causally associated molecular changes integral to the pathogenesis of pancreatic cancer from pancreatic cyst fluids.
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BACKGROUND: Recognizing aggressive tumor biology is essential to optimizing patient management for papillary thyroid carcinomas (PTC). Aggressive lymph node (ALN) status is one feature that influences decision-making. We evaluated genomic deletions in regions of tumor suppressor genes, detected by loss of heterozygosity (LOH) analysis, to understand causal alterations linked to thyroid cancer aggressiveness and to serve as a molecular diagnostic biomarker for ALN status. METHODS: We analyzed 105 primary PTC enriched for patients with ALN (64% with, 36% without). We also analyzed 39 positive lymph nodes (79% with, 21% without ALN). LOH was determined using a panel of 25 polymorphic microsatellite alleles targeting 10 genomic loci harboring common tumor suppressor genes. Additionally, ThyGeNEXT® and ThyraMIR® assays were performed. RESULTS: LOH was detected in 43/67 primary PTC from patients with ALN status, compared with only 5/38 primary PTC without ALN (minimal metastatic burden) (P=0.0000003). This is further supported by post hoc analyses of paired primary and metastatic samples. Paired samples from patients with ALN are more likely to harbor LOH, compared to the ALN negative group (P=0.0125). Additionally, 12/31 paired samples from patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. No association was seen between ALN and mutational, translocation, or microRNA data. CONCLUSIONS: LOH detected in primary PTC significantly predicts ALN status. Analysis of paired primary and metastatic samples from patients with / without ALN status further supports this relationship. The acquisition of LOH at additional loci is common in lymph nodes from patients with ALN status. SIMPLE SUMMARY: A subset of patients with papillary thyroid carcinoma (PTC) will develop recurrent disease. One known predictor of recurrence is the American Thyroid Association category "Aggressive Lymph Node" (ALN) disease, considering metastatic burden. Loss of heterozygosity (LOH) - chromosomal loss in regions of tumor suppressor genes - has yet to be investigated as a possible mechanism driving ALN status in PTC. The ability to predict ALN status prior to surgery can guide the extent of surgery and postoperative treatment options. We found that paired samples from patients with ALN are more likely to harbor LOH, compared to patients without ALN disease. 38% of patients with ALN demonstrated additional or different LOH loci in metastatic samples compared to the primary tumor samples. LOH complements current molecular analysis of thyroid cancer when searching for evidence of aggressive biology.
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Perda de Heterozigosidade , Neoplasias da Glândula Tireoide , Humanos , Câncer Papilífero da Tireoide/genética , Perda de Heterozigosidade/genética , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Mutação , Genes Supressores de TumorRESUMO
Background: The addition of genetic analysis to the evaluation of thyroid nodule fine-needle aspiration biopsy samples improves diagnostic accuracy of cytologically indeterminate thyroid nodules (ITNs) with Bethesda III or IV cytopathology. We previously reported the performance of a multiplatform molecular test, referred to in this study as MPTXv1, that includes a mutation panel (ThyGeNEXT®) plus an algorithmic microRNA (miRNA) risk classifier (ThyraMIR®). Complex interactions of growth-promoting and -suppressing miRNAs affect the phenotype. We previously demonstrated that accounting for these interactions with pairwise miRNA expression analysis improves the diagnosis of medullary thyroid carcinoma. In this study, we assess the impact of pairwise miRNA expression analysis on risk stratification of ITNs. Methods: Pairwise expression analysis of 11 miRNAs was performed on a training cohort of histopathology-proven benign nodules (n = 50) to define the mean and standard deviation of each pairwise analysis and create a Benign/Malignant Profiler (MPTXv2), deviations from which predicted the malignancy risk. Clinical validation of MPTXv2 was assessed using a cohort of 178 ITN (Bethesda III and IV) samples from a multicentered, blinded retrospective study, previously evaluated by MPTXv1. Results: Compared with MPTXv1, MPTXv2 significantly improved the test performance. The receiver operating characteristic (ROC) areas under the curve (AUC) increased from 0.85 to 0.97 (p < 0.001), and the diagnostic accuracy at the positive threshold increased significantly (p < 0.05) from 83% [95% confidence interval (CI) = 76-88] to 93% [CI = 89-96]. The significant improvement in the ROC AUC and the diagnostic accuracy was due to a strong statistical trend for improvement in specificity at the positive threshold. At the positive threshold, the specificity for MPTXv1 was 90% [CI = 84-95] and improved to 98% [CI = 94-99] for MPTXv2. Using the MPTXv2, the Moderate-Risk cohort decreased from 50 samples (28% of the cohort) to 24 samples (13% of the cohort). This 52% decrease is statistically significant (p < 0.001) and clinically meaningful. Conclusion: As compared with MPTXv1, pairwise miRNA expression analysis used in MPTXv2 significantly improved the diagnostic accuracy of ITN risk stratification and reduced the size of the Moderate-Risk group. Prospective trials are indicated to confirm these findings in a clinical practice setting.
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MicroRNAs , Neoplasias da Glândula Tireoide , Nódulo da Glândula Tireoide , Humanos , Nódulo da Glândula Tireoide/diagnóstico , Nódulo da Glândula Tireoide/genética , Nódulo da Glândula Tireoide/patologia , Estudos Retrospectivos , Estudos Prospectivos , Neoplasias da Glândula Tireoide/diagnóstico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , MicroRNAs/genética , MicroRNAs/análise , MutaçãoRESUMO
BACKGROUND: Molecular testing of thyroid fine-needle aspirates has demonstrated value in cases of indeterminate cytology (Bethesda categories III, IV, and V) enabling optimized individual patient management leading to better outcomes with health economic benefits. For most molecular testing modalities, including mutational panels and classifier analyses, part or all of a dedicated needle aspiration pass is required to obtain an adequate sample for testing. Our analysis, which is based on a combination approach (mutation detection and microRNA classifier status), has documented clinical validity and utility when performed on thyroid fine-needle aspirates placed directly into RNA preservative fluid. Here we show that the combination approach can be extended to microdissected stained cytology slides provides the physician greater opportunity to resolve cytological indeterminacy. METHODS: Extracted nucleic acid from needle aspirate and corresponding cytology preparations of 47 thyroid nodules were analyzed using identical methodology and results were compared. RESULTS: Of 94 molecular analyses (47 mutational analyses, 47 microRNA classifier assessments based on a validated 10 marker panel) only 5 samples showed discordant results. CONCLUSION: These findings, together with supplementary work using archival specimens shows that the combination approach can be effectively applied to both direct aspirated thyroid nodule aspirates or to nucleic acid extracted from macrodissected and microdissected cytology slide smears, with the expectation of equivalent results. The advantages of both specimen sources, direct aspirate, and cytology slide smears are discussed.
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Biomarcadores Tumorais/genética , Microdissecção/métodos , Neoplasias da Glândula Tireoide/diagnóstico , Humanos , MicroRNAs/classificação , MicroRNAs/genética , Mutação , Neoplasias da Glândula Tireoide/genéticaRESUMO
BACKGROUND: Limited data are available on the analysis of somatic mutations in metastatic lymph nodes in adult and pediatric patients with papillary thyroid carcinomas. METHODS: A total of 92, microdissected, formalin-fixed, paraffin-embedded tissue specimens from 39 patients were analyzed for the presence of somatic mutations utilizing the ThyGenX next-generation sequencing test. RESULTS: Somatic mutations were detected in 67% of papillary thyroid carcinoma specimens. The majority of patients with synchronous and all 6 patients with radioactive iodine-resistant (metachronous) metastatic lymph nodes contained BRAF mutations. Four patients had mutations detected in their metastatic lymph nodes that were not detected in their primary tumors. For the most part, BRAF mutations were seen in adults, and RAS mutations were seen in children. CONCLUSION: Findings of different mutations in metastatic lymph nodes compared with the primary papillary thyroid carcinomas are probably the result of tumor heterogeneity. The presence of the BRAF mutation in metastatic lymph nodes might be responsible for the recurrence of papillary thyroid carcinomas and resistance to radioactive iodine therapy. The good prognosis observed in papillary thyroid carcinomas found in pediatric and young adult patients might be explained by the predominance of RAS rather than BRAF mutations.
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Carcinoma/genética , Análise Mutacional de DNA , Linfonodos/patologia , Recidiva Local de Neoplasia/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias da Glândula Tireoide/genética , Adulto , Fatores Etários , Idoso , Biópsia por Agulha , Carcinoma/patologia , Carcinoma Papilar , Criança , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática/genética , Masculino , Pessoa de Meia-Idade , Mutação , Recidiva Local de Neoplasia/patologia , Prognóstico , Medição de Risco , Estudos de Amostragem , Fatores Sexuais , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/patologia , Tireoidectomia/métodos , Adulto JovemRESUMO
Bacterial biofilms are microbial communities held together by an extracellular polymeric substance matrix predominantly composed of polysaccharides, proteins and nucleic acids. We had previously shown that Borrelia burgdorferi sensu stricto, the causative organism of Lyme disease in the United States is capable of forming biofilms in vitro. Here, we investigated biofilm formation by B. afzelii and B. garinii, which cause Lyme disease in Europe. Using various histochemistry and microscopy techniques, we show that B. afzelii and B. garinii form biofilms, which resemble biofilms formed by B. burgdorferi sensu stricto. High-resolution atomic force microscopy revealed similarities in the ultrastructural organization of the biofilms form by three Borrelia species. Histochemical experiments revealed a heterogeneous organization of exopolysaccharides among the three Borrelia species. These results suggest that biofilm formation might be a common trait of Borrelia genera physiology.